Cellular and Molecular Physiology of Melatonin Receptors in Xenopus

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Jun 5, 1999 - M-T Teh ▫ Cellular & Molecular Studies on XenopusMelanophores ▫ Thesis 2000. 1 ...... The first melatonin receptor subtype was identified by expression cloning ..... position within individual cells, viewed under the microscope, based on the ... (Chapter 6) and effects of light on melanophores (Chapter 7).
Thesis Title

Cellular and Molecular Studies on Pigment-Granule Translocation in Xenopus laevis Melanophores* A thesis presented by

MUY-TECK TEH for the degree of Doctor of Philosophy in the University of London.

Physiology Division, Guy's, King's and St. Thomas' School of Biomedical Sciences, King's College London, University of London. MARCH 2000

* The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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Acknoledgements

Acknowledgements I owe a debt of gratitude to my supervisor Dr. David Sugden for his constant guidance and support throughout my PhD research. Without his enthusiasm in my work, his ingenious ideas and suggestions for solving obstacles encountered during my research, this thesis would not have materialized. His daily supervision and discussion has helped me to 'finetune' my foundation and skills in scientific research. My gratitude also extends to Dr. Kevin Pedley for his help with immunocytochemistry and confocal imaging on the melanophores. Without his help, the interesting Chapter 8 would not have appeared in this thesis! This work enabled us to visualize the dynamic organization of cytoskeleton during melanosome translocation. An image worth a thousand words! I am indebted to Mr. Gennaro Ruocco who 'entrains' my lifestyle to a healthy daily-cycle and maintains my social interactions. I cannot be more grateful for his constant support, and allowing me to use his multimedia PC for writing and printing this thesis. Finally, I am especially indebted to my Mum who gives love, my Dad whose love is silent and my Sis who understands us all.

In remembrance of Mr. Teh Siew Hooi March 2000

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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Table of Contents

Table of Contents Acknowledgements ...............................................3 Table of Contents...................................................3 List of Figures ........................................................7 List of Tables..........................................................10 Abbreviations.........................................................11 Thesis Abstract.......................................................13

Chapter 1: General Introduction

15

1.1. Background..................................................................................................................15 1.2. The Pineal Gland ........................................................................................................16 1.2.a. Non-Mammalian Vertebrates.............................................................................16 1.2.b. Mammals..............................................................................................................16 1.3. Control of Mammalian Pineal Melatonin Synthesis............................................17 1.3.a. Neural Innervation...............................................................................................17 1.3.b. Sympathetic Control............................................................................................17 1.4. Biosynthesis of Melatonin..........................................................................................18 1.4.a. The Role of the Pineal.........................................................................................18 1.4.b. Biosynthetic Pathway..........................................................................................19 1.5. Function of Melatonin................................................................................................21 1.5.a. Seasonal Physiology............................................................................................21 1.5.b. Circadian Rhythms..............................................................................................22 1.5.c. Retinal Physiology...............................................................................................24 1.5.d. Others ...................................................................................................................24 1.6. Melatonin Receptors ..................................................................................................25 1.6.a. Second-Messenger Signal Transduction Mechanisms.....................................26 1.6.a.i. cAMP ...........................................................................................................26 1.6.a.ii. cGMP ..........................................................................................................26 1.6.a.iii. Phospholipids ...............................................................................................27 1.6.a.iv. Calcium .......................................................................................................27 1.7. Melatonin Action on Xenopus Melanophores.......................................................28 1.7.a. Color Change .......................................................................................................28 1.7.a.i. Physiological Colour Change in Amphibia ........................................................29 1.7.b. Signal Transduction of Melanosome Translocation.........................................30 1.7.b.i. Melanosome Dispersion ..................................................................................30 1.7.b.ii. Melanosome Aggregation...............................................................................32 1.8. Melanophore as a Functional Model System........................................................34

Chapter 2: Materials and Methods

37

2.1. Introduction .................................................................................................................37 2.2. Cell Culture..................................................................................................................37 2.2.a. Xenopus Fibroblasts and Melanophores............................................................37 2.2.a.i. Growth of Melanophores .................................................................................37 2.2.a.ii. Subculture of Melanophores............................................................................38 2.2.a.iii. Density-Gradient Partition of Melanophores ....................................................38 2.2.a.iv. Cryopreservation...........................................................................................39 2.2.b. Mammalian Cells.................................................................................................40 2.2.b.i. Subculturing ..................................................................................................40 2.2.b.ii. Cryopreservation ...........................................................................................40 2.3. 2-[125I]-Iodomelatonin Binding Assay .....................................................................41 2.3.a. Membrane Preparation........................................................................................41 2.3.b. Protein Concentration Determination ................................................................41 2.3.c. 2-[125I]-Iodomelatonin Saturation Assay............................................................42 2.3.d. 2-[125I]-Iodomelatonin Competition Assay........................................................42 2.4. Quantification of Melanosome Translocation.......................................................43 2.5. Cyclic AMP Assay ......................................................................................................43 2.5.a. Sample Collection and Assay .............................................................................43 2.5.b. Radioimmunoassay (RIA) ..................................................................................45

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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Table of Contents

2.6. Polymerase Chain Reaction (PCR) .........................................................................45 2.6.a. Poly A+ mRNA Isolation ....................................................................................46 2.6.b. Reverse Transcription (RT)................................................................................46 2.6.c. Design of Primers ................................................................................................46 2.6.d. PCR Analysis.......................................................................................................47 2.6.e. Verification of PCR Products.............................................................................47 2.7. Data Analysis ...............................................................................................................48 2.7.a. 2-[125I]-Iodomelatonin Binding Analysis...........................................................48 2.7.a.i. Determination of KD........................................................................................48 2.7.a.ii. Determination of IC50 .....................................................................................50 2.7.a.iii. Determination of Binding Affinity (Ki) ............................................................50 2.7.b. Analysis of Melanosome Translocation ............................................................50 2.7.b.i. Determination of Agonist Potency (EC50) ..........................................................51 2.7.b.ii. Determination of Antagonist Potency...............................................................51

Chapter 3: Validation of Melanosome Translocation Assay

54

3.1. Introduction .................................................................................................................54 3.2. Materials and Methods..............................................................................................55 3.3. Results ...........................................................................................................................55 3.3.a. Granule Translocation as a Reporter System for Receptor Activation ...........55 3.3.a.i. Cell Density and Absorbance ...........................................................................55 3.3.a.ii. Growth Profile...............................................................................................56 3.3.a.iii. Time-Course, Ligand Concentration and Melanosome Translocation..................58 3.3.a.iv. Cell Density and Melatonin Potency................................................................61 3.3.a.v. Light Sensitivity and Melatonin Potency ...........................................................61 3.3.a.vi. Variations in the Medium and Melanosome Translocation .................................64 3.3.a.vii. Temperature and Melanosome translocation....................................................66 3.4. Discussion .....................................................................................................................69

Chapter 4: Molecular Physiology of Melatonin Receptors

72

4.1. Introduction .................................................................................................................72 4.1.a. Molecular Aspects...............................................................................................72 4.1.b. Receptor Signalling .............................................................................................77 4.1.c. Experimental Aims..............................................................................................77 4.2. Materials and Methods..............................................................................................78 4.2.a. Intracellular Calcium ([Ca2+]i) Measurements...................................................78 4.2.a.i. Spectrofluorimetry ..........................................................................................78 4.2.a.ii. [Ca2+]i-Fluorescence Imaging ..........................................................................79 4.2.b. Drugs ....................................................................................................................79 4.3. Results ...........................................................................................................................83 4.3.a. Melatonin Receptor Pharmacology....................................................................83 4.3.a.i. Native Xenopus Melanophore Melatonin Receptor Binding Studies .....................83 4.3.a.ii. Recombinant Human Melatonin Receptor Binding Studies.................................86 4.3.a.iii. Functional Correlation of Melatonin Receptors.................................................86 4.3.a.iv. Molecular Biology Evidence ..........................................................................91 4.3.b. Signal Transduction of Melatonin-Induced Pigment Aggregation .................92 4.3.b.i. G-Protein Coupling.........................................................................................92 4.3.b.ii. Cyclic AMP..................................................................................................92 4.3.b.iii. Calcium.......................................................................................................99 4.3.b.iv. Protein Kinase C...........................................................................................105 4.3.b.v. Others ..........................................................................................................108 4.4. Discussions....................................................................................................................112 4.4.a. Characterization of Melatonin Receptors in Melanophores.............................112 4.4.b. Melatonin Signal Transduction in Melanophores.............................................113 4.4.b.i. Melatonin Receptor is Coupled to a Gi/o Protein..................................................113 4.4.b.ii. The Role of cAMP and PKA ..........................................................................113 4.4.b.iii. The Role of Calcium .....................................................................................114 4.4.b.iv. The Role of PKC ..........................................................................................116 4.4.b.v. The Role of cGMP.........................................................................................116 4.4.b.vi. The Role of Membrane Ca2+-activated K+ Channels..........................................117 4.4.b.vii. The Role of Small G Proteins ........................................................................117 4.4.b.viii. The Role of Protein Phosphorylation and Dephosphorylation ...........................117 4.4.c. Conclusion............................................................................................................118

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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Table of Contents

Chapter 5: Attenuation of The Melatonin Signal

121

5.1. Introduction .................................................................................................................121 5.1.a. Experimental Aims..............................................................................................122 5.2. Materials and Methods..............................................................................................122 5.2.a. 2-[125I]-Iodomelatonin Binding Assay ...............................................................122 5.2.b. Melanosome Translocation Assay.....................................................................123 5.2.c. Metabolism of Melatonin....................................................................................123 5.2.d. Drugs ....................................................................................................................123 5.3. Results ...........................................................................................................................124 5.3.a. Prolonged Melatonin Treatment.........................................................................124 5.3.a.i. Changes in Receptor Density............................................................................124 5.3.a.ii. Changes in Melatonin Potency ........................................................................125 5.3.b. Deacetylation of [3H]-Melatonin........................................................................126 5.4. Discussion .....................................................................................................................130

Chapter 6: Characterization of Serotonin Receptor

134

6.1. Introduction .................................................................................................................134 6.1.a. Experimental Aims..............................................................................................135 6.2. Methods and Materials..............................................................................................135 6.2.a. Melanosome Translocation Assay .....................................................................135 6.2.b. cAMP Assay........................................................................................................135 6.2.c. RT-PCR Analysis ................................................................................................135 6.2.d. Drugs ....................................................................................................................136 6.3. Results ...........................................................................................................................137 6.3.a. High Doses of Melatonin Activate the Melanophore 5-HT Receptor ............137 6.3.b. Time- and Concentration-Dependent Melanosome Dispersion......................138 6.3.c. Stimulatory Gs-Protein Coupling........................................................................139 6.3.d. Pharmacological Characterization of 5-HT receptors......................................141 6.3.d.i. 5-HT Agonists ...............................................................................................141 6.3.d.ii. 5-HT Antagonists ..........................................................................................142 6.3.e. Expression of 5-HT7 mRNA in Melanophores.................................................142 6.4. Discussion .....................................................................................................................145

Chapter 7: Photoreception in Melanophores

149

7.1. Introduction .................................................................................................................149 7.1.a. Experimental Aims..............................................................................................150 7.2. Methods ........................................................................................................................150 7.2.a. Irradiation of Melanophores ...............................................................................150 7.2.a.i. White and Monochromatic Light ......................................................................150 7.2.a.ii. Ultraviolet (UV) Light ....................................................................................151 7.2.a.iii. Conversion of Irradiance Energy to Photons .....................................................153 7.2.b. cAMP Assay........................................................................................................153 7.2.c. RT-PCR Analysis ................................................................................................153 7.3. Results ...........................................................................................................................154 7.3.a. Effect of Darkness on Melanosome Translocation...........................................154 7.3.a.i. Inhibitory Gi/o-Protein Coupling ........................................................................156 7.3.b. Phototransduction Mechanism...........................................................................156 7.3.b.i. Stimulatory Gs-Protein Coupling ......................................................................156 7.3.b.ii. Light-Stimulated Pigment Dispersion Does Not Involve a Gt Protein ..................157 7.3.b.iii. Light-Stimulated Pigment Dispersion Does Not Involve a Gq Protein..................159 7.3.c. Effect White Light on Melanosome Dispersion................................................160 7.3.c.i. White Light and Melatonin Potency ..................................................................160 7.3.c.ii. Irradiance-Response of White Light on Melanosome Dispersion .........................161 7.3.d. Spectral Sensitivity of Melanosome Dispersion ...............................................161 7.3.d.i. Action Spectrum.............................................................................................161 7.3.d.ii. UV Sensitivity...............................................................................................162 7.3.e. Expression of Melanopsin mRNA in Melanophores .......................................162 Discussion.............................................................................................................................165

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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Table of Contents

Chapter 8: Imaging Melanosome Translocation

169

8.1. Introduction .................................................................................................................169 8.1.a. Experimental Aims..............................................................................................171 8.2. Methods ........................................................................................................................171 8.2.a. Culture of Depigmented Melanophore..............................................................171 8.2.b. Pseudo-Darkfield Illumination Imaging............................................................171 8.2.c. Immunocytochemistry and Confocal Imaging..................................................172 8.3. Results ...........................................................................................................................173 8.3.a. Physiological Responses of Depigmented Melanophores ...............................173 8.3.a.i. Reversible Depigmentation ..............................................................................173 8.3.a.ii. Functional Response of Depigmented Melanophores .........................................173 8.3.a.iii. Melanosome Aggregation in Depigmented Melanophores .................................175 8.4. Confocal Imaging on Melanophores.......................................................................175 8.4.a. Depigmented versus Pigmented Melanophores................................................175 8.4.b. Imaging of F-Actin and α-Tubulin.....................................................................177 8.4.b.i. Dispersed (Control) Melanophores ...................................................................177 8.4.b.ii. Aggregated (Melatonin-treated) Melanophores .................................................177 8.5. Discussion .....................................................................................................................180

