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Journal of Neurochemistry, 2001, 78, 88±99

Expression and role of phosphodiesterase 6 in the chicken pineal gland Fabrice Morin,* Claire Lugnier,² Jacques Kameni² and Pierre Voisin* *Laboratoire de Neurobiologie Cellulaire, UMR CNRS 6558, UFR Sciences, Poitiers, France ²Pharmacologie et Physico-Chimie des Interactions Cellulaires et MoleÂculaires, UMR CNRS 7034, Universite Louis Pasteur, Faculte de Pharmacie, Illkirch, France

Abstract The chicken pineal gland is directly photosensitive, with light causing an inhibition of melatonin synthesis. A possible role of phosphodiesterase 6 (PDE6, the primary effector of retinal phototransduction) in mediating this response was investigated. RT-PCR, DNA sequencing and northern blots revealed the presence of RNA encoding both catalytic and regulatory subunits of PDE6 in the chicken pineal gland. Both rod and cone forms of PDE6 subunits mRNA were detected. The concentration of the transcripts encoding PDE6 catalytic subunits peaked at night. Western blot analysis of chicken pineal proteins with an antibody directed against the catalytic subunits of bovine rod PDE6 identi®ed a single immunoreactive protein of 97 kDa. Anion exchange chromatography of chicken pineal soluble proteins revealed a peak of PDE6

activity that accounted for about 30% of cyclic GMPhydrolysis. In cultured chick pineal glands, arylalkylamine N-acetyltransferase (AA-NAT), the rate-limiting enzyme of melatonin synthesis, was protected from inhibition by light when selective PDE5/6 inhibitors (zaprinast, DMPPO) were added to the culture medium. PDE5/6 inhibitors did not affect AA-NAT activity in the dark. In contrast, a general PDE inhibitor (IBMX) increased AA-NAT in a light-independent manner. Together, the data indicate that rod and cone forms of PDE6 are expressed in chick pineal cells and that this enzyme plays a role in the inhibition of melatonin synthesis by light. Keywords: chicken pineal gland, melatonin, phosphodiesterase 6. J. Neurochem. (2001) 78, 88±99.

The pineal gland regulates circadian activity and seasonal breeding in different species through the production of an indolic hormone, melatonin (Zimmerman and Menaker 1979; Reiter 1980). Melatonin synthesis is organized on a daily basis, with high levels at night and low levels during daytime (Klein et al. 1981). In chicken and other nonmammalian vertebrates, the pineal gland is directly photosensitive in vitro, with light causing an inhibition of arylalkylamine N-acetyltransferase (AA-NAT), the ratelimiting enzyme of melatonin synthesis (Binkley et al. 1978; Deguchi 1981; Robertson and Takahashi 1988; Zatz et al. 1988; Bernard et al. 1997). The mechanism of this extra-retinal light perception remains ill-de®ned; however, there is increasing evidence that a phototransduction cascade similar to that previously described in retinal rods might operate in the chicken pineal gland. In rod outer segments, photolyzed rhodopsin activates the G protein, transducin, which in turn activates a cyclic GMP-phosphodiesterase (phosphodiesterase 6, PDE6), thus causing a rapid drop in the concentration of this cyclic nucleotide and the

closure of cyclic GMP-gated cation channels (see Yar®tz and Hurley 1994 for review). Studies in the chicken pineal gland have revealed that this organ contains several components of a phototransduction cascade, including opsins, transducin and cyclic GMP-gated cation channels (Okano et al. 1994; Max et al. 1995; Van Veen et al. 1986; Dryer and Henderson 1991; BoÈnigk et al. 1996; Kasahara et al. 2000). Noticeably, at least three photopigments

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Received December 19, 2000; revised manuscript received March 15, 2001; accepted March 22, 2001. Address correspondence and reprint requests to Pierre Voisin, Laboratoire de Neurobiologie Cellulaire, UMR CNRS 6558, UFR Sciences., 40 Avenue du Recteur Pineau, 86022 Poitiers, France. E-mail: [email protected] Abbreviations used: AA-NAT, arylalkylamine N-acetyltransferase; BSA, bovine serum albumin fraction V; DMPPO, 1,3-dimethyl-6-(2propoxy-5-methanesulphonylamidophenyl)pyrazolo[3,4-d]pyrimidin-4(5H )-one; DTT, dithiothreitol; IBMX, 1-methyl-3-isobutylxanthine; PDE, phosphodiesterase; SDS±PAGE, sodium dodecyl sulfate±polyacrylamide gel electrophoresis; ZT, Zeitgeber time.