Chapter 9: General Discussion

185

9.1. A Functional Model System for Receptor Activation..........................................185 9.1.a. Molecular Aspects of the Melanophore Melatonin Receptors ........................185 9.1.b. Signal Transduction of Melatonin-Induced Pigment Aggregation .................186 9.1.c. Attenuation of Melatonin Signal in Melanophores...........................................190 9.1.d. Characterization of a 5-HT7 Receptor in Melanophores..................................191 9.1.e. Photoreception in Melanophores........................................................................192 9.2. An In Vivo Model System for Cytoskeletal and Motility Studies ......................195 9.3. Concluding Remarks..................................................................................................197

References

199

List of Publications

214

Thesis Information

215

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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List of Figures

List of Figures Chapter 1 Figure 1.1

The chemical structure of melatonin ......................................................................................................15

Figure 1.2

The neuronal control of diurnal pineal melatonin synthesis and the modulation of circadian and seasonal rhythms...............................................................................................................................18

Figure 1.3

Effect of pinealectomy on human plasma melatonin............................................................................19

Figure 1.4

Schematic biosynthetic pathway of melatonin ......................................................................................20

Figure 1.5

Schematic representation of a typical organization of the dermal chromatophore unit in anuran (frogs and toads) skin..................................................................................................................31

Figure 1.6

A schematic diagram summarising the signal transduction cascades of Xenopus melanophore pigment dispersion............................................................................................................32

Figure 1.7

A current model of melatonin-signal transduction cascade mediating Xenopus melanophore pigment aggregation ................................................................................................................................34

Chapter 2 Figure 2.1

Quantification of melanophore pigment aggregation ...........................................................................44

Figure 2.2

Quantification of melanophore pigment dispersion..............................................................................44

Chapter 3 Figure 3.1

The relationship between melanophore cell density and absorbance..................................................56

Figure 3.2

The morphology and size of melanophores in culture..........................................................................57

Figure 3.3

The growth profile of melanophores cultured in a 96-well plate.........................................................59

Figure 3.4

Bright-field images of melanophores in vitro before and after melatonin treatement........................59

Figure 3.5

Time- and concentration-dependent melanosome aggregation and dispersion triggered by melatonin and α-MSH, respectively ......................................................................................................60

Figure 3.6

Effect of melanophore cell density on potency of melatonin...............................................................62

Figure 3.7

Effect of single or repeated exposure to 630 nm monochromatic light used by the platereader for absorbance measurements on melanophores...................................................................................63

Figure 3.8

Effect of room light (~70 µW/cm2) on melatonin potency...................................................................63

Figure 3.9

Effects of changing pH, osmolality and BSA concentration on melanosome translocation. ............65

Figure 3.10 Diurnal changes in melanophore absorbance in constant darkness.....................................................67 Figure 3.11 Changes in melanophore absorbance in constant darkness and temperature......................................68 Figure 3.12 Effect of temperature on melanosome translocation.............................................................................69

Chapter 4 Figure 4.1

Comparison of Xenopus Mel1c, Mel1cα-short & -long and Mel1cβ-short & -long receptor cDNA sequences......................................................................................................................................75

Figure 4.2

Alignment of deduced amino acid sequences of xMel1c, xMel1cα and xMel1cβ receptor subtypes ....................................................................................................................................................76

Figure 4.3

Alignment of deduced amino acid sequences of xMel1c, xmt1 and xMT2 receptor fragments..........76

Figure 4.4

Chemical structure of compounds 1-19 .................................................................................................82

Figure 4.5

Specific 2-[125I]-iodomelatonin binding on Xenopus melanophore nuclei, melanosome, membrane and cytoplasmic fractions.....................................................................................................83

Figure 4.6

Time-course and saturation binding of 2-[125I]-iodomelatonin on Xenopus-melanophore membranes ...............................................................................................................................................84

Figure 4.7

2-[125I]-Iodomelatonin saturation binding studies on recombinant hmt1 and hMT2 receptors expressed in NIH3T3 cells......................................................................................................................85

Figure 4.8

A representative 2-[125I]-iodomelatonin competitive inhibition-binding curves for melatonin, SV448 and K185 on recombinant hmt1 and hMT2 receptors..............................................................87

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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List of Figures

Figure 4.9

A representative determination of melatonin agonist (causes melanosome aggregation) and antagonist (prevents aggregation) activities on Xenopus melanophores by melatonin, SV448 and K185 as indicated .............................................................................................................................88

Figure 4.10 A representative determination of melatonin antagonist potency (pKB) on Xenopus melanophores ...........................................................................................................................................89 Figure 4.11 Pharmacological correlation of recombinant hmt1 and hMT2 receptor subtype binding affinity with antagonist potency on Xenopus melanophores................................................................90 Figure 4.12 An ethidium bromide-stained digitised agarose-gel image of melatonin receptor-subtypes PCR products in Xenopus melanophores. .............................................................................................91 Figure 4.13 Restriction digestion analysis of xmt1, xMT2 and xMel1c PCR products............................................92 Figure 4.14 Effect of pertussis toxin (PTX) on melatonin-stimulated melanosome aggregation..........................93 Figure 4.15 Concentration-dependent melanosome dispersion and increase in cAMP levels triggered by α-MSH, 5-HT and forskolin in melanophores......................................................................................94 Figure 4.16 Effect of Ro 31-8220 on α-MSH, 5-HT and 4β-PDBu concentration-response curves...................95 Figure 4.17 Concentration-response curves of various pharmacological agents that increase intracellular cAMP levels.............................................................................................................................................96 Figure 4.18 Effect of agents expected to inhibit intracellular cAMP signalling on melanosome aggregation ...............................................................................................................................................97 Figure 4.19 Effect of Rp- and Sp-isomers of 8-Br-cAMPs on melanosome translocation....................................98 Figure 4.20 Effect of H89 on α-MSH, 5-HT and melatonin concentration-response curves................................98 Figure 4.21 Effect of H89 on the time course of melatonin-stimulated pigment aggregation...............................99 Figure 4.22 Time- and concentration-dependent melatonin-stimulated melanosome aggregation and inhibition of basal cAMP levels in melanophores.................................................................................100 Figure 4.23 Ca2+ measurements in depigmented melanophores ..............................................................................101 Figure 4.24 A example of digitised fluorescence image of depigmented melanophores loaded with Fura-2-AM ...............................................................................................................................................102 Figure 4.25 Effect of removing extracellular calcium ([Ca2+]o) on melatonin-stimulated pigment aggregation ...............................................................................................................................................103 Figure 4.26 The role of [Ca2+]o on pigment translocation.........................................................................................104 Figure 4.27 Effect of depleting [Ca2+]i stores or quenching [Ca2+]i on pigment aggregation.................................104 Figure 4.28 Effect of PKC inhibition by Ro 31-8220 on the time-course of melanosome aggregation and dispersion..................................................................................................................................................106 Figure 4.29 Effect of phospholipase C inhibitor, U73122 on the time-course of melanosome aggregation and dispersion...........................................................................................................................................107 Figure 4.30 Effect of PKC, PLC and CaM inhibition on the potency of melatonin-stimulated pigment aggregation ...............................................................................................................................................107 Figure 4.31 Effect of increasing intracellular concentration of cGMP on melanosome translocation..................109 Figure 4.32 Effect of BKCa2+ channel activator NS-1619 and blocker TEA on melanosome translocation. .......110 Figure 4.33 Effect of various phosphatase inhibitors on melanosome translocation..............................................111

Chapter 5 Figure 5.1

Effect of prolonged melatonin incubation on receptor density determined by specific 2-[125I]-iodomelatonin binding................................................................................................................124

Figure 5.2

Effect of eserine on melatonin potency after prolonged melatonin incubation ..................................125

Figure 5.3

Effect of eserine on melatonin potency as a function of incubation time............................................126

Figure 5.4

Eserine prevented the degradation of melatonin by melanophores .....................................................127

Figure 5.5

Metabolic pathway of melatonin ............................................................................................................128

Figure 5.6

Metabolism of [3H]-melatonin by Xenopus melanophores..................................................................129

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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List of Figures

Chapter 6 Figure 6.1

Effect of high concentrations of melatonin on melanosome translocation..........................................137

Figure 6.2

Time- and concentration-dependent melanosome dispersion stimulated by 5-HT............................138

Figure 6.3

Effect of ascorbic acid, imipramine and pargyline on the 5-HT concentration-response curve .......139

Figure 6.4

5-HT stimulated pigment dispersion and elevation of melanophore cAMP ......................................140

Figure 6.5

Effect of PKC and PKA inhibitors, Ro 31-8220 and H89, respectively, on 5-HT-stimulated pigment dispersion...................................................................................................................................140

Figure 6.6

Examples of 5-HT agonist concentration-response curves on pigment dispersion at 60 min...........141

Figure 6.7

Examples of 5-HT antagonism by the three most potent antagonists tested, risperidone, methiothepin and mesulergine................................................................................................................143

Figure 6.8

The detection of Xenopus 5-HT7 receptor subtype mRNA expression in melanophores by RT-PCR....................................................................................................................................................144

Chapter 7 Figure 7.1

A diagram illustrating the irradiation of melanophores grown in a 96-well culture plate..................152

Figure 7.2

Effect of prolonged culture (7 days) of melanophores in growth medium in the dark on pigment position.......................................................................................................................................155

Figure 7.3

Effect PTX on dark-stimulated pigment aggregation and light-stimulated dispersion ......................156

Figure 7.4

Effect of room light on the time-course of cAMP levels in melanophores.........................................157

Figure 7.5

No evidence of Gt-protein coupled mechanism in light-stimulated melanosome dispersion............158

Figure 7.6

No evidence of Gq-protein coupled mechanism in light-stimulated melanosome dispersion. ..........159

Figure 7.7

Effect of white light on melatonin potency............................................................................................160

Figure 7.8

Potency of white light on melanosome dispersion................................................................................161

Figure 7.9

Action spectrum of melanophore pigment dispersion ..........................................................................163

Figure 7.10 Effect of UV light on melanosome translocation..................................................................................164 Figure 7.11 The detection of melanopsin mRNA expression in melanophores by RT-PCR................................164

Chapter 8 Figure 8.1

Scanning electron micrographs of a dispersed and an aggregated Xenopus melanophore ...............169

Figure 8.2

A diagram depicting the inhibition of melanin synthesis by the copper chelator, PTC .....................170

Figure 8.3

Principle of pseudo-darkfield illumination microscopy........................................................................172

Figure 8.4

Depigmentation of melanophores by PTC is reversible and repigmented cells respond to melatonin ..................................................................................................................................................174

Figure 8.5

Time- and concentration-dependent melatonin-stimulated inhibition of basal cAMP levels in depigmented melanophores ....................................................................................................................174

Figure 8.6

Pseudo-darkfield illumination imaging on a single depigmented melanophore cell before and after the addition of melatonin.........................................................................................................175

Figure 8.7

Depigmentation reveals fluorescence staining of the melanophore cytoskeleton ..............................176

Figure 8.8

Example of an actively protruding cell with a concentric cortex of F-actin........................................178

Figure 8.9

Confocal images of melatonin-stimulated pigment aggregation in partially depigmented melanophores showing aggregated melanosomes, F-actin and α-tubulin ..........................................179

Figure 8.10 Confocal imaging of pigmented melanophores showing pigment granules, F-actin and nuclei at two different focal planes I (~1µm) and II (~12 µm) from the coverslip.............................180

Chapter 9 Figure 9.1

A schematic diagram summarising the signal transduction of melatonin-mediated melanosome aggregation in the cultured Xenopus melanophore cell line ..........................................189

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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List of Tables

List of Tables Table 2.1

Primer sequences used in PCR analysis for selected Xenopus-receptor genes...................................49

Table 3.1

The potency of melanosome aggregation (pEC50) by melatonin and dispersion (pIC50) by α-MSH at various time points. ...............................................................................................................61

Table 4.1

Membrane equilibrium constant (KD) of recombinant hmt1 & hMT2 and native Xenopus melanophore melatonin receptors...........................................................................................................86

Table 4.2

Melatonin-antagonist binding affinity on recombinant hmt1 & hMT2 and potency on Xenopus melanophores. ..........................................................................................................................90

Table 4.3

Dispersion potency (pIC50) of various receptor ligands on Xenopus melanophores..........................95

Table 6.1

5-HT agonist potency (pIC50) on melanosome dispersion. ..................................................................142

Table 6.2

5-HT antagonist potency (estimated pKB) on melanosome dispersion...............................................143

Table 6.3

Inactive 5-HT antagonists. ......................................................................................................................144

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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Abbreviations

Abbreviations α-MSH [Ca2+]i [Ca2+]o 1F 1R 2F 2R 3F 3R 4R 7F 7R 5-HT 5-HT1-7 5-MIAA 5-ML 5-MT A A/Ala AA AC ADP Af Ai AMP AMV-RT ANOVA AR Arg/R Asn/N Asp/D ATP BB bGM biGM BKCa2+ Bmax bp BSA C C/Cys Ca2+ CaM cAMP cDMEM cGMP CHO CNS COS-7 cpm CREB cRNA CT

alpha-melanocyte stimulating hormone intracellular calcium extracellular calcium forward primer of xmt1 receptor cDNA reverse primer of xmt1 receptor cDNA forward primer of xMT2 receptor cDNA reverse primer of xMT2 receptor cDNA forward primer of xMel1c receptor cDNA reverse primer of xMel1c receptor cDNA reverse primer of xMel1cα and β receptor cDNAs forward primer of 5-HT7 receptor cDNA reverse primer of 5-HT7 receptor cDNA 5-hydroxytryptamine or serotonin subtypes of 5-HT receptors 5-methoxyindoleacetic acid 5-methoxytryptophol 5-methoxytryptamine adrenaline or epinephrine alanine arachidonic acid adenylate cyclase adenosine diphosphate final absorbance initial absorbance adenosine monophosphate avian moloney virus-reverse transcriptase analysis of variance adrenergic receptor(s) arginine asparagine aspartic acid adenosine triphosphate blocking buffer boiled growth medium boiled incubated growth medium large conductance Ca2+-activated K+ channel maximal binding base pair bovine serum albumin cytosine cysteine calcium calmodulin adenosine 3',5'-cyclic monophosphate complete DMEM guanosine 3',5'-cyclic monophosphate Chinese hamster ovary central nervous system monkey fibroblasts counts per minute cAMP-responsive element binding protein complementary ribonucleic acid circadian time