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(pinopsin, chicken red opsin and chicken green opsin) appear to be expressed in the chicken pineal, as well as rod and cone forms of the cyclic GMP-gated cation channel (Okano et al. 1994; Max et al. 1995; BoÈnigk et al. 1996). This may be indicative of a heterogeneous population of pineal photoreceptors. At this point, however, the primary effector of phototransduction, PDE6, has not been described in the chicken pineal gland. In the retina, this enzyme is a heterotetramer-abgr2 in rods and a 0 2gc2 in cones; where a, b and a 0 are catalytic subunits and gr and gc are regulatory subunits that mediate activation by light (Baehr et al. 1979; Deterre et al. 1988; Gillespie and Beavo 1988). The ®rst objective of the present study was to examine the expression of both catalytic and regulatory subunits of PDE6 in the chicken pineal gland, because this enzyme remained the missing link of a retina-like phototransduction cascade. In addition it was anticipated that an analysis of PDE6 isoforms could provide further information on the diversity of photoreceptor subtypes in the chicken pineal. The second objective of this study was to examine a possible role of PDE6 in mediating the inhibition of AA-NAT by light in the chicken pineal gland. Materials and methods Animals One-day-old chicks (Gallus domesticus) were purchased from Rumolo Cie (QuincËay, France). They were kept for 2 weeks in a

12-h light/12-h dark (12L/12D) lighting schedule [lights on at `Zeitgeber time' (ZT) 0, lights off at ZT12, 500 lux] with food and water ad libitum. Chickens were killed by decapitation at the times indicated in the ®gure legends. Isolation of total and polyA1 RNA Total RNA was isolated as described by Bothwell et al. (1990). Tissues were sonicated 30 s in ice-cold 3 m LiCl/6 m Urea. After standing overnight at 48C, total RNA was obtained by centrifugation at 6000 g for 30 min. Pellets were resuspended in TE/SDS buffer (10 mm Tris-HCl pH 7.4, 1 mm EDTA pH 8, 0.5% sodium dodecylsulphate), washed by phenol/chloroform extraction and RNA was precipitated with ethanol. PolyA1 RNA was puri®ed from total RNA with Dynabeads oligo-dT25 (Dynal, Oslo, Norway), following the manufacturer's instructions. Reverse transcription±polymerase chain reaction (RT-PCR) Pineal or retinal total RNA (3 mg) was reverse transcribed (2 h, 428C) in 20 mL containing 100 ng oligo-dT25230 primer, 0.5 mm of each dNTP, 200 U of Reverse Transcriptase (M-MLV RT, Promega, Madison, WI, USA) and the manufacturer's buffer. The PCR was performed on 1/20th of an RT reaction, in 50 mL containing 50 pmol of each primer, 0.2 mm of each dNTP, 1.5 units of Taq DNA polymerase (Pharmacia, Saclay, France) and the manufacturer's buffer. The PCR reaction was: 958C for 50 s (Tm2 28C) for 50 s, 728C for 50 s; 25±35 cycles. The reaction products were analyzed by electrophoresis (1.5% agarose gel containing 0.7 mg/mL ethidium bromide) and visualized under UV light. PCR products were gel-puri®ed (QIA Quick Gel Extraction, Qiagen, Valencia, CA, USA) and pGEM-Tw-cloned (Promega) according to the manufacturer's instructions.

Table 1 Oligonucleotides used in this study Chick-a 0 -PDE6 S 2 53 Chick-a 0 -PDE6 AS 1179 Chick-a 0 -PDE6 S 1015 Chick-a 0 -PDE6 AS 2303 Degen-cat-PDE6 S #1 Degen-cat-PDE6 AS #1 Degen-cat-PDE6 S #2 Chick-rod-cat-PDE6 AS #1 Chick-rod-cat-PDE6 AS #2 Mam-g-c S 26 Mam-g-c AS 252 Chick-g AS Chick-g S Chick-g-c S 86 Chick-g-c S 2 99 Chick-g-c AS 45 Chick-g-r S 2 149 Chick-g-r AS 44 Chick-g-r AS 364 Amplimer Anchor-poly dG AP1 (Clontech) AP1-dT

5 0 -TGCTTTGATGGTGATGAAGTTC-3 0 5 0 -GACAATAGGCAATGAAAGGACA-3 0 5 0 -GCAGATCACTGGTGTCTTAT-3 0 5 0 -TTGTTTCTGTCCATCATGGG-3 0 5 0 -(C/T)CTAGA(A/G)GCCTT(G/C/T)GCCATGGC-3 0 5 0 -TTGTT(C/T)C(G/T)GTCCATCAT(A/G/C)GGAAT-3 0 5 0 -TTTGA(A/G)GAGCT(A/G/C)AC(A/G)GA(C/T)AT-3 0 5 0 -ATCAGTCCAGGCAGTTGTATTG-3 0 5 0 -GGCCTACTGAGTACCTGTCAC-3 0 5 0 -CTCCAACTTCAAACCAGGGTC-3 0 5 0 -TCAGATGATCCCAAACTGAGC-3 0 5 0 -CAGCTCGTGCAGTTCCAGATG-3 0 5 0 -TCCCAAGTTCAAGCAGAGACA-3 0 5 0 -AGCAGAGACAGACAAGACAG-3 0 5 0 -CTACCCAGGACGCAGGACAG-3 0 5 0 -GGGAGCATCTCCAGTGGTAAG-3 0 5 0 -AGAGCACCCAGAGGACGCGT-3 0 5 0 -CTGGTGGCTGACTTGAGCTCC-3 0 5 0 -GGTTTTAATGAGTGTTTCTCCAG-3 0 5 0 -GTCGGTAGGCTGGCGGCCGCT-3 0 5 0 -CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3 0 5 0 -CCATCCTAATACGACTCACTATAGGGC-3 0 5 0 -CCGCCCGGGCAGGTTTTTT-3 0

S, sense; AS, antisense; numbering from ®rst ATG codon.

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Fig. 1 Partial nucleotide and deduced amino acid sequences of chicken b-PDE6. The 5 0 -untranslated region is in lower case letters; the coding region is in capital letters; the underlined portion of the sequence indicates the b-PDE6 cDNA probe used for northern blot analysis (see Fig. 3); the deduced amino acid sequence is in

one-letter code. The primers used for RT-PCR and 5 0 -RACE are indicated as arrows (see Table 1 for details). All these primers were also used for sequencing, along with one more sequencing primer indicated as a dotted arrow.

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DNA sequencing Plasmid DNA preparations (Wizard Plus SV Minipreps, Promega) and gel-puri®ed PCR products (QIA Quick Gel Extraction, Qiagen) were sequenced using Big Dye reagent (Applied Biosystems, Courtaboeuf, France) and analyzed by a sequencing company (Genome Express, Grenoble, France). Chicken b-PDE6 cloning procedure Degenerated primers that did not discriminate the rod and cone forms of PDE6 catalytic subunits (degen-cat-PDE6 S #1 and degen-cat-PDE6 AS #1, see Table 1 and Fig. 1) were designed after alignment of human, murine and bovine sequences. These primers were used for RT-PCR ampli®cation of chicken pineal RNA. The 537 bp PCR product was digested with Dra1 to eliminate the cDNA ampli®ed from the a 0 subunit (a Dra1 site, conserved in all a 0 sequences, is absent from all a and b sequences). The 537 bp Dra1-resistant RT-PCR product was gel-puri®ed and pGEM-Twcloned. Sequence analysis of eight clones gave the same sequence, with high identity to mammalian a and b subunits. Based on this information, a chicken-speci®c antisense primer was designed (chick-rod-cat-PDE6 AS #1, see Table 1 and Fig. 1) and set aside for later use. 5 0 -RACE was performed to obtain more sequence information on this transcript. Chicken pineal cDNA, immobilized on oligo-dT Dynabeads and 5 0 -anchored as previously described (Grechez-Cassiau et al. 1998), was PCR-ampli®ed between `amplimer' (a primer complementary to the anchor) and `degencat-PDE6 AS #1' (35 cycles, annealing at 508C), followed by a nested PCR (on 1/50th of the ®rst reaction product) between `degen-cat-PDE6 S #2' (see Table 1 and Fig. 1) and `chick-rodcat-PDE6 AS #1' (35 cycles, annealing at 558C). The nested PCR product was gel-puri®ed and sequenced and the information was used to design a chicken-speci®c antisense primer (chick-rod-catPDE6 AS #2, see Table 1 and Fig. 1). Using this primer and the `amplimer', a 5 0 -RACE product of 900 bp was obtained from the 5 0 -anchored chicken pineal cDNA on Dynabeads (PCR 35 cycles, annealing at 608C). This PCR product was pGEM-Tw-cloned and sequenced. Chicken g-PDE6 cloning procedure A pair of primers, chosen within conserved regions of human and bovine g-cone PDE6 subunits (mam-g -c S 26 and mam-g -c AS 252, see Table 1), was used for RT-PCR on chicken retinal RNA. A 350-bp product was obtained and sequenced. It appeared later on that this RT-PCR product corresponded to a rare splice variant of the 5 0 end that was not found in the 5 0 -RACE reactions described below. Nevertheless, sequence information over 190 nucleotides upstream of the antisense primer revealed 86% identity with mammalian g-cone PDE6 subunit and 80% identity with mammalian g-rod PDE6 subunit. This information allowed us to design a chicken-speci®c antisense primer (chick-g AS, see Table 1) for 5 0 -RACE on chicken retinal RNA. This 5 0 -RACE yielded both g-rod and g-cone cDNA extremities. 5 0 -RACE protocol Total RNA (2 mg) of chicken retina was reverse transcribed with oligo-dT25230 and dC-tailed (5 0 -RACE kit; Life Technologies, Grand Island, NY, USA). The PCR was performed with an antisense primer (chick-g AS, see Table 1 and Fig. 2) and an `anchor-poly dG' primer for the dC-tail. We obtained two PCR products of 320 and 370 bp that were cloned in pGEM-Tw vector.