Cys /C D/Asp DAG DMEM DNA dp E/Glu ET-3 ETC F/Phe FBS fGM FSH G Gα α Gβ βγ G/Gly G418 GC Gi/o Gln/Q Glu/E Gly/G GM GnRH Golf GPCR Gq Gs Gt h H/His HEK293 HeLa hmt1 hMT2 HPD I/Ile ICN iGM IML IP3 K/Lys K+ Ki L/Leu L-15 LH Lys/K M/Met MAO MAP MAPK

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

cysteine aspartic acid diacylglycerol Dulbecco’s modified Eagles medium deoxyribonucleic acid depigmented glutamic acid endothelin type-3 endothelin receptor subtype C phenylalanine foetal bovine serum fresh growth medium follicle stimulating hormone guanosine alpha subunit of guanine-binding protein beta-gamma subunit of guanine-binding protein glycine Geneticin guanylate cyclase inhibitory guanine-binding protein glutamine glutamic acid glycine growth medium gonadotropin releasing hormone olfactory-excitatory guanine-binding protein guanine-binding protein coupled receptor inhibitory excitatory guanine-binding protein transducin-excitatory guanine-binding protein hour(s) histidine human embryonic kidney-293 Henrietta Lacks/Helen Lane cervical cancer cells human melatonin receptor subtype-1 human melatonin receptor subtype-2 hypothalamic-pituitary-disconnected isoleucine internal carotid nerve incubated growth medium intermediolateral cell column inositol-1,4,5-trisphosphate lysine potassium inhibition constant leucine Leibovitz-15 medium luteinizing hormone lysine methionine monoamine oxidase mitogen-activated protein mitogen-activated protein kinase

11

Abbreviations

MBH MCH Mel MFB min MopsF MopsR mRNA MT myr N/Asn NA NAT NC ND NIH3T3 p p P P/Pro PCR PD PDE Phe/F PKA PKC PLA PLC PLD PP1 PP2A PP2B PT PTC PTX

mediobasal hypothalamus melanin-concentrating hormone melatonin median forebrain bundle minute(s) forward primer of melanopsin cDNA reverse primer of melanopsin cDNA messenger/polyadenylated ribonucleic acid microtubule myristoylated asparagine noradrenaline or norepinephrine N-acetyltransferase nervii conarii not determined or not done mouse fibroblasts negative logarithm of pigmented statistical probability proline polymerase chain reaction pars digitalis phosphodiesterase phenylalanine protein kinase A protein kinase C phospholipase A phospholipase C phospholipase D protein phosphatase type-1 protein phosphatase type-2A protein phosphatase type-2B or calcineurin pars tuberalis phenylthiocarbamide pertussis toxin

PVN Q/Gln R/Arg RF RHT RIA RNA RNase RNasin rp RPE RT s S/Ser SCG SCL-1 SCN SEM sGC T/Thr TM TRITC Trp/W Tyr/Y V/Val VIP W/Trp XM xMel1c XMmemb xmt1 xMT2 Y/Tyr YL 1/2

M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

paraventricular nucleus glutamine arginine reticular formation retinohypothalamic tract radioimmunoassay ribonucleic acid ribonuclease ribonuclease inhibitor repigmented retinal pigment epithelium reverse transcription second(s) serine superior cervical ganglion orphan receptor for MCH suprachiasmatic nucleus standard error of the mean soluble guanylate cyclase threonine transmembrane domain tetramethylrhodamine isothiocyanate tryptophan tyrosine valine vasoactive intestinal peptide tryptophan Xenopus melanophores Xenopus melatonin receptor subtype-1c membranes of Xenopus melanophores Xenopus homologue of mt1 receptor subtype Xenopus homologue of MT2 receptor subtype tyrosine rat-monoclonal anti-Tyrosinated-α-tubulin

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Abstract

Abstract Xenopus laevis melanophores utilize a variety of G-protein coupled receptors (GPCRs) to rapidly translocate their pigment granules (melanosomes) in response to external stimuli such as hormones and photons to cause physiological colour changes important for courtship and display, camouflage and self defence, photoprotection and thermoregulation. A primary aim of the experiments in this thesis is to identify and characterize the signal transduction mechanisms activated by melatonin, serotonin and light in Xenopus melanophores. The molecular mechanisms involved in the bi-directional melanosome translocation triggered by these GPCRs were also investigated. All three known melatonin receptor-subtype mRNAs were detected in Xenopus brain and melanophores using RT-PCR. The potency of a series of melatonin antagonists in melanophores was compared with their affinity at recombinant hmt1, hMT2 and xMel1c receptors to identify the subtype responsible for melanosome aggregation. Functional correlation results argue against the involvement of hmt1 or hMT2, hence strengthening the role of xMel1c in pigment aggregation. Melatonin activated a pertussis toxin-sensitive Gi/o-coupled receptor which decreases cAMP levels. While elevation of cAMP is a signal for pigment dispersion, attenuation of the AC-cAMP-PKA pathway failed to stimulate pigment aggregation. Agents inhibiting PLC, intracellular Ca2+ mobilization or calmodulin, prevented melatonin-stimulated aggregation. These results show that in addition to cAMP inhibition, melatonin also activated [Ca2+]i mobilization through a Gq-coupled pathway. The role of other intracellular messengers including cGMP, PKC, membrane potential, small G proteins and protein phosphatases was investigated using pharmacological tools. The potency of melatonin declined slowly, but progressively, with prolonged incubation. The role of receptor down regulation in this loss of potency was determined using melanophore membrane binding studies. The loss of potency was found to be entirely due to degradation of melatonin by an eserine-sensitive deacetylase enzyme within the melanophores, not by receptor desensitization or downregulation. At high concentrations, melatonin stimulated an endogenous melanophore 5-HT receptor causing pigment dispersion by elevation of cAMP. Pharmacological, signal transduction and RT-PCR studies indicate that a 5-HT7 receptor subtype was responsible. Light stimulated melanosome dispersion by elevation of cAMP. Neither inhibition of cGMP-dependent PDE, elevation of intracellular cGMP, inhibition of PLC, removal of [Ca2+]o, inhibition of [Ca2+]o influx, elevation of [Ca2+]i nor inhibition of PKC affected light-stimulated dispersion, indicating no role for a Gt- or Gq-protein coupling phototransduction mechanisms. The action spectrum of light on melanosome dispersion had a peak at ~500 nm suggesting the involvement of a vitamin A-based photopigment. In addition, UV light also stimulated pigment dispersion indicating the presence of a distinct UV photoreceptor. Although melanopsin mRNA was detected in the melanophores by RT-PCR, its role as a photoreceptor remains to be elucidated. The dense melanin pigmentation renders fluorescence microscopy in melanophores impossible. A method of reversible depigmentation was developed and used to visualize the cytoskeleton using immunofluorescence and confocal microscopy. Depigmented cells were physiologically and morphologically identical to the pigmented melanophores. Preliminary experiments showed that melanosome aggregation is accompanied by a gross reorganization of actin filaments and cell morphology.

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Chapter 1: General Introduction

Chapter 1

GENERAL INTRODUCTION

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Chapter 1: General Introduction

Chapter 1: General Introduction 1.1. Background The rapid and reversible blanching (lightening) of pigmented amphibian skin by pineal extracts has been known since the early 20th century (McCord and Allan, 1917). However, it was not until the late 1950s when Aaron Lerner and his co-workers (Lerner et al., 1958) isolated the most potent amphibian skin lightening substance ever found  melatonin. These authors fractionated the extracts of hundreds of thousands of bovine pineal glands through a series of separation procedures, and each fraction was then assayed for its biological activity on amphibian skin. The chemical structure of melatonin, N-acetyl-5-methoxytryptamine (see Figure 1.1), was subsequently confirmed by chemical analysis and synthesis of candidate molecules (Lerner et al., 1959).

Figure 1.1 The chemical structure of melatonin with a molecular weight of 232.3. The order of the indoleamine-ring carbons is signified numerically, whereas, side-chain carbons are named alphabetically in Greek letters.

The primary function of pineal melatonin is thought to be a hormonal signal conveying information about the environmental light-dark cycle, received by the gland either by direct photoreception (lower vertebrates) or indirectly via the eye (mammals and humans). The pineal gland of all vertebrates synthesizes and releases melatonin into the bloodstream only during the hours of darkness (Wurtman et al., 1963; Sugden, 1989). Being neutral and highly lipophilic, melatonin is able to diffuse into all parts of an organism. Melatonin may also be synthesized in the retina (in some species) during the night but here it acts as a local hormone important for various aspect of retinal physiology (reviewed in Iuvone, 1995). There is an extensive literature on the importance of pineal melatonin in the control of circadian rhythms and photoperiodic adaptation and reproduction in many seasonal species (reviewed in Cassone, 1990 & 1998).

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1.2. The Pineal Gland 1.2.a. Non-Mammalian Vertebrates In lower vertebrates such as fish, amphibia, reptiles and some avian species, the pineal complex (consisting of the frontal/parietal organ and the pineal gland), lies just below the skin on the dorsal surface of the cranium. It has been called the ‘third eye’ as it possesses a light-sensing apparatus and an endogenous clock machinery, and also shares molecular functions with the ocular eye including nocturnal melatonin synthesis. Hence, in lower vertebrates the endogenous pineal melatonin rhythm, like that found in the eyes, can be directly entrained by the environmental lightdark cycle (reviewed in Alder, 1976; Arendt, 1995a; Cassone, 1990 & 1998). There is evidence from pinealectomy and melatonin administration experiments that the pineal organ of lower vertebrates is closely associated with the circadian oscillator (biological clock) of the animal. It has been shown that the circadian activity rhythm of amphibia can be shifted by changes in the light-dark cycle provided that the pineal organ, not the eyes, is intact (Demian and Taylor, 1977). Pinealectomy in some fish and amphibia tends to disrupt or abolish circadian rhythms altogether, whereas, pinealectomy of reptiles and avian species alters, rather than abolishes, circadian rhythms (reviewed in Cassone, 1990 & 1998). Similarly, melatonin administration has a profound effect on the circadian rhythms of many lower vertebrates.

1.2.b. Mammals In mammals including humans, the pineal gland is connected by a stalk to the habenular commissure which forms part of the posterior roof of the third ventricle (reviewed in Oksche, 1982; Arendt, 1995a). During evolution, the mammalian pineal gland appears to have lost its photoreceptive and endogenous pacemaker functions. However, its secretory function is retained and constitutes its primary role  melatonin synthesis. Nevertheless, the mammalian pineal does express some typical retinal proteins such as the rod-opsin, an apoprotein of the visual pigment rhodopsin, as well as other rod phototransduction components such as S-antigen and rhodopsin kinase (Lolley et al., 1992; Blackshaw and Snyder, 1997). However, due to the absence of the 11-cis and all-trans retinaldehyde chromophores, the pineal rod-opsin is unable to initiate the phototransduction cascade (Korf et al., 1991). Instead, the diurnal rhythm of pineal melatonin synthesis is driven via a neuronal pathway from a master circadian oscillator in the hypothalamus, the suprachiasmatic nucleus (SCN; Moore, 1983; Klein et al., 1991). The SCN oscillator is entrained by a neural connection with the retina (reviewed in Foulkes et al., 1997; Cassone, 1998; see Figure 1.2 and Section 1.5). Unlike lower vertebrates, the physiological role of the pineal in mammalian circadian rhythms is controversial, given the fact that pinealectomy has little or no effect on the circadian rhythms. However, accumulating evidence suggests that the pineal may have a modulatory role in mammalian circadian rhythms (see Section 1.5.b). On the other hand, there is no doubt that pineal melatonin is essential for photoperiodic adaptation in many seasonal mammals (discussed further in Section 1.5.a). M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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1.3. Control of Mammalian Pineal Melatonin Synthesis 1.3.a. Neural Innervation In lower vertebrates, the pineal organ is reciprocally connected to the central nervous system (CNS). However, during phylogenesis, the direct efferent innervation of the mammalian pineal gland is lost, making the pineal an endocrine gland. The most important central afferent pathway is a postganglionic sympathetic innervation arising from the superior cervical ganglion (SCG), which consists of noradrenergic fibers dispersed throughout the interstitial space of the gland (Kappers, 1965; Vollrath, 1981; see Figure 1.2). This sympathetic innervation is the main input controlling rhythmic pineal synthesis (reviewed in Klein, 1993; Arendt, 1995b). Although there is also evidence for parasympathetic (Kenny, 1961; Møller and Liu, 1999), peptidergic (reviewed in Møller et al., 1996), habenular (Ronnekleiv and Møller, 1979) and commissural (Dafny, 1980a & 1980b) innervation, their functional significance remain to be determined.

1.3.b. Sympathetic Control The postganglionic sympathetic fibers originating from the SCG are the major control on pineal melatonin synthesis. Interruption of nerve impulses reaching the gland by section of the preganglionic nerve or ganglionectomy completely abolishes the light-dark control of rhythmic melatonin synthesis. Although other neurotransmitters, have from time to time been suggested to have a role in the control of melatonin synthesis, noradrenaline (NA) is by far the most important (reviewed in Klein, 1985; Arendt, 1995b). Through evolution, the mammalian pineal has lost both the pacemaker and photoreceptive functions found in the pineal of lower vertebrates, whilst its secretory function has been retained. Nevertheless, the rhythmic secretion of pineal melatonin in mammals is still under the control of light-dark changes via an indirect multi-synaptic neuronal pathway (see Figure 1.2). Light acting on retinal photoreceptors is transmitted via the retinohypothalamic tract (RHT) to the SCN, a paired nucleus located above the optic chiasm in the hypothalamus (Moore, 1983; Klein et al., 1991). Nerve impulses from the SCN are relayed to the paraventricular nucleus (PVN) and leave the hypothalamus via the medial forebrain bundle (MFB) and reticular formation (RF) to enter the thoracic spinal cord through the intermediolateral cell column (IML). From here, preganglionic fibres project to the sympathetic chain and synapse with the SCG. Postganglionic noradrenergic fibres from the SCG travel along the internal carotid nerve (ICN) and enter the perivascular space of the pineal gland via the nervii conarii (NC). Light, received by the eye, is known to entrain the circadian rhythms generated by the SCN, as well as 'switching' off the neural signals emerging from the SCN responsible for melatonin synthesis. Hence, the secretion of melatonin is minimal during the day. During the night in absence of light, the postganglionic sympathetic pathway is released from inhibition which causes the release of NA into the perivascular space of the pineal gland. NA acts on both α1- and β1-adrenergic

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receptor subtypes on the plasma membrane of pineal cells (pinealocytes). Stimulation of β1-adrenergic receptors alone has been shown to elevate the activity of a rate-limiting enzyme, N-acetyltransferase (NAT; see Section 1.4 and Figure 1.4) in melatonin synthesis (Klein and Weller, 1970; Klein, 1993). Elegant studies using receptor subtype-selective agonists have shown that although an α1-adrenoceptor selective agonist has little effect on its own, activation of α1-adrenoceptors greatly potentiate the effect of β1 stimulation (Sugden et al., 1985; Sugden, 1989 & 1991).

Figure 1.2 The neuronal control of diurnal pineal melatonin synthesis and the modulation of circadian and seasonal rhythms (modified from Arendt, 1995c & 1995e). Light signal received by the eye (probably by a non-rod, non-cone photoreceptor in the retinal ganglion cells, see Freedman et al., 1999; Lucas et al., 1999) is transmitted via the retinohypothalamic tract (RHT) to the suprachiasmatic nucleus (SCN) in the hypothalamus. Impulses pass from the SCN to the paraventricular nucleus (PVN) and leave the hypothalamus via the medial forebrain bundle (MFB) and reticular formation (RF) then enter the thoracic spinal cord through the intermediolateral cell column (IML). Preganglionic fibres originate in the IML, project to the sympathetic chain and synapse with the superior cervical ganglion (SCG). Postganglionic noradrenergic fibres from the SCG travel along the internal carotid nerve (ICN) and enter the perivascular space of the pineal gland via the nervii conarii (NC). Circulating melatonin influences seasonal and circadian rhythms by acting on the basal hypothalamus and pituitary gland. Pineal melatonin may also modulate retinal function (see text for details).

1.4. Biosynthesis of Melatonin 1.4.a. The Role of the Pineal The major source of circulating melatonin is the pineal gland. Although some melatonin may be synthesized in the eyes in some species, it has been shown that retinal melatonin is metabolized rapidly by a deacetylase and a monoamine oxidase (Cahill and Besharse, 1989; Grace et al., 1991; see also Section 1.5.c and Chapter 5). This may explain the relatively small contribution of ocular

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melatonin to circulating levels (Reppert and Sagar, 1983; Underwood et al., 1984) and the low melatonin content in mammalian retinae (Reiter et al., 1983; Dubocovich et al., 1985). The fact that removal of the pineal gland abolished the nocturnal rise in plasma melatonin levels in rodents, primates and ungulates illustrates the contribution of the pineal to plasma melatonin (reviewed in Arendt, 1995d). The same applies to humans, i.e., pinealectomy abolishes the diurnal plasma melatonin rhythm (Figure 1.3).

Figure 1.3 Effect of pinealectomy on human plasma melatonin. An example of a human subject’s plasma melatonin before (closed symbols) and after (open symbols) removal of a tumour involving the healthy pineal gland. Melatonin concentrations were measured by gas chromatography-mass spectrometry with a limit of detection of ~1 pg/ml. The shaded area indicates the dark phase. This figure was modified from Neuwelt and Lewy, 1983.

1.4.b. Biosynthetic Pathway The first step in melatonin biosynthesis is the active uptake of the dietary essential amino acid tryptophan from the blood circulation into the pinealocytes (refer to Figure 1.4). The rate-limiting enzyme for serotonin (5-hydroxytryptamine, 5-HT) synthesis, tryptophan hydroxylase, adds a hydroxy group to tryptophan at position 5 to give 5-hydroxytryptophan, which is then decarboxylated by 5-hydroxytryptophan decarboxylase to produce serotonin (Sugden, 1989). Although the level of tryptophan hydroxylase is already high in the rat pineal, it has been shown that it exhibits a diurnal rhythm with 2- to 3-fold higher activity at night (Sitaram and Lees, 1978; Shibuya et al., 1978; reviewed in Bernard et al., 1999). In contrast, 5-hydroxytryptophan decarboxylase shows little daily variation (Snyder et al., 1965). Pineal serotonin exhibits a dramatic circadian variation (Quay, 1963; reviewed in Arendt, 1995b).

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Figure 1.4 Schematic biosynthetic pathway of melatonin (modified from Sugden, 1989) with corresponding 24 hrhythms (data from the rat; modified from Klein, 1974 & 1979) of substrates (right panels) and enzymes (left panels). Shaded area represents the dark phase.

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The rate limiting pineal-specific enzyme, N-acetyltransferase (NAT; also named arylalkylamine-N-acetyltransferase), transfers an acetyl group from acetyl coenzyme A to the amino group of serotonin (Weissbach et al., 1960 & 1961). This enzyme has been shown to exhibit a dramatic circadian rhythm in the rat pineal with a 70- to 100-fold increase in activity during the night (Klein and Weller, 1970). This nocturnal increase in NAT activity is a direct response to noradrenergic stimulation of β1- and α1-adrenoceptors on the pinealocytes when the sympathetic activity is high during the night (see Section 1.3.b). As a consequence, N-acetylserotonin also exhibits a diurnal rhythm. The final step involves another pineal-specific enzyme, hydroxyindole-Omethyltransferase (HIOMT), which catalyzes O-methylation at the 5 position of N-acetylserotonin using the methyl donor S-adenosyl methionine (Weissbach et al., 1960), to form melatonin (N-acetyl-5-methoxytryptamine). The rat HIOMT enzyme activity shows only a small diurnal variation but its mRNA levels exhibit a genuine circadian rhythm with a peak at night (Gauer and Craft, 1996). The nocturnal rise in melatonin synthesis in the pinealocytes is directly reflected by plasma levels (see Figure 1.3) as the newly synthesized lipophilic melatonin rapidly diffuses from pinealocytes into the extensive network of fenestrated pineal capillaries.

1.5. Function of Melatonin 1.5.a. Seasonal Physiology Many non-equatorial mammals exhibit seasonal changes in their physiology and behaviour, such as timing of reproduction, change in coat colour and thickness, body weight and fat deposition and hibernation. These changes are vital for adapting the animal to the changing environment which is essential for its survival and reproduction. As seasonal changes are directly related to the length of day (photoperiod), the most direct and reliable seasonal time cue is therefore the annual change in photoperiod. In fact most seasonal animals use photoperiod as an environmental time cue (zeitgeber) for the organization of their seasonal rhythms (reviewed in Arendt, 1986, 1995d & 1998). Pineal melatonin plays a major role in the photoperiodic timing of mammalian seasonal physiology. It is now clear that the pineal gland, through the secretion of melatonin, is essential for photoperiodic time measurement. Pinealectomy in animals such as sheep and hamsters abolishes their ability to respond to photoperiodic changes, and administration of melatonin can mimic darkness and drive seasonal rhythms (reviewed in Arendt, 1995d & 1998). As pineal melatonin secretion is directly controlled by light impacting on the retina, the duration of nocturnal melatonin secretion serves to signify the duration of darkness. The changing profile of melatonin secretion conveys photoperiodic information for the control of seasonal rhythms in animals. The existence of seasonality in humans is controversial. There is evidence from population studies in Western Europe that there tends to be a major peak of conception in the summer (MayJune) and a minor peak in the winter (December). However, a reverse pattern was found in comparable latitudes in the USA and over many years the patterns may shift (reviewed in Arendt,

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1995f). A 10-year statistical study on a large human population (>500,000 male subjects) in Austria showed that height at age 18 is dependent on the month of birth exhibiting a bimodal variation with a 1.0-year period with maxima in spring and minima in autumn differing by 0.6 cm (Weber et al., 1998). There is evidence that the phase of the nocturnal melatonin peak (acrophase) has a slight seasonal difference, i.e., phase delay during the winter and phase advance during the summer (reviewed in Arendt, 1995f & 1998). Furthermore, it has been shown that bright light pulses (2500 lux or ~680 µW/cm2; 1 lux ≈ 0.27 µW/cm2) given as a spring photoperiod can phase advance the delayed-melatonin phase during the of winter in the Antarctic (Broadway et al., 1987). Another convincing study has shown that human subjects kept for 2 months in a short-day photoperiod (8 h light:16 h dark) exhibited slightly longer duration of sleep and nocturnal melatonin secretion when compared to long-day photoperiod (16 h light: 8 h light) exposure (Wehr, 1991). Patients with seasonal affective disorder (SAD) tend to be more seasonal than the general population in terms of their reproduction (conception time) and body weight (reviewed in Arendt, 1995f). Collectively, the evidence suggests that in humans, as in other mammals the melatonin profile does change with season. However, seasonal changes in human physiology are generally minor or difficult to discern, suggesting, perhaps, a phylogenetic remnant of photoperiodism in man. The target sites through which melatonin regulates seasonal reproduction has been a topic of intense research. Evidence from lesion and implant/infusion studies points towards the involvement of the mediobasal hypothalamus (MBH) and pituitary (Lincoln, 1992; Maywood and Hastings, 1995). However, not all effects on seasonal physiology involve the hypothalamus. An ingenious study by Lincoln and Clarke (1994) demonstrated that the seasonal prolactin rhythm persisted and remained responsive to changing artificial photoperiod and melatonin administration in hypothalamic-pituitary-disconnected (HPD) Soay rams. It is known that the photoperiodicallyinduced testicular activity cycle in intact rams is controlled by changes in the pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus (Lincoln and Short, 1980; Rhim et al., 1993). As secretion of gonadotropic hormones [i.e., luteinizing (LH) and follicle stimulating (FSH) hormones] from gonadotrophs in the pars digitalis (PD) were disrupted in HPD sheep (Clarke et al., 1983) and HPD rams, these animals were permanently hypogonadotrophic (Lincoln and Clarke, 1994). These experiments suggest that the action of melatonin on seasonal rhythms is specific to the type of seasonal effect and may involve the hypothalamus and pituitary independently.

1.5.b. Circadian Rhythms There is no doubt that the pineal gland of many lower vertebrates has a central role in the regulation of circadian rhythms. For example, pinealectomy in birds can lead to arrhythmicity in constant darkness (Lu and Cassone, 1993; reviewed in Cassone, 1998). However, the role of pineal (melatonin) in mammalian and human circadian physiology is less clear. Studies on pinealectomized rodents (rats, Quay, 1968; hamsters, Aschoff et al., 1982) have failed to show any effect on freerunning activity rhythms in constant darkness. Nevertheless, it has been shown that pinealectomized

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rodents (rats, Quay, 1970; hamsters, Finkelstein et al., 1978; Anguilar-Roblero and Vega-Gonzalez, 1993) re-entrained more rapidly to large shift of artificial photoperiod than intact or sham-operated animals. A more recent study showed that pinealectomized rats with free-running rhythms were sensitive to incremental increase in light intensity and entrained more readily than their shamoperated controls (Warren and Cassone, 1995). Furthermore, numerous studies have shown that arrhythmic or free-running rats in constant conditions can be entrained by exogenous melatonin (e.g., Cassone et al., 1986; Chesworth et al., 1987). Consistent with the mechanisms found in nonmammalian vertebrates, these studies suggest a role of the pineal melatonin in the regulation of photic sensitivity of the mammalian circadian oscillator (reviewed in Rusak, 1982; Cassone, 1998). An increasing number of studies have also shown that melatonin is important in the regulation of human circadian rhythms (Lewy et al., 1991; Deacon and Arendt, 1994; Oren et al., 1997; Cagnacci, 1997). Although melatonin appears not to be important for endogenous circadian organization, it appears to reinforce many physiological functions (e.g., inactivity or sleep, drop in body temperature) associated with darkness (reviewed in Arendt et al., 1999). Furthermore, administration of melatonin at an appropriate time can advance or delay the timing of sleep in humans as well as entrain free-running circadian rhythms (Lockley et al., 2000). However, the role of the melatonin on circadian sleep-wake rhythm in humans remains controversial as many of these studies involved pharmacological doses of melatonin. The SCN is known to be a primary site of melatonin action in mammals. Surgical ablation of the rat SCN abolishes endogenous circadian rhythms (Moore, 1983) and blocks the entraining effects of melatonin (Cassone et al., 1986). This effect is not dependent on rhythmicity per se as arrhythmic rats in constant light can be re-entrained by exogenous melatonin (Chesworth et al., 1987). The SCN of many mammalian species, including humans, show high-affinity 2-[125I]iodomelatonin binding sites (Vanecek, 1988; Reppert et al., 1988; Cassone, 1998). In species such as mink (Mustela vison) where 2-[125I]-iodomelatonin fails to bind to the SCN, exogenous melatonin does not entrain their free-running activity rhythm (Bonneford et al., 1993). This supports the view that melatonin receptors within the SCN may be required for its circadian effects. In vitro studies using rat SCN slices have shown that melatonin produced a phase advance of the endogenous rhythm of electrical-firing. The response is restricted to two specific circadian times, one near dusk (CT10-14; Starkey et al., 1995; McArthur et al., 1997) and the other near dawn (CT23-0) (McArthur et al., 1997). Both groups also demonstrated that the melatonin-induced phase advance at CT10 is concentration-related with low picomolar potency values (pEC50 ~12, Starkey et al., 1995; pEC50 ~13, McArthur et al., 1997). Recently, a high-affinity melatonin receptor agonist, GR196429, was found to have similar phase-advance potency (pEC50 ~12, Beresford et al., 1998) at CT10. The melatonin-stimulated phase advance of SCN firing was shown to be sensitive to pertussis toxin pre-treatment (McArthur et al., 1997) and could be blocked by the melatonin receptor antagonist, luzindole (Starkey et al., 1995) suggesting the involvement of melatonin receptor. However, the subtype(s) of melatonin receptor responsible for the phase shifting in the SCN remains

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controversial. A study found that the Mel1b (now MT2; see Section 1.6) receptor gene of the Siberian hamster, Phodopus sungorus, cannot encode a functional receptor but its seasonal and circadian responses to melatonin remain (Weaver et al., 1996). This suggests that the Mel1a (now mt1; see Section 1.6), not Mel1b, receptor may be responsible for the reproductive and circadian responses to melatonin. Interestingly, the same investigators later found melatonin can no longer cause the acute inhibition of neuronal-firing in SCN of Mel1a receptor-knockout mice, whereas, the phase-shifting effect of melatonin remains intact (Liu et al., 1997). Disruption of the Mel1a receptor gene eliminates all detectable 2-[125I]-iodomelatonin binding in the SCN of knockout mice. The authors suggest that the Mel1a receptor subtype is responsible for the acute neuronal inhibition of the SCN, whilst, the remaining Mel1b receptor may be important for the phase-shifting response to melatonin. Nevertheless, this theory remains speculative. Evidence from simultaneous disruption of both receptor subtypes is required to confirm the role these receptor subtypes in circadian physiology.