The 320 bp insert, sequenced from six clones, had the 5 0 end of g-cone cDNA, with a 107 bp 5 0 -untranslated region and 216 bp of the coding region. The 370 bp insert, sequenced from nine clones, had the 5 0 end of g-rod cDNA, with a 151-bp 5 0 -untranslated region and 222 bp of the coding region. 3 0 -RACE protocol Total RNA (2 mg) of chicken retina was reverse transcribed with oligo-dT25230 primer (Marathon cDNA Ampli®cation kit; Clontech, Palo Alto, CA, USA). The second strand was synthesized with Escherichia coli DNA pol I and a sense primer (chick-g S, see Table 1 and Fig. 2), chosen in a conserved region of g-rod and g-cone. A double-stranded anchor was then ligated at the blunt end and PCR was performed with chick-g S (see Table 1) and an anchor antisense primer (AP1, see Table 1). The PCR reaction was Genecleanw-puri®ed and pGEM-Tw-cloned. We isolated one clone containing the 3 0 end of g-rod cDNA, with 160 bp of the coding region (121 bp overlap with the 5 0 -RACE sequence) and a complete 111 bp 3 0 -untranslated region. The complete g-rod cDNA sequence was veri®ed by direct sequencing of an RT-PCR product obtained from chicken retinal RNA (®ve animals) with primers chick-g -r S 2149 and chick-g -r AS 364 (see Table 1 and legend to Fig. 2 for details). In order to isolate the 3 0 end of the g-cone transcript, the anchored template was submitted to a semi-nested PCR protocol: ®rst reaction with the primer pair chick-g S/AP1-dT (see Table 1 and Fig. 2), second reaction (on 1/50th of the ®rst reaction) with the primer pair chick-g -c S 86/AP1-dT (see Table 1 and Fig. 2). The PCR reaction was Genecleanw-puri®ed and pGEM-Tw-cloned. We isolated three identical clones containing the 3 0 end of g-cone cDNA, with 150 bp of the coding region (111 bp overlap with the 5 0 -RACE sequence) and a partial 164 bp 3 0 -untranslated region. cDNA probes The human b-actin cDNA probe was produced by PCR ampli®cation of lymphocyte cDNA (Delfau et al. 1990). The chicken a 0 -PDE6 cDNA probe was produced by PCR ampli®cation of a pGEM-Tw insert covering bp 1790 to bp 2303 of the coding sequence. The chicken b-PDE6 cDNA probe (see Fig. 1) was produced by PCR ampli®cation of a pGEM-Tw insert corresponding to the DraI-resistant RT-PCR product obtained from chicken pineal RNA (see `Chicken b-PDE6 cloning procedure' for details). The chicken g-rod cDNA probe (see Fig. 2) was produced by PCR ampli®cation of a pGEM-Tw clone of the g-rod 5 0 -RACE product (see `Chicken g-PDE6 cloning procedure'), with the primer pair chick-g -r S 2149 and AS 44 (see Table 1). The chicken g-cone cDNA probe (see Fig. 2) was produced by PCR ampli®cation of a pGEM-Tw clone of the g-cone 5 0 -RACE product (see `Chicken g-PDE6 cloning procedure'), with the primer pair chick-g -c S 299 and AS 45 (see Table 1). Northern blot analysis Total and polyA1 RNA were fractionated by electrophoresis on 1% agarose/0.7 m formaldehyde gels containing 0.7 mg/mL ethidium bromide and blotted overnight in 10  SSC buffer (1.5 m NaCl, 0.15 m sodium citrate, pH 7) on nitrocellulose membranes. Nonspeci®c binding sites were blocked for 2 h at 428C with hybridization buffer composed of 40% formamide, 10% dextran sulphate, 4  SSC, 1  Denhardt (0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin fraction V (BSA) and 0.02% ®coll

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Fig. 2 Nucleotide and deduced amino acid sequences of chicken g-rod and g-cone PDE6. The 5 0 - and 3 0 -untranslated regions are in lower case letters; the coding regions are in capital letters; stop codons are labelled with an asterisk; the g-rod polyadenylation signal is framed; the deduced amino acid sequences are in one-letter code, with boldface letters indicating sequence identity with mammalian counterparts. The primers used for 5 0 - and 3 0 -RACE are

indicated as arrows (see Table 1 for details). These primers were also used for sequencing. The g-rod sequence was veri®ed on an RT-PCR product obtained from retinal RNA with the primers indicated as dotted arrows (see Table 1 for details). These primers were also used for sequencing. The shaded portions of the sequences indicate g-rod-and g-cone-speci®c cDNA probes used for northern blot analysis (see Fig. 4).