1.5.c. Retinal Physiology Melatonin, synthesized in the retina (reviewed in Iuvone, 1995), serves as a local hormone, and is known to regulate various aspects of retinal physiology such as retinomotor movement of rods and cones and melanosomes in retinal pigment epithelial cells (Burnside and Nagle, 1983; Besharse et al., 1988), shedding and phagocytosis of photoreceptor outer segments (Besharse and Dunis, 1983), and release of dopamine from amacrine cells (Dubocovich, 1983). Recently, Tosini and Menaker (1996) found a circadian clock in Syrian golden hamster (Mesocricetus auratus) eyes which generates a photo-entrainable diurnal ocular melatonin rhythm in vitro. A similar endogenous melatonin rhythm was found in the mouse eye (Tosini and Menaker, 1998). This suggests that the mammalian eye possesses an endogenous oscillator which regulates ocular melatonin synthesis as in non-mammalian species.

1.5.d. Others Although melatonin receptors have been identified in a number of peripheral tissues (reviewed in Morgan et al., 1994), the functional significance in many of these sites is largely unknown. Nevertheless, the first and the best recognized peripheral function of melatonin is its ability to cause physiological colour change by stimulating pigment aggregation in Xenopus melanophores, which led to the discovery of melatonin (discussed in Section 1.7). One of the better characterized melatonin functions is its effect on thermoregulation in animals. In poikilothermic (or ectothermic) vertebrates, the pineal organ is vital for physiological (e.g., skin colour change) and behavioural (e.g., selection of shade or basking in the sun, body contact with cold/warm surface, panting rate) thermoregulation as metabolic rate and locomotor activity are dependent on the body temperature. Shielding of the pineal organ, pinealectomy and administration of melatonin all have profound effects on thermoregulation (reviewed in Hutchison

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and Dupre, 1992). Melatonin may also have a role in mammalian thermoregulation in some species. Melatonin produces vasoconstriction of the rat tail (caudal) artery through the mt1 melatonin receptor subtype (Ting et al., 1999). In nocturnal rats, melatonin may have a role in reducing primary heat loss from the rat tail by enhancing caudal artery vasoconstriction at night (Viswanathan et al., 1990). In humans, data demonstrate that melatonin decreases body temperature regardless of the time of day of administration (Krauchi et al., 1997a & 1997b), as well as phase advancing the circadian body temperature rhythm when given in the early evening (Krauchi et al., 1997b). The increase in circulating melatonin at night contributes to the circadian fall in core body temperature which occurs at night, and may have a role in initiating sleep (Krauchi et al., 1997a & 1999). Although some studies have used doses of melatonin which produce circulating blood levels equivalent to those seen at night (e.g., Dollins et al., 1994; Deacon and Arendt, 1995), many other studies (e.g., Krauchi et al., 1997a & 1997b) have used doses expected to give blood levels well above the physiological nighttime peak (30-120 pg /ml) (Waldhauser et al., 1984; Sanders et al., 1999). Such studies may reveal interesting pharmacological effects of melatonin, but must be interpreted cautiously with regard to potential physiological roles of the pineal hormone. Many studies (reviewed in Blask et al., 1999) have reported an antiproliferative action of melatonin on tumor cells, and its ability to act as an antioxidant scavenging free-radicals, has been observed in many model systems (reviewed in Reiter et al., 1999).

1.6. Melatonin Receptors The physiological effects of melatonin are thought to be elicited through its binding to highaffinity GPCRs. There are currently three subtypes of melatonin receptor which have been cloned: mt1 (previous name Mel1a)*, MT2 (Mel1b)* and Mel1c. *[for receptor nomenclature see IUPHAR classification in Dubocovich et al., 1998; the new (mt1 and MT2) receptor nomenclature has been used throughout this thesis, however on occasion when discussing older articles the previous nomenclature has been used]. Studies have shown that these are high affinity (KD: 40-160 pM) receptors and belong to the seven-transmembrane guanine-nucleotide (G-protein) coupled receptor superfamily (reviewed in Dubocovich, 1995; Reppert et al., 1996a). The first melatonin receptor subtype was identified by expression cloning from Xenopus melanophores (Ebisawa et al., 1994), and was later designated Mel1c. Two other receptor subtype mRNA fragments have been identified in the Xenopus genome, but it is not known if they are expressed in Xenopus melanophores. The Mel1c receptor has also been found in chicken and zebrafish, but it has not been identified in mammals including humans. The mt1 and MT2 receptor subtypes have been identified in birds and mammals, including humans (reviewed in Reppert et al., 1996a; Reppert, 1997). The mt1 receptor is expressed in the rodent SCN and pituitary pars tuberalis (PT), presumed sites of melatonin action on the circadian 'clock' and neuroendocrine regulation of seasonal breeding, respectively (Reppert et al., 1994). Although the MT2 receptor mRNA has been identified in human M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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retina and brain by reverse-transcription polymerase chain reaction (RT-PCR), its mRNA could not be detected by in situ hybridization in the brain suggesting low abundance (Reppert et al., 1995a).

1.6.a. Second-Messenger Signal Transduction Mechanisms 1.6.a.i. cAMP In cells expressing recombinant melatonin receptor subtypes and tissues expressing high affinity melatonin binding, melatonin reduces forskolin-stimulated cAMP production. This action is blocked by pertussis toxin (PTX) treatment of cells suggesting the involvement of a Gi/o protein which is negatively coupled to adenylate cyclase (AC) (reviewed in Vanecek, 1998). However, there are exceptions. Melatonin does not decrease forskolin-stimulated cAMP in the SCN (Cutler et al., 1997). Native hmt1 receptors (Bmax 1.1 fmol/mg protein; Conway et al., 1997b) in the human embryonic kidney (HEK293) cell line fail to inhibit forskolin-stimulated cAMP accumulation (Yung et al., 1995; Conway et al., 1997b). Melatonin does not inhibit forskolin stimulated cAMP production in rat tail artery (Ting et al., 1999). Although melatonin does inhibit GnRH-stimulated cAMP production in neonatal rat gonadotrophs, the inhibition of GnRH-induced luteinizing hormone (LH) release by melatonin was not abolished in the presence of forskolin or nonhydrolyzable cAMP analogues [e.g., 8-bromo-cAMP (8-Br-cAMP), dibutyryl-cAMP (db-cAMP)] suggesting that melatonin-mediated cAMP inhibition could be a parallel or secondary signal for LH release (Vanecek and Klein, 1995; reviewed in Vanecek, 1999). Similarly, there is evidence that melatonin does not mediate circadian phase-shifting via an inhibition of cAMP in the rat SCN (McArthur et al., 1997).

1.6.a.ii. cGMP The role of cGMP in melatonin signalling is controversial. Melatonin has been found to inhibit GnRH-stimulated cGMP production in the cultured neonatal rat pituitary cells (Vanecek and Vollrath, 1989). Although cGMP was found to potentiate LH release in adult rat gonadotrophs (Naor et al., 1978), a membrane permeable cGMP analogue, 8-bromo-cGMP (8-Br-cGMP), failed to antagonize melatonin-mediated inhibition of LH release in neonatal gonadotrophs suggesting the inhibition of cGMP has no role in this response (Vanecek, 1998). In murine melanoma M2R cells (Bubis and Zisapel, 1999) and human prostate epithelial cells (Gilad et al., 1997), physiological concentrations of melatonin suppressed cGMP levels. On the other hand, melatonin was found to, stimulate cGMP production in cultured rat medial basal hypothalamus (Vacas et al., 1981) and, potentiate vasoactive intestinal peptide (VIP)-stimulated cGMP production in human lymphocytes (Lopez-Gonzalez et al., 1992). Recently, it has been shown that the recombinant xMel1cβ receptor isoform is preferentially coupled (negatively) to the cGMP pathway, whereas the xMel1cα isoform couples (negatively) preferentially to the cAMP pathway (Jockers et al., 1997). Such differential signalling is also found in HEK293 cells expressing either recombinant hmt1 or hMT2 subtype receptors. Both subtypes inhibited forskolin-stimulated cAMP levels, but only the hMT2 subtype was M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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found to inhibit cGMP (Brydon et al., 1999; Petit et al., 1999). Thus, the differential cGMP signalling found in native tissues could be due to the expression of different melatonin receptor subtypes and/or isoforms.

1.6.a.iii. Phospholipids A similar inconsistent action of melatonin on membrane phospholipids exists. Melatonin has been shown to inhibit the GnRH-stimulated diacylglycerol (DAG) formation and arachidonic acid (AA) release via a PTX-sensitive pathway in cultured neonatal rat pituitary cells (Vanecek and Vollrath, 1990). Although GnRH is known to activate the phospholipase C (PLC)-DAG-Ca2+protein kinase C (PKC) pathway in gonadotrophs to release LH (Andrews and Conn, 1986; Huckle and Conn, 1987; Zheng et al., 1995; reviewed in Vanecek, 1999), there is no evidence that melatonin inhibits GnRH-induced IP3 formation (Vanecek, 1998). Hence, it is possible that melatonin inhibits GnRH-stimulated DAG/AA levels via a PLD rather than a PLC pathway (Zheng et al., 1994; Vanecek, 1998). The fact that inhibition of LH release by melatonin was not affected in the presence of phorbol esters, which are known to activate PKC directly, suggests that the melatonin-inhibited LH release is not mediated by decreasing DAG levels (Vanecek and Klein, 1995). Although PLA and PLD pathways have been shown exist in the ovine PT cultures, melatonin does not stimulate these pathways (McNulty et al., 1994). However, in NIH3T3 cells expressing recombinant hmt1 receptors, melatonin potentiates the effects of PGF2α-stimulation of phosphoinositide hydrolysis (PLC activation) and subsequent activation of a DAG-PKC-phospholipase A2 (PLA2) pathway resulting in AA release (Godson and Reppert, 1997). In addition, there is evidence that melatonin mediates circadian phase-shifting via activation of PKC in the rat SCN (McArthur et al., 1997), as specific inhibitors of PKC, such as calphostin C and chelerythrine chloride, blocked the melatoninstimulated phase advance at both windows of sensitivity (CT10 and CT23). Furthermore, activation of PKC using TPA mimics the melatonin-induced phase advance only at the two windows of melatonin sensitivity. These authors also demonstrated that melatonin administration transiently elevated PKC phosphotransferase activity at CT10 and CT23 but not at CT6.

1.6.a.iv. Calcium The effects of melatonin on intracellular calcium ([Ca2+]i) has been shown to be tissuedependent. In the neonatal rat pituitary, there is no doubt that [Ca2+]i plays a central role in the regulation of LH release (reviewed in Vanecek, 1998 & 1999). In this tissue, melatonin inhibits GnRH-induced Ca2+ release from endoplasmic reticulum (ER) as well as Ca2+ influx via voltagesensitive calcium channels. This effect is PTX sensitive suggesting that melatonin acts through a Gi/o protein. The fact that not all gonadotrophs in the neonatal rat pituitary are sensitive to melatonin (Vanecek and Klein, 1993) indicates a heterologous distribution of melatonin receptors within the gonadotrophs. It is well characterized that melatonin inhibits the calcium-dependent release of dopamine by the retina (Dubocovich, 1993; reviewed in Dubocovich, 1995). As the effect of melatonin on M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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dopamine release requires the presence of extracellular calcium, it has been suggested that melatonin may act to decrease [Ca2+]i in the presynaptic nerve terminals (Krause and Dubocovich, 1991). However, it is not clear if calcium is part of the melatonin receptor signalling cascade or simply a requirement for granule exocytosis. In arteries of rat cerebrum (Geary et al., 1997) and tail (Ting et al., 1997 & 1999; Geary et al., 1998; Doolen et al., 1999; Lew and Flanders, 1999; Bucher et al., 1999), melatonin inhibits Ca2+dependent-large conductance K+ channels (BKCa2+) to either cause or potentiate vasoconstriction. One study showed that inhibition of L-type calcium channels by nifedipine prevented the vasoconstriction effect of melatonin in isolated rat tail arteries (Lew and Flanders, 1999). Other studies have demonstrated that elevation of [Ca2+]i can induce vasoconstriction in rat tail artery (Sulpizio and Hieble, 1991; Capdeville Atkinson et al., 1993). These results indicate that [Ca2+]i could be involved in the vascular effect of melatonin. There are reports suggesting direct in vitro binding of melatonin to the Ca2+-activated protein, calmodulin (CaM ) with high affinity (KD 0.1 nM, Benitez-King et al., 1993; Anton-Tay et al., 1998a) and the in vitro inhibition of the CaM-dependent protein kinase II (CaM-Kinase II) (BenitezKing et al., 1996). In addition to being a CaM antagonist, melatonin was shown, by the same authors, to be capable of activating PKC by direct interaction with the kinase in the presence of Ca2+(Anton-Tay et al., 1998b). A separate detailed study confirms that the in vitro binding of melatonin to CaM was indeed Ca2+-dependent but with KD values in the mM range (Ouyang and Vogel, 1998) which cast doubts on the physiological relevance of melatonin as a CaM antagonist.

1.7. Melatonin Action on Xenopus Melanophores 1.7.a. Color Change Many lower vertebrates such as fish, amphibia and reptiles, are capable of altering their integument colouration by morphological and/or physiological colour change in their pigmentcontaining cells, termed chromatophores (see Section 1.7.a.i and Figure 1.5; reviewed in Rollag and Adelman, 1992; Fujii, 1993; Nery and Castrucci, 1997). Morphological colour change involves modulation of gene expression, in response to prolonged environmental stimuli (e.g., seasonal background adaptation) resulting in a slow but long-term (days to months) change in the density of pigments within each cell and/or the number of chromatophores. Whereas, physiological colour change, a process important for courtship and display, camouflage and self-defense, thermoregulation and photoprotection, refers to a rapid, short-term (within seconds to minutes) and readily reversible colour change as a result of intracellular translocation of pigment-containing organelles (see Section 1.7.a.i). Although physiological colour change does not involve gene expression itself, a prolonged stimulation may initiate morphological colour change.

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There are two types of skin pigment cells, the dermal and epidermal chromatophores. In general, dermal chromatophores are responsible for physiological colour change in response to nervous, humoral or environmental (e.g., light and temperature) stimuli, whereas, epidermal chromatophores are only susceptible to morphological colour regulation which may be responsible for stable external markings and/or gross hue (colour intensity) of the skin (reviewed in Rollag, 1988; Fujii, 1993). Physiological colour change is not known in birds and mammals including humans perhaps because of the absence of dermal chromatophores. Nevertheless, their epidermal chromatophores remain under the control of morphological colour change, which is responsible for the regulation of coat or skin colour in response to prolonged environmental stimuli such as seasonal background adaptation. Although a direct effect of melatonin in morphological colour change remains unclear, it is no doubt that melatonin has an indirect effect on mammalian coat colour through actions on the hypothalamus and pituitary. Comprehensive reviews of the neuroendocrinological effects of melatonin on mammalian coat colour exist (Rollag and Alderman, 1992; Arendt , 1995d) and therefore not discussed further in this thesis.

1.7.a.i. Physiological Colour Change in Amphibia There are three types of neural-crest derived dermal chromatophores, classified by the type of pigments they contain, which are involved in physiological colour change in amphibian skin (reviewed in Bagnara et al., 1968 & 1979; Bagnara and Hadley, 1969; Bagnara, 1976). The disc-like xanthophores form the uppermost layer of the dermis under the basal lamina (see Figure 1.5). Each xanthophore contains many bright-yellowish pteridine pigment granules (pteriosomes) and reddish carotenoid lipid droplets (carotenoid vesicles). The degree of yellow to red is therefore dependant on the proportion of the pteriosomes and carotenoid vesicles within xanthophores. The cone-shaped iridophores, found below the xanthophore layer, contain thin sheets (~5 nm) of orderly stacked platelets which consist of light-reflecting insoluble purine crystals (e.g., guanine, hypoxanthine and uric acid; reviewed in Bagnara et al., 1968; Fujii, 1993). The deepest dermal chromatophore is the melanophore which contains many brown-black melanin pigment granules (melanosomes). The cell bodies of melanophores are either directly below the iridophores or separated by an interstitial space, depending on the species (Bagnara et al., 1968). However, in most anuran (frog and toad) species, the processes of melanophores wrap around the iridophores as shown in Figure 1.5. The distal finger-like processes of melanophores terminate between the xanthophore and iridophore layers. Melatonin has a direct role in causing physiological colour change in amphibian skin. Activation of melatonin receptors found on Xenopus melanophores (Ebisawa et al., 1994) causes a rapid translocation of pigment granules within the 'fingers' of melanophores towards the cell body to accumulate around the nucleus. This process reveals the light-reflecting iridophores and hence lightens the amphibian skin. In reverse, neuronal and many other humoral factors such as alphamelanocyte-stimulating hormone (α-MSH), catecholamines (such as NA, adrenaline), serotonin, and environmental stimuli such as light, can cause melanophore dispersion such that pigment granules become evenly distributed throughout the cell body and processes (reviewed in Lerner, 1994). In M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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dispersed melanophores, the melanin-filled fingers mask the iridophores causing less reflection of light and thereby darkens the skin. The 'fingers' of melanophores have been shown to form stable associations with the iridophores (but not xanthophores) and are therefore unperturbed by melanosome translocation, i.e. they remain in position between the xanthophores and iridophores during pigment aggregation and dispersion. This permanent contact between the melanophore processes and the iridophores is thought to be important for the rapid and reversible melanosome translocation accomplishing physiological colour change (Bagnara et al., 1968).

1.7.b. Signal Transduction of Melanosome Translocation 1.7.b.i. Melanosome Dispersion Cyclic AMP is one of the earliest studied intracellular signalling candidates for melanosome translocation. Its role in causing amphibian and reptile skin-darkening has been known for a long time (Bitensky and Brustein, 1965). In vitro cell culture and direct microinjection studies on fish and amphibian melanophores confirm the melanosome dispersing action of cAMP (reviewed in Nery and Castrucci, 1997). The most well-known vertebrate skin darkening substance, α-MSH, was found to cause melanophore dispersion by receptor-coupled stimulation of cAMP production in cultured fish (reviewed in Fujii, 1993) and amphibian (reviewed in Lerner, 1994; Lerner et al., 1996) melanophores. Catecholamines, such as NA, adrenaline, and serotonin (see Table 4.3), have also been shown to stimulate pigment dispersion by increasing the production of cAMP in isolated Xenopus melanophores (reviewed in Lerner, 1994). Whilst there is no doubt that cAMP is important in causing melanosome dispersion mediated by several ligands, pigment dispersion can also be triggered by activating PKC (Sugden and Rowe, 1992; Graminski, 1993). Thus cAMP is not the only second messenger capable of causing pigment dispersion in melanophores. Indeed, activation of an endogenous Xenopus melanophore endothelin (ETc) receptor, coupled to a Gq protein, causes pigment dispersion through activation of PKC via phosphoinositide hydrolysis (Karne et al., 1993; summarized in Figure 1.6). As pigment dispersion involves activation of protein kinases, protein phosphorylation in various species of fish and amphibian melanophores has been investigated (reviewed in Nery and Castrucci, 1997). A consensus pattern suggests that, regardless of the melanophores of different species, activation of protein kinases (A or C) leads to the phosphorylation of a granule-bound 53-57 kDa protein (reviewed in Nery and Castrucci, 1997; Tuma and Gelfand, 1999). A recent study comparing the phosphorylation of melanosome proteins isolated from Xenopus melanophores during pigment aggregation or dispersion, found (in addition to a 57 kDa protein) proteins between 85-97 kDa which were more highly phosphorylated in dispersed than aggregated cells (Reilein et al., 1998). However, in all species of melanophores studied, the identity of the phosphorylated proteins is unknown and how these proteins regulate granule translocation remains to be elucidated.

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Figure 1.5 Schematic representation of a typical organization of the dermal chromatophore unit in anuran (frogs and toads) skin. The top figure shows that the chromatophore units are embedded in the dermis, which is separated by the basal lamina from the epidermis (adapted from Bagnara et al., 1968). The structural interaction of xanthophore, melanophore and iridophore in a chromatophore unit is depicted in the bottom figure [created using the computer software Microstation (V5.07; Bentley Systems Incorporated) by Mr. Shoong Goh, School of the Built Environment, University of Westminster, London, UK.] where the iridophore is enfolded by the melanophore fingers (cell body of the melanophore is not shown here). Melanophores with dispersed pigment are represented in these figures for illustration purposes. Pigment aggregation or dispersion causes melanosomes within the fingers to translocate (arrows) toward the cell centre or into the fingers which remain associated with the iridophore throughout granule translocation. M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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Figure 1.6 A schematic diagram summarising the signal transduction cascades of Xenopus melanophore pigment dispersion mediated by Gs or Gq protein-coupled receptors such as α-MSH (Potenza and Lerner, 1992; McClintock et al., 1996) and ETc (Karne et al., 1993; McClintock et al., 1996) receptors, respectively. The α-MSH receptor (MC1) has been shown to couple to a Gs protein which stimulates an adenylate cyclase (AC) that catalyzes the production of cAMP. cAMP can also be elevated using an AC activator, forskolin or phosphodiesterase (PDE) inhibitor, IBMX. Elevation of cAMP in turn activates protein kinase A (PKA) which leads to protein phosphorylation and pigment dispersion. Alternatively, pigment dispersion can be activated by a Gq-coupled ETc receptor. Receptor stimulation leads to the activation of a phospholipase C (PLC) which in turn catalyses the hydrolysis of phosphatidylinositol 4,5bisphosphate (PIP2). Protein kinase C (PKC) could be activated by diacylglycerol (DAG), elevation of [Ca2+]i via inositol-1,4,5-trisphosphate (IP3). The pigment dispersion triggered by activation of PKC is mimicked by phorbol esters and inhibited by specific PKC inhibitor, Ro 31-8220. It is unclear how protein phosphorylation by PKA or PKC leads to melanosome dispersion.

1.7.b.ii. Melanosome Aggregation The signal transduction mechanism triggering pigment aggregation in Xenopus melanophores is less-well characterized due to the lack of aggregating ligands other than melatonin. As stimulation of cAMP production causes pigment dispersion, it is perhaps logical and convenient to assume that decreasing the production of cAMP causes pigment aggregation. This concept is probably too simplistic. It implicitly assumes that the opposing phenomena of aggregation and dispersion are controlled by a single intracellular signal regulating a single molecular motor mechanism. Accumulating evidence indicates that multiple molecular motors (kinesin II, cytoplasmic dynein and myosin V) make use of both actin filament and microtubule systems for intracellular motility

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(reviewed in Kelleher and Titus, 1998; Brown, 1999). While a reduction in intracellular cAMP may inhibit dispersion (which would seem to be a sensible prerequisite before aggregation is activated), additional signal(s) may be needed to activate the motor(s) or mechanism(s) responsible for moving pigment granules to the cell centre. Many species of fish and amphibian melanophores do show a correlation between reduction in cAMP level and pigment aggregation (reviewed in Nery and Castrucci, 1997). In melanophores of various fish species, catecholamines such as NA stimulate pigment aggregation via a PTX-sensitive Gi/o protein coupled receptor which decreases cAMP synthesis (reviewed in Nery and Castrucci, 1997). The same mechanism is found in amphibian melanophores with melatonin as an agonist (reviewed in Lerner, 1994; summarized in Figure 1.7). However, reduction of intracellular cAMP is not the only signal triggering melanophore aggregation. In melanophores of some but not other species of crustacean and fish an increase in [Ca2+]i is responsible for pigment aggregation. Melanophores of amphibian and reptile are thought to be insensitive to [Ca2+]i changes (reviewed in Nery and Castrucci, 1997). The most potent pigment aggregating peptide, melanin-concentrating hormone (MCH; Wilkes et al., 1984a & 1984b; Castrucci et al., 1989), is known to cause pigment aggregation in fish melanophores through activation of PKC (Abrao et al., 1991). However, a separate study shows that in isolated fish (Lebistes ossifagus) melanophores, pigment aggregation was correlated with an inhibition of cAMP production in response to MCH treatment (Svensson et al., 1991). The recent cloning of the MCH receptor protein identifies this receptor as a PTX-sensitive Gi/o protein-coupled receptor (orphan receptor SCL-1; Lembo et al., 1999; Chambers et al., 1999; Saito et al., 1999). One of these groups demonstrate that receptor (in a recombinant HEK293 cell system) activation not only leads to a reduction of forskolin-elevated cAMP levels but also mobilization of [Ca2+]i which is PTX sensitive (i.e., independent of Gq protein activation) (Lembo et al., 1999). However, another group showed that activation of the recombinant MCH receptor in CHO cells can only mobilize [Ca2+]i in the presence of a chimeric Gq/i3 protein suggesting that MCH receptor can couple to different G proteins given a different cellular environments (Saito et al., 1999). Interestingly, MCH causes a full pigment dispersion (rather than aggregation) in both amphibian and reptile melanophores (Wilkes et al., 1984a & 1984b; Ide et al., 1985; Castrucci et al., 1989; reviewed in Nery and Castrucci, 1997). Given that MCH can activate PKC (Abrao et al., 1991), perhaps it is not surprising that MCH causes pigment dispersion in amphibian melanophores as activation of PKC in these cells is known to cause pigment dispersion (see Figure 1.6). As pigment dispersion involves protein phosphorylation, it has been suggested that dephosphorylation by protein phosphatase is required for pigment aggregation. Early experiments suggested the involvement of a Ca2+/CaM-dependent phosphatase, calcineurin or PP2B, in pigment aggregation in the melanophores of the African cichlid (Tilapia mossambica) (Thaler and Haimo, 1990). However, two separate studies have shown that calcium is not required for pigment aggregation in melanophores of the squirrel fish (Holocentrus ascensionis Sammak et al., 1992) and

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angelfish (Pterophyllum scalare; Kotz and McNiven, 1994). In Xenopus melanophores, a phosphatase inhibitor, okadaic acid, at a concentration that inhibits both phosphatase 2A (PP2A) and PP2B, blocked pigment aggregation (Cozzi and Rollag, 1992; Reilein et al., 1998). Expression of specific phosphatase inhibitor in Xenopus melanophores provided evidence that PP2A, but not PP1 or PP2B, is required for pigment aggregation (Reilein, 1998).

Figure 1.7 A current model of melatonin-signal transduction cascade mediating Xenopus melanophore pigment aggregation. Melatonin receptor activation causes the dissociation of the PTX-sensitive Gαi/o from the βγ subunit. The active Gαi/o in turn inhibits the action of AC thereby reducing the production of cAMP. The diminution of cAMP leads to the inactivation of PKA which is thought to cause protein dephosphorylation required to drive pigment aggregation.