(400), 20 mm Tris-HCl pH 7.4 and 0.3 mg/mL salmon sperm DNA. Probes used for hybridization were labelled by random priming (Feinberg and Vogelstein 1983) with [a-32P]dCTP (3000 Ci/mmol) and added (80 ng, 10±20 mCi) to 5±10 mL of

hybridization buffer. Hybridization on the northern blots was performed overnight at 428C. The nitrocellulose membranes were then washed in 2  SSC, 0.1% SDS at room temperature for 45 min and in 0.1  SSC, 0.1% SDS at 528C for 2 h. The

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membranes were then exposed 1±7 days on the screen of a PhosphorImager (Storm 820, Molecular Dynamics, Sunnyvale, CA, USA). Densitometric analysis of the autoradiograms was performed with the ImageQuant program (Molecular Dynamics). RNase H digestion of retinal RNA The conditions used for the reaction were adapted from those described by Donis-Keller (1979). Retinal total RNA (5 mg) and the sensitizing chick-g AS oligonucleotide (10 pmol) (see Table 1) were mixed in 20 mL containing 20 mm Tris HCl pH 8.4, 50 mm KCl, 2.5 mm MgCl2, 0.1 mg/mL BSA and 10 mm dithiothreitol (DDT). Hybridization was performed at 558C for 5 min. E. coli RNase H (0.2 U) was then added and the reaction was allowed to proceed for 15 min at 558C. Total RNA was then extracted by phenol/chloroform and ice-cold ethanol precipitation before northern analysis with the g-cone probe. Organ culture Two-week-old chickens raised in a 12L/12D lighting schedule (lights on at ZT0 and lights off at ZT12) were killed between ZT10 and ZT12 and the pineal glands were rapidly removed. Pineals were placed in 24-well culture plates (1 pineal per well), with 200 mL of BGJb medium (Life Technologies, Rockville, MD, USA), supplemented with 2 mm glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin. Organ culture was performed in a CO2 incubator at 378C, 5% CO2 in air and 70% humidity. The same 12L/12D lighting schedule as in vivo was provided in the incubator by a ¯uorescent lamp, with an illumination intensity of 500 lux during daytime. The medium was replaced after 1 day of culture. Pharmacological treatments Drugs were solubilized in dimethylsulphoxide and further diluted in water (1% dimethylsulphoxide ®nal concentration in culture medium). Treatments [i.e. IBMX 100 mm, zaprinast 100 mm, 1,3-dimethyl-6-(2-propoxy-5-methanesulphonylamidophenyl)pyrazolo[3,4-d]pyrimidin-4-(5H)-one (DMPPO) 10 mm] were performed after 36 h of organ culture, from ZT0 to ZT6, either in the dark or in the light. Control groups received 1% dimethylsulphoxide. At ZT6, pineal glands (eight in each experimental group) were frozen in liquid nitrogen and processed for NAT activity assay. NAT activity assay The assay was performed on individual pineal glands as previously described (Voisin et al. 1993), with 0.5 mm [acetyl-3H]acetyl-CoA (®nal speci®c activity, 4 Ci/mol) and 1 mm tryptamine. The N-[acetyl-3H]tryptamine formed was extracted into chloroform and counted. Anion exchange chromatography of chicken pineal phosphodiesterases Pineal glands from 24 chicks (2 weeks old) raised in 12L/12D (lights off at ZT12) were removed at ZT24, after 2 h of unexpected light, and frozen on dry ice before homogenization by ultra-turrax (5  10 s) in 1.25 mL buffer A (20 mm Tris-HCl pH 7.5, 2 mm Mg21 acetate, 1 mm DTT, 1 mm EGTA and protease inhibitors at 10 mg/l: soybean trypsin inhibitor, pefabloc, aprotinin and leupeptin). The 105 000 g (1 h) supernatant was applied onto a Mono Q (Pharmacia) column equilibrated in 20 mm Tris-HCl, 2 mm Mg21 acetate, 1 mm DTT, pH 7.5, and then eluted with a linear 0.05±0.45 m NaCl gradient in buffer in test tubes in which BSA was at 0.5 mg/mL ®nal concentration. Each fraction

(0.75 mL) was assessed for PDE activity at 0.4 mm cyclic AMP or cyclic GMP by a radioenzymatic assay (Lugnier et al. 1993). PDE inhibitors were added as freshly prepared stock solution in dimethylsulphoxide (®nal concentration 1%, which did not interfere with PDE activity). Western blot analysis of PDE6 Chickens raised in 12L/12D for 2 weeks were killed at the end of the night after 2 hours of unexpected light exposure. Pineals or retinas from ®ve animals were homogenized in electrophoresis sample buffer (Laemmli 1970) at 0.2 mg of protein/mL (dry weight estimate). The homogenates (30 mL) were analyzed by sodium dodecyl sulfate polyacrylamide (8%) gel electrophoresis (SDS± PAGE) and bovine rod PDE6 (30 ng), puri®ed according to Virmaux et al. (1971) was analyzed as a positive control. Proteins were transferred onto PVDF membrane and probed with the antibody against PDE6 a, b subunits diluted 1/10 000 (antibody 63F raised against amino acids 397±417 in the noncatalytic domain of PDE6 and recognizing with equal intensities both a and b subunits; kind gift of Dr Rick H. Cote, University of New Hampshire, Durham, USA; this antibody did not recognize PDE2 nor PDE5). The antibody±antigen complexes were detected using 1/3000 dilution goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) and ECL reagent (Amersham). The immunoblots were then exposed using Kodak X-ray ®lm (Eastman Kodak Company, Rochester, NY, USA).