1.8. Melanophore as a Functional Model System The rapid and reversible pigment translocation within melanophores in response to melatonin serves as an excellent model system for the study of melatonin receptors and the intracellular signalling mechanisms they activate. The colour change as a result of pigment translocation can be quantified using various techniques (reviewed in Rollag, 1988; see also Chapter 3). In brief, the simplest, but very time-consuming and subjective technique is by manual scoring of pigment

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position within individual cells, viewed under the microscope, based on the melanophore index (Hogben and Slome, 1931). Alternatively, pigment position can be more accurately determined by using a computer-assisted digital image analysis but this is applicable only to small numbers of cells (Sugden, 1994; Sugden et al., 1995). A more accurate technique using light transmittance (or absorbance used in this thesis, see Chapter 3) through a monolayer of cultured melanophores has been developed recently (reviewed in Lerner, 1994; McClintock and Lerner, 1997). This technique was originally used on melanophorecontaining skin explants (Mori and Lerner, 1960; Bitensky and Burstein, 1965). As an immortalized melanophore cell line was successfully established from Xenopus embryos (Daniolos et al., 1990), a monolayer of pure melanophore cells can be used instead of skin pieces. The technique was modified to use a microtitre platereader to measure the transmittance (or absorbance) of monochromatic light (wavelength >600) through a layer of melanophores in a 96-well plate. This melanophore assay system has been used extensively and successfully in this laboratory for the study of structure-activity relationships of the melatonin receptor using systematically designed melatonin analogues (Sugden et al. 1997 &1999b; Davies et al. 1998; Teh and Sugden, 1998 & 1999). This bioassay has also been used to screen other compounds such as αMSH-peptide libraries (Jayawickreme et al., 1994a & 1994b; Quillan et al., 1995; Carrithers and Lerner, 1996). On the other hand, the melanophore assay system has also been widely employed for functional studies of other endogenous Xenopus melanophore GPCRs, such as MSH (melanocortin) receptor (Jayawickreme et al., 1994a & 1994b; Ollmann and Barsh, 1999), β adrenergic receptor (Potenza and Lerner, 1992), endothelin receptor (Karne et al., 1993), 5-HT receptor (Potenza and Lerner, 1994; see Chapter 6) and VIP receptor (Marotti et al., 1999), or exogenous receptors using transgenic expression (transfection) techniques, for example the human β2-adrenergic, murine substance P and bombesin receptors (McClintock et al., 1993); human-dopamine D2 and D3 receptors (Potenza et al., 1994); murine platelet-derived growth factor β (PDGF-β) receptor (Graminski and Lerner, 1994); chimeric olfactory-adrenergic receptor (McClintock et al., 1997); human CX-chemokine (CXCR4) receptor (Chen et al., 1998); human galanin (GALR2 and GALR3) receptors (Kolakowski et al., 1998); human GABAB receptor (Ng et al., 1999a & 1999b); murine and human leukotriene B4 receptors (Martin et al., 1999); human cysteinyl leukotriene CysLT1 receptor (Lynch et al., 1999); murine µ-opioid receptor (Potenza et al., 1999); human calcitonin (CTR2), neuropeptide Y (NPY1, 2 and 4) and chemokine C (CCR5) receptors (Chen et al., 2000). The melanophore assay system provides a direct, rapid and accurate quantification of colour change in response to receptor activation. This technique is used in subsequent chapters for the study of melatonin receptors and signal transduction mechanisms (Chapter 4), metabolism of melatonin by deacetylation (Chapter 5), functional characterization of a Xenopus melanophore 5-HT7 receptor (Chapter 6) and effects of light on melanophores (Chapter 7).

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Chapter 2: Materials & Methods

Chapter 2:

MATERIALS AND METHODS

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Chapter 2: Materials & Methods

Chapter 2: Materials and Methods 2.1. Introduction This chapter describes the general techniques used throughout this thesis. More detailed applications of the techniques as well as other specific methods will be discussed later in subsequent chapters. Most media and reagents were obtained from Sigma Aldrich Company Ltd., Poole, Dorset, UK., unless otherwise stated. As a large number of drugs were used in this thesis, they will be listed and explained in each specific chapter for ease of referencing.

2.2. Cell Culture 2.2.a. Xenopus Fibroblasts and Melanophores Xenopus laevis fibroblast and melanophore clonal cell lines were invaluable gifts (February 1997) from Dr. Michael Lerner (University of Texas, Dallas, USA.). These cells were originally isolated from Xenopus embryos (Daniolos et al., 1990) between stages 30 and 35 (Nieuwkoop and Farber, 1956). As amphibia are poikilothermic, the melanophores and fibroblasts were grown optimally at room temperature (20-27°C). As these cells were grown in Leibovitz L-15 based medium (diluted to 0.7× to give osmolality ~230 mOsm/Kg H2O appropriate for amphibian cells; Balinsky, 1981) containing high concentration of amino acids, pyruvate (3.5 mM) and D-galactose (3.5 mM), the usual 5% CO2/95% air-gaseous exchange is not needed (Leibovitz, 1963) and flasks could be closed tight to prevent evaporation and contamination by any air-born infectious agents. Cells were fed twice a week with growth medium. Unlike fibroblasts, the majority of melanophores do not survive cryopreservation due to their heavy melanin pigmentation (see Chapter 8). However, a method of reversible depigmentation has been developed to allow melanophores to survive cryopreservation (see Section 2.2.a.iv).

2.2.a.i. Growth of Melanophores As specific growth factors for melanophores have not yet been identified, Xenopus fibroblasts were grown to produce conditioned-growth medium for melanophores. 0.7×L-15 medium containing 100 i.u./ml penicillin, 0.1 mg/ml streptomycin, 4 mM L-glutamine and 15% heatinactivated (56°C for 30 min) fetal calf serum ('Myoclone'; Gibco/BRL, Paisley, UK), known as fibroblast growth medium, was conditioned by fibroblasts for feeding melanophores. Five 175 cm2-tissue culture flasks (T175; Falcon; Becton Dickinson and Co., Lincoln Park, New Jersey, USA.) were seeded with 4-6 million fibroblast cells/flask (20-30% confluent) and were fed twice weekly with 50 ml fibroblast growth medium. The media in which the fibroblasts grew was collected when cells were ~50% (2-4 days) and 100% (6-8 days) confluent. One of the five confluent flasks was subcultured (see Section 2.2.a.ii) into five T175 flasks. This procedure was M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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repeated once a week producing 500 ml conditioned growth medium. This medium was filtered (0.2 µm-surfactant free cellulose acetate filter; Nalgene, Rochester, USA) to avoid fibroblast contamination while feeding melanophores. It is important to prevent cross-contamination of melanophores as fibroblasts have a much higher growth rate. In case of fibroblast contamination, 'unpigmented' colonies of fibroblasts would appear on a 'black' confluent layer of pigmented melanophores. Purifying contaminated melanophores by density-gradient separation (see Section 2.2.a.iii) eradicates fibroblast cells from the melanophore population. Normally 2 million melanophores were seeded (30-40% confluent) in a T175 flask and fed once or twice weekly with 50 ml conditioned growth medium collected from fibroblasts. Melanophores duplicated about once in 2-3 days (see Figure 3.3B) at ~25°C, but because of the low survival rate after cryopreservation, a continuous culture of melanophores had to be maintained to ensure a constant supply of cells. To minimize the use of conditioned growth medium and hence the task of culturing fibroblasts, some melanophores were kept at 14-18°C where cells duplicate only once in 2-3 weeks. These cells remain viable and could be fed once a month until confluent (usually after 2-3 months). In fact, the original melanophore stock was kept for more than 2 years at ~16°C. Subcultures originating from this stock show the same physiological responses and no morphological differences to their sister cells maintained at room temperature.

2.2.a.ii. Subculture of Melanophores A confluent T175 flask of melanophores (or fibroblasts) was rinsed with 10-15 ml PBS (0.7×; 2+

Ca

and Mg2+ free-Dulbecco’s Phosphate Buffered Saline) and 3 ml of trypsin [1×trypsin

(EC 3.4.21.4; 2.5 mg/ml porcine trypsin in 0.9% NaCl) in 0.7×PBS containing 3 mM EDTA at pH 8] was added to detach cells from the flask. The flask was agitated to expedite cell detachment as prolonged action of trypsin would cause cell damage. Ten ml conditioned growth medium was added to terminate the action of trypsin. The melanophore cell-suspension was pelleted by centrifugation (MSE Minor Centrifuge, Maedowrose Scientific Ltd., Oxford, Oxfordshire, UK.) at 200×g for 1-2 min (400×g 1-2 min for fibroblasts due to smaller cell size compared to melanophores). The supernatant was removed and the pellet was harvested. Usually, a confluent T175 flask was subcultured into 3-5 T175 flasks, each containing 45-50 ml growth medium. For other smaller flasks or plates, the cell pellet was diluted and an appropriate volume of growth medium added according to the type of culture flask/plate used: 20-25 ml/T80 (Nunc; Kamstrup, Roskilde, Denmark); 4-6 ml/T25 (Nunc); 2-4 ml/well of a 6-well plate (9.6 cm2/well, Nunc); 0.5-1.0 ml/well of a 24-well plate (2.0 cm2/well; Linbro Flow Laboratories, Inc., Virginia, USA.); 100-200 µl/well of a 96-well plate (0.30 cm2/well; Nunc).

2.2.a.iii. Density-Gradient Partition of Melanophores A reduction in melanin-pigment synthesis in some melanophores was obvious in long-term culture. Percoll selection was used to isolate the less pigmented cells from the heavily pigmented M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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ones. This procedure also helped to eradicate fibroblast contamination although this problem rarely occurred. A melanophore pellet from a confluent T175 flask was obtained as described in Section 2.2.a.ii. The pellet was resuspended in 4 ml conditioned growth medium and laid gently over an 8 ml 60% percoll-conditioned growth medium layer in a 15 ml-centrifuge tube. The tube was left standing upright. Heavily pigmented cells would sink rapidly within 10 minutes while less pigmented cells or fibroblasts would stay above the percoll gradient. After 1 h the percoll gradients were aspirated leaving the heavily-pigmented melanophores at the bottom of the tube, which were then resuspended with 10 ml conditioned growth medium. Cells were pelleted once again to remove remaining percoll from the cells before resuspending in conditioned growth medium for culturing.

2.2.a.iv. Cryopreservation Fibroblasts A pellet of fibroblast cells was obtained from a confluent T175 flask as described in Section 2.2.a.ii. The number of cells in the pellet was estimated by counting on a haemocytometer. A confluent flask of fibroblasts usually contained 18-20 million cells and was aliquoted into 6 cryotubes (Nunc) with ~3 million cells suspended in 1 ml fetal bovine serum (FBS; Imperial Laboratories, England, UK.) containing 10% DMSO (dimethyl sulfoxide). The cryotubes of cells were initially cooled to -70°C in a freezing container ('Mr. Frosty', Nalgene) containing isopropyl alcohol (BDH Limited, Poole, England, UK.) to ensure a constant cooling rate of 1°C/min, before transferring to liquid nitrogen for cryopreservation at -196°C. This two-step freezing procedure, is essential for cells to survive cryopreservation. When growing from frozen stocks, an aliquot of cells was thawed quickly at 25°C and the cell suspension then added to a T175 flask containing 50 ml fibroblast growth medium. Fibroblasts were then grown to produce conditioned growth medium as described in Section 2.2.a.i. Melanophores Cryopreservation of melanophores was not successful because pigment granules in the melanophores serve as nucleation sites for ice crystal formation during the cooling process and hence kill the cells (personal communication, Dr. Michael Lerner). Melanophores can be depigmented using a specific copper chelator phenylthiourea (phenylthiocarbamide; PTC; Jimenez-Cervantes et al., 1994) at a concentration (10-3 M) that inhibits tyrosinase but allows other critical copperdependent systems to function within the melanophores (see Figure 8.2). Tyrosinase is a key copperdependent enzyme essential for the synthesis of melanin from tyrosine (Furumura et al., 1998). As depigmentation took about 2-3 weeks to complete, melanophores were initially grown in a small flask (T25). Cells were fed once a week with PTC-containing conditioned growth medium until confluent and subsequently subcultured into larger flasks to obtain more depigmented cells for cryopreservation. When completely depigmented and confluent in a T175 flask (~6 million cells), melanophores could then be pelleted and cryopreserved as described for the fibroblasts. M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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The depigmentation of melanophores before cryopreservation dramatically increased the cell survival rate from less then 1% to more than 50%. To further maximize the number of cells surviving, each aliquot of cryopreserved melanophores (~1 million cells) were seeded into a small T25 flask rather than the usual T175 flask and fed twice weekly until confluent. Repigmentation was more rapid than depigmentation and was usually completed in about 1-2 weeks. As a small fraction of cells (600 nm) through a cell monolayer grown in a 96-well plate. This method allows M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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simultaneous measurements of 96 samples in less than 30s and is therefore particularly suitable for rapid, comparison of drug potency and kinetic studies. The aim of this chapter is to establish the use of a 96-well microtitre platereader to quantify melanosome translocation which serves as a reporter system for the study of melatonin receptor activation and transduction mechanisms.

3.2. Materials and Methods The basic methods for quantifying melanosome translocation and data analysis are described in Section 2.4. Any methodological variations from the standard protocols will be detailed in each figure legend. Melatonin, N-acetyl-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (α-MSH; stock 3×10-5 M in H2O), 7β-acetoxy-1α,6β,9α-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin, isolated from Coleus forskohlii; DMSO) and 1-(β-D-arabinofuranosyl)cytosineyHCl (Ara-C or arabinosylcytosine; H2O) were obtained from Sigma Aldrich Company Ltd., Poole, Dorset, UK. All compounds were freshly diluted with 0.7×L-15 medium (containing 100 i.u./ml penicillin, 0.1 mg/ml streptomycin and 1 mg/ml BSA) before use from stock solutions kept at -20°C.

3.3. Results 3.3.a. Granule Translocation as a Reporter System for Receptor Activation As illustrated in Figure 2.1 and Figure 2.2 (p44), melanosome translocation could be accurately quantified by measuring the absorbance change using a platereader. However, the change in melanophore absorbance could be due to at least three factors either alone or in combination: 1. a spatial change in melanosome position as a result of granule translocation, 2. a change in the total quantity of melanin pigment as a result of synthesis or degradation, and, 3. cell growth or cell lysis. To address these questions, a series of experiments designed to investigate and verify the use of platereader for quantifying receptor mediated melanosome translocation is discussed.

3.3.a.i. Cell Density and Absorbance To determine if melanophore cell number is correlated with cell absorbance (due to melanin pigment), a varying number of cells were seeded in a 96-well plate. After 3 days, the absorbance of the fully attached and dispersed cells was measured before performing a cell count (Figure 3.1). It has proven impossible to count the melanophores using the standard haemocytometer method as the pigmented cells are black and cell size varied dramatically from a diameter of ~30 to 208 µm (cell area of ~700-34,000 µm2; Figure 3.2) hence making it difficult to differentiate between clumps of small cells and single large cells. Thus, a different method was employed for counting melanophore cells. Melanophores in each well were harvested by trypsinization and a series of dilutions were performed before re-seeding into a 96-well plate. Melanophores were left to attach over a 2-4 h period and wells with countable number of cells (200-400 cells/well) were counted with the aid of M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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square grids drawn under each well. The total cell number was then calculated based on the dilution factor made to each well. This ensured identification of only individual melanophore cells for counting. This method makes the assumption that harvesting cells by trypsinization is equally efficient at each cell density. Examination of the wells after trypsinization suggested this was the case as virtually no melanophores remained in the well in all cases.

Figure 3.1 The relationship between melanophore cell density and absorbance. Cell number was determined as described in the text. The approximate cell confluence level (%) of each data point is depicted by a representative image of a well captured digitally as for Figure 2.1 in p44. Each data point represents a mean ± SEM of n=6 determinations. This experiment has been repeated twice on separate occasions with similar results.

Figure 3.1 shows that melanophore cell density is highly correlated with cell absorbance. This confirms that melanophore cell number is directly proportional to the amount of melanin pigment contained within the cells. Based on this linear relationship, the number of melanophores in any given well could be estimated by measuring the absorbance of dispersed melanophores.

3.3.a.ii. Growth Profile It was found that melanophores grew equally well in non-fibroblast-conditioned growth medium containing 15% FBS compared to fibroblast-conditioned growth medium containing 15% Myoclone-fetal calf serum (see Section 2.2.a.i). Because of the extra cost, time and effort needed to prepare fibroblast-conditioned medium which provided little advantage over non-conditioned growth medium, melanophores were subsequently grown in non-conditioned growth medium [0.7×L-15 medium containing 100 i.u./ml penicillin, 0.1 mg/ml streptomycin, 4 mM L-glutamine and 15% heat-inactivated (56°C for 30 min) FBS].

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A

i

ii

15 00 35 00 55 00 75 00 95 00 11 50 13 0 50 15 0 50 17 0 50 0 19 50 21 0 50 0 23 50 0 25 50 0 27 50 29 0 50 0 31 50 0 33 50 0

Number of Cells

B

Cell Size (µm2) Figure 3.2 The morphology and size of melanophores in culture. A, a randomly chosen bright-field image of melanophores grown in a 35-mm culture dish viewed (10× objective) using a BioRad MRC 600 laser scanning confocal microscope attached to a Nikon Diaphot microscope. i indicates a small cell with an area of ~1,018 µm2, whilst, ii is a large cell with an area ~16,617 µm2. Scale bar represents 100 µm. B, a frequency histogram of melanophore cell area distribution. The dispersed pigment area of 300 cells was measured using the NIH Image software (V 1.59). Note that the fitted curve shows a positive skew with a mode value of ~3500 µm2. Arrows indicate the approximate size of cell i and ii in A.

Figure 3.1 shows that melanophore cell number is linearly correlated with its absorbance, therefore the change in absorbance in dispersed melanophores over time (days/weeks) was used to study cell proliferation. A low number of cells (~1,500 cells/well) were seeded into a 96-well plate. M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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Cells were fed with either serum-free medium (0.7×L-15 medium containing 100 i.u./ml penicillin, 0.1 mg/ml streptomycin, 4 mM L-glutamine and 1 mg/ml BSA) or one of the three growth media with different concentrations of FBS added (see Figure 3.3), at room temperature (24-26°C). Absorbance was measured over a period of 34 days. Melanophores cultured without serum neither increased nor decreased their absorbance values over this period. Observations through the microscope showed no formation of melanophore colonies. This suggests that there is little or no change in the total melanin pigment concentration within the melanophores and that cell proliferation is minimal. Remarkably, after 24 days of incubation in serum-free medium, 40-60% of cells were able to aggregate their melanosomes in response to melatonin (data not shown) and growth resumed when cells were fed with growth medium containing serum (Figure 3.3A). This indicates that melanophores can survive prolonged serum deprivation. Increasing concentration of FBS caused a concentration-dependent growth of melanophores (Figure 3.3A). Optimal growth could be obtained by feeding melanophores twice weekly with growth medium containing 10-20% FBS. In these conditions, cells replicate once in every 2-3 days (Figure 3.3B). Based on this observation, all melanophores were routinely fed with growth medium containing 15% FBS. Melanophore growth rate is also highly dependent on temperature. Cells cultured in growth medium containing 15% FBS at ~16°C duplicate only once every 2-3 weeks (data not shown; see also Section 2.2.a.i).

3.3.a.iii. Time-Course, Ligand Concentration and Melanosome Translocation Melanosome translocation, either aggregation towards the perinuclear region or dispersion evenly throughout melanophores, is a rapid response resulting in a dramatic colour change within minutes (Figure 3.4 and Figure 3.5). The change in pigment position can accurately be quantified by measuring the absorbance change of the melanophores as described in Section 2.4. As discussed earlier (Section 3.3.a.ii) melanophores maintained in serum-free medium did not change absorbance over a period of 24 days, and, melanophores only proliferate very slowly (doubling time ~2.5 days; Figure 3.3) in the presence of serum in the culture medium. Therefore, the rapid change in absorbance which is completed within 60-90 min after melatonin or α-MSH treatment cannot be due to a change in cell density. Melanosome aggregation triggered by melatonin was completely reversible upon agonist removal, and, aggregation resumed with reapplication of melatonin (data not shown). Similarly, dispersion triggered by α-MSH (as well as other dispersing agents such as forskolin, 5-HT, NA, etc.), could be reversed by replacing the medium with fresh 0.7×L-15 medium containing melatonin (10-9 M) (data not shown). The return of absorbance to the original values suggests that there is no significant synthesis or degradation of melanin within this period, and that the mechanism for causing physiological colour change in vitro (reviewed in Rollag, 1988; see Section 1.7.a.i) is preserved in this clonal melanophore line. M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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*** ***

5

10

20

Figure 3.3 The growth profile of melanophores cultured in a 96-well plate. A, melanophores were fed twice a week with media containing the indicated percentage of FBS at ~25°C. Cells maintained in serum-free medium were fed after 24 days with growth medium containing 20% FBS as indicated (filled circles). Each data point represents a mean value of n=24 wells. Error bars (SEM) were omitted as they were all less than (600 CPM) of radioactivity was also detected in section 1, but this is likely due to an impurity of the stock. Figure 5.6B (solid bars) shows that after 96h of [O-methyl-3H]-melatonin (containing vehicle, 1% methanol) incubation, very little [3H]- melatonin was detected (TLC section 6, red arrows) in either the medium or cell extract suggesting a degradation of melatonin. Consistent with the metabolism pathway, the two final metabolites, [3H]- 5-ML and [3H]-5-MIAA, appeared (TLC section 8 & 9) in the medium and cell extract. A moderate amount of [3H]- 5-MT was detected only in the cell extract.

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Metabolism of Melatonin NHCOCH3 H3CO N H

Melatonin Melatonin Deacetylase

Eserine NH2

H3CO N H

5-Methoxytryptamine Monoamine Oxidase

Pargyline CHO

H3CO N H

Alcohol dehydrogenase OH H3CO

COOH

H3CO N H

N H

Figure 5.5 Metabolic pathway of melatonin (modified from Grace et al., 1991). The first and second enzymes, melatonin deacetylase and monoamine oxidase, are inhibited by eserine and pargyline, respectively. See text for more details.

In the presence of eserine (10-4 M; see Figure 5.6B, crossed bars), most of the [3H]-melatonin remained in the medium and little or virtually no metabolites were detected in either the medium or cell extract. Interestingly, in the presence of pargyline (10-4 M; Figure 5.6B, stripped bars), [3H]-5-MT appeared only in the cell extract. None of the other [3H]-5-methoxyindoles including melatonin was detected in either the medium or cell extract.

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A

Solvent front

Origin

1

2

3

4

5

6

7

8

9

10

B

Figure 5.6 Metabolism of [3H]-melatonin by Xenopus melanophores. A, a typical thin layer chromatogram (TLC) plate showing the separation of [3H]-5-methoxyindoles visualized by exposure to iodine vapour. The TLC plate was cut into ten1-cm sections and each section then assayed for [3H]-CPM. 5-MT, 5-methoxytryptamine; Mel, melatonin; 5-ML, 5-methoxytryptophol; 5-MIAA, 5-methoxyindoleacetic acid. B, cells were incubated with [O-methyl-3H]melatonin in 0.7×L-15 medium containing either vehicle (1% methanol), eserine (10-4 M) or pargyline (10-4 M) for 96h. [3H]-5-Methoxyindole metabolites were separated and identified in cell extract and medium by TLC as described in A and Section 5.2.c. TLC sections containing 5-MT and melatonin are indicated by green and red arrows, respectively. This experiment has been repeated twice with similar results.

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5.4. Discussion The potency of melatonin in activating melanosome aggregation in melanophores was found to diminish progressively with prolonged incubation up to 4 days tested. The decline in melatonin potency was slow; a 2-fold loss of potency occurred every 13 h during 96 h of incubation. This decline in potency could be due to several factors such as desensitization of the melanophore melatonin receptor, downregulation of the receptor population and/or agonist degradation. 2-[125I]-Iodomelatonin binding studies showed that prolonged melatonin incubation (up to 2 days) did indeed cause a progressive decline in receptor density. A significant receptor loss was first detected after 4 h of incubation with a concentration of melatonin sufficient to occupy all of the receptors on the surface of melanophores. Maximal loss (38% of control) was observed after 6 h of incubation. A decrease in the affinity of the melanophore receptors for 2-[125I]-iodomelatonin may also have occurred. As a saturating concentration of 2-[125I]iodomelatonin was used in the radioligand binding assay, the loss of binding clearly involves a decline in the density of receptors available for radioligand binding. The time-course of receptor loss is consistent with a mechanism of receptor downregulation (reviewed in Bohm et al., 1997). It is clear that the kinetics of receptor loss did not match the time-course of diminishing potency, i.e. when receptor loss was maximal (6-8 h), there was negligible loss of melatonin potency. As the density of melatonin receptors is very high in these cells (1224 fmol/mg membrane protein; Teh and Sugden, 1999), it seems likely that only a small proportion of the melanophore receptor population is needed to produce a maximal pigment aggregation response. Although there is a strong correlation (r2=0.93) between the affinity (Ki) of a series of melatonin agonists at recombinant Xenopus Mel1c receptors (determined in 2-[125I]iodomelatonin binding assays) and their potency (pEC50) in melanosome aggregation (Pickering, 1997), the agonists are ~10-fold more potent than expected from the binding affinity data. This suggests that not all of the receptors expressed on the melanophore are needed for a full aggregation response. Taken together these observations suggest that it is unlikely that the downregulation of receptors observed contributes to the loss of melatonin potency. Furthermore, longer melatonin incubation (>10 h) caused receptor density to return to its original level, yet the potency of melatonin continued to decline. An alternative explanation for the diminishing melatonin potency on prolonged incubation is receptor desensitization. GPCR desensitization is usually a rapid process, occurring within seconds to minutes of agonist exposure (reviewed in Chuang et al., 1996; Freedman and Lefkowitz, 1996; Lefkowitz, 1998). In the present study, after 6-10 h of melatonin incubation with melanophores, at which a maximal receptor down regulation is expected, there was virtually no loss of melatonin aggregation potency. Furthermore, the fact that melatonin potency remained constant throughout the 4-day melatonin incubation period in the presence of eserine argues strongly against any receptor desensitization. As receptor downregulation or desensitization cannot explain the loss of melatonin potency, another explanation would be the loss of melatonin from the medium. There are two possible M-T Teh „ Cellular & Molecular Studies on Xenopus Melanophores „ Thesis 2000

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mechanisms. First, melatonin could be unstable chemically at room temperature. This was not the case as no loss of melatonin aggregation potency was found even after 10 days of incubation (no cells) at room temperature. Second, melatonin could be degraded by the melanophores themselves in culture. This was found to be the case as the aggregating potency of media containing melatonin incubated with melanophores for 96 h was the same before (pEC50=8.27) and after it was transferred to naïve cells (pEC50=8.48) confirming the degradation of melatonin by melanophores. The loss of melatonin potency could be completely prevented by the addition of a cholinesterase inhibitor, eserine, an inhibitor of a deacetylase enzyme previously found in Xenopus retina and skin which can degrade melatonin (Cahill and Besharse, 1989; Grace et al., 1991; Grace and Besharse, 1993). In the presence of eserine, the aggregating potency of melatonin remained virtually unchanged (pEC50=10.51 at 90 min and pEC50=10.42 at 96h) throughout the 4-day incubation period. Also the naïve cells gave the same potency (pEC50=10.43) when treated with medium transferred from the melanophores incubated for 96 h with eserine suggesting a mechanism involving melatonin deacetylation. Using [3H]-melatonin incubated with melanophores in the presence or absence of eserine or pargyline (an inhibitor of monoamine oxidase) it was confirmed that after 96h little [3H]-melatonin could be detected in the medium unless melanophores were incubated with eserine. In the absence of eserine, it appears that melatonin is degraded to 5-MT, but this is then further metabolized to 5-MIAA and 5-ML by an enzyme sensitive to pargyline. 5-MT accumulates in cells if melanophores are incubated with pargyline (as it has poor permeability across the plasma membrane). In radioligand binding assays and functional models, 5-MT and 5-ML are either inactive or very weak melatonin receptor agonists (reviewed in Morgan et al., 1994). In melanophores, 5-MT, 5-MIAA and 5-ML are all unable to cause pigment aggregation (data not shown). This degradative metabolic pathway for melatonin has been shown previously to occur in the eyecups of teleost fish (Carassius), amphibia (Xenopus), reptiles (Anolis), birds (Gallus), and also in Xenopus skin (Grace et al., 1991). There is also evidence for the existence of such a metabolic pathway for melatonin in mammalian skin (Slominski et al., 1996). In contrast with the present results, Rollag and Lynch (1993) reported a rapid (