Results MRNA encoding PDE6 catalytic subunits in the chicken pineal gland RT-PCR protocols were designed to examine the expression of both cone and rod forms of PDE6 catalytic subunits in the chicken pineal. Based on the previously published sequence of chicken a 0 -PDE6 (Semple-Rowland and Green 1994), two sets of speci®c primers (chick-a 0 -PDE6 S 2 53 and AS 1179, chick-a 0 -PDE6 S 1015 and AS 2303, see Table 1) were designed to amplify this transcript from chicken pineal RNA. Agarose gel electrophoresis revealed RT-PCR products of the expected sizes and gave no indication of alternative splicing (single band patterns, data not shown). Sequence analysis revealed 99.7% identity between the RT-PCR products and chicken a 0 -PDE6 (data not shown). Minor changes in the sequence could be due to allelic variation or to Taq errors. To examine the expression of a rod-like form of PDE6 in the chicken pineal gland, a cloning step had to be taken because neither a- nor b-subunits of PDE6 had been previously characterized in this species. The cloning procedure from chicken pineal RNA (RT-PCR and 5 0 -RACE, see `Materials and methods') yielded a 2362-bp sequence comprising 154 bp of 5 0 -untranslated region and 2208 bp of incomplete coding region (Fig. 1). The deduced amino acid sequence showed 84% identity (96% similarity) with mouse b-PDE6. This sequence was more distant from mouse a-PDE6 (74% identity, 91% similarity) or from chicken a 0 -PDE6 (67% identity, 87% similarity). Therefore this cDNA was tentatively named `chicken b-PDE6'.

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Fig. 3 Northern blot analysis of PDE6 catalytic subunits mRNA levels in the chicken pineal gland and retina. Two-week-old chickens raised in 12L/12D lighting schedule (lights on at ZT0, lights off at ZT12) were killed either at the end of the night or at the indicated `Zeitgeber times' (ZT). PolyA1 RNA was extracted from pools of 25 pineals or retinas, fractionated on denaturing agarose gels (4 mg/lane), blotted on nitrocellulose membranes and analyzed with a 0 -PDE6 or b-PDE6 cDNA probes. A b-actin cDNA probe was used for normalization purpose. (a) a 0 -PDE6 mRNA levels (two mRNA species of 3.4 and 4.4 kb) are 50-fold higher in the retina than in the pineal gland. (b) b-PDE6 mRNA levels (4.2 kb) are three-fold higher in the retina than in the pineal gland. (c) Day/night changes in a 0 -PDE6 mRNA levels in the chicken pineal gland. (d) Day/night changes in b-PDE6 mRNA levels in the chicken pineal gland.

A fragment of chicken a 0 -PDE6 cDNA (bp 1790 to bp 2303 of the coding sequence) was used to probe northern blots of pineal and retinal polyA1 RNA. Two transcripts of 3.4 and 4.4 kb could be detected in both tissues (Fig. 3a), in agreement with a previous study in the retina (SempleRowland and Green 1994). Quantitative analysis of the hybridization signals indicated the concentration of a 0 -PDE6 mRNA was approximately 50-fold higher in the retina than in the pineal (Fig. 3a). A cDNA fragment encompassing bp 1695 to bp 2231 of the coding sequence of chicken b-PDE6 was used to hybridize on northern blots of pineal and retinal polyA1 RNA. As illustrated in Fig. 3(b), a single band of approximately 4.2 kb was detected in both tissues. Quantitative analysis indicated that b-PDE6 mRNA levels were about three-fold higher in the retina than in the pineal. Day/night rhythm in PDE6 mRNA abundancy in the chicken pineal gland. Chickens raised in a 12L/12D lighting schedule were killed at ZT6, ZT11, ZT18 or ZT23 and pineal polyA1 RNA was analyzed on northern blot, with a 0 -PDE6 and with b-PDE6 cDNA probes. As illustrated in Fig. 3(c and d), a 0 -PDE6 mRNA, as well as b-PDE6 mRNA could be detected only at night in the chicken pineal gland. MRNA encoding PDE6 regulatory subunits in the chicken pineal gland In the absence of previous information on the sequence of PDE6 regulatory subunits in chicken, our ®rst step was to clone the corresponding cDNAs from chicken retina. The highly conserved sequences of mammalian g-PDE6 subunits

were used to design PCR primers, then 5 0 -RACE and 3 0 -RACE protocols were applied to chicken retinal RNA (see Materials and methods). A cDNA encoding chicken g-rod PDE6 was obtained. It contained 151 bp of 5 0 -untranslated region, 111 bp of 3 0 -untranslated region and an open reading frame of 261 bp (Fig. 2a). The 87 amino acid predicted sequence was 90% identical to mammalian g-rod PDE6 (Fig. 2a). A cDNA encoding chicken g-cone PDE6 was also obtained. It contained 107 bp of 5 0 -untranslated region, 164 bp of an incomplete 3 0 -untranslated region and an open reading frame of 255 bp (Fig. 2b). The predicted sequence of 85 amino acids was 89% identical to mammalian g-cone PDE6 (Fig. 2b). Speci®c probes for g-rod and g-cone were designed at the 5 0 ends of the cDNAs and used on northern blots of pineal and retinal polyA1 RNA (Figs 2a and b). In both tissues, the g-rod transcript was detected as a single band of 0.6 kb (Fig. 4a). The concentration of g-rod mRNA was about two-fold higher in the pineal than in the retina (Fig. 4a). Two forms of g-cone mRNA (1.1 kb and 3.4 kb) were detected in both tissues (Fig. 4b). Both forms of the g-cone mRNA were about 50-fold more abundant in the retina than in the pineal (Fig. 4b). No signi®cant day/night difference could be observed in the abundance of g-rod and g-cone mRNAs in the chicken pineal gland. The origin of the two mRNA species encoding g-cone was further investigated by digesting retinal RNA with Rnase H, in the presence of a g-cone antisense oligonucleotide located at the 3 0 end of the open reading frame (chick-g AS, see Table 1 and Fig. 2). After this treatment, northern blot analysis revealed a single 5 0 fragment of about 0.35 kb (Fig. 4c). A result indicating

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Fig. 4 Northern blot analysis of PDE6 regulatory subunits mRNA levels in the chicken pineal gland and retina. Two-week-old chickens raised in 12L/12D lighting schedule were killed at the end of the night. Total and polyA1 RNA was extracted from pools of 25 pineals or retinas, fractionated on denaturating agarose gels and blotted on nitrocellulose membranes and analyzed with the indicated probes. (a) PolyA1 RNA (4 mg/lane) hybridized with g-rod PDE6 cDNA (one mRNA species of 0.6 kb) and with b-actin cDNA (g-rod mRNA levels are two-fold higher in the pineal than in the retina). (b) PolyA1 RNA

(4 mg/lane) hybridized with g-cone PDE6 cDNA (two mRNA species of 1.1 and 3.4 kb) and with b-actin cDNA (g-cone mRNA levels are 50-fold higher in the retina than in the pineal). (c) Total RNA from chicken retina (5 mg) was digested with RNase H, in the presence of the sensitizing oligonucleotide chick-g -AS (see Table 1), before northern blot analysis with the g-cone probe. Lane 1: test sample with both RNase H and oligonucleotide; lane 2: control without RNase H; lane 3: control without oligonucleotide.

that the chicken g-cone mRNA species of 1.1 kb and 3.4 kb contain 3 0 -untranslated regions of 0.75 kb and 3.1 kb, respectively.

previously shown for the enzyme from bovine retina (Miki et al. 1973).

PDE6 protein and activity in the chicken pineal gland. Chicken pineal and retinal proteins were analyzed on western blot, with an antiserum raised against bovine rod PDE6. Chicken retinal PDE6 was resolved as two protein bands that migrated like the a and b subunits of bovine PDE6 (Fig. 5). In contrast, chicken pineal PDE6 showed only the protein band of higher molecular weight (Fig. 5), previously identi®ed as the a subunit in mammals (Baehr et al. 1979). Chicken pineal supernatant was submitted to anion exchange chromatography and a pro®le of cyclic AMPand cyclic GMP-PDE activities was obtained (Fig. 6). A double peak of cyclic GMP-PDE activity was of special interest because it eluted at the same ionic strength as authentic PDE6 from bovine rod outer segments (Fig. 6). The PDE6 peak (cyclic GMP-PDE activity 93% inhibited by 10 mm zaprinast, data not shown) contained little cyclic AMP-PDE activity (about one-®fth that of cyclic GMP-PDE activity, Fig. 6) and this activity was inhibited 70% by rolipram 10 mm (data not shown), suggesting it was mostly due to contamination by PDE4, a cyclic AMP-speci®c enzyme that peaked in the next fractions (Fig. 6). Nevertheless, after treating the PDE6 fractions by rolipram, the remaining cyclic AMP-PDE activity was further inhibited by 15% in the presence of a PDE5/6 inhibitor (zaprinast 10 mm, data not shown), thus con®rming that chicken pineal PDE6 has some cyclic AMP-hydrolyzing activity, as

Effect of PDE5/6 inhibitors on AA-NAT activity To examine a possible role of PDE6 in mediating the inhibition of AA-NAT activity by light, chick pineal glands were placed in organ culture on a 12L/12D lighting schedule and treated with phosphodiesterase inhibitors from ZT0 to ZT6, either in the dark or in the light. This timing was chosen because it was observed above that a 0 - and b-PDE6 mRNA levels peaked at the end of the night. In the absence of PDE inhibitors, AA-NAT activity was 50% to 70% lower in the light than in the dark (Fig. 7). In a ®rst experiment,

Fig. 5 Western blot analysis of chicken pineal PDE6. Chicken pineal and retinal proteins (6 mg) were fractionated on SDS±PAGE (8% acrylamide) and blotted on a PVDF membrane. A similar treatment was applied to 30 ng of authentic bovine PDE6. The PVDF membrane was probed with anti-PDE6 antiserum (rabbit) at 1/10 000 dilution, followed by goat anti-rabbit peroxidase conjugate at 1/3000 dilution. Immunoreactions were revealed with the ECL reagent (Amersham, Saclay, France). Kodak X-ray ®lm exposure was for 1 min. Molecular weights were determined by comparison with kaleidoscope standards (Bio-Rad).

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Fig. 6 Anion exchange chromatography pro®le of cyclic AMP-and cyclic GMP-PDEs in chicken pineal supernatant. Two-week-old chickens raised in a 12L/12D lighting schedule were killed at the end of the night, after two hours of unexpected light exposure. A pool of 24 pineals was homogenized in 1250 mL of buffer A and centrifuged at 105 000 g 48C for 1 h. A 1100 mL aliquot of the supernatant containing 1700 mg of protein was submitted to anion exchange chromatography on a monoQw column, with a 0.05±0.45 M NaCl gradient (dotted line). The fractions were assayed for PDE activity with either 0.4 mM cyclic AMP (W) or 0.4 mM cyclic GMP (B) as

substrate (no activity before fraction 15). Pineal PDE6 was identi®ed in fractions 37±42 (same elution fractions as authentic bovine PDE6 and 93% inhibition by 10 mM zaprinast, data not shown). The peak of cyclic AMP-PDE activity in fractions 46±49 was identi®ed as a mixture of pineal PDE4 (70% inhibition by rolipram 10 mM, data not shown) and PDE3 (50% inhibition by 4 mM cyclic GMP, data not shown). Although not fully characterized, fractions 20±35 appeared to contain a mixture of zaprinast-sensitive PDE5 and/or PDE9 as well as Ca21/calmodulin-activated PDE1.

Fig. 7 Effects of PDE5/6 inhibitors on AA-NAT activity in cultured chick pineal glands. Two-week-old chickens raised in a 12L/12D lighting schedule (lights on at ZT0 and lights off at ZT12) were killed between ZT10 and ZT12 and their pineal glands were placed in organ culture under the same lighting schedule. After 36 h in organ

culture, the pineal glands were treated with the indicated drugs between ZT0 and ZT6, either in the light or in the dark. Pineal glands were collected at ZT6 and assayed for AA-NAT activity. Data are means ^ SEM (n ˆ 8). Statistical signi®cance was estimated by double-tailed Student's t-test at the indicated probability level.

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the PDE5/6 inhibitor zaprinast (100 mm) signi®cantly protected AA-NAT activity from inhibition by light, without affecting enzyme activity in the dark (Fig. 7). Lower concentrations of zaprinast (10, 30 mm) were ineffective (data not shown). To con®rm these data, a second experiment was performed using DMPPO (10 mm), a PDE5/6 inhibitor of similar selectivity but 100-fold higher potency than zaprinast (Stoclet et al. 1995). Again, AA-NAT activity was protected from inhibition by light, while it remained unchanged in darkness (Fig. 7). In both experiments, the general PDE inhibitor, IBMX, increased AA-NAT activity in a light-independent manner (Fig. 7). Discussion The present study indicates that PDE6 is expressed in the chicken pineal gland, where it appears to play a role in the inhibition of melatonin synthesis by light. By-products of this study are the previously uncharacterized sequences of b, g-rod and g-cone subunits of chicken PDE6. The presence of PDE6 in the chicken pineal gland combines, with previous information on opsins (Okano et al. 1994; Max et al. 1995), transducin (Van Veen et al. 1986; Kasahara et al. 2000) and cyclic GMP-gated cation channels (Dryer and Henderson 1991; BoÈnigk et al. 1996), to establish that this organ contains all the basic components of a phototransduction cascade of the retinal type. Day/night variation in a 0 - and b-PDE6 mRNA levels was phase-opposed to that of pinopsin mRNA (Takanaka et al. 1998). Although the physiological signi®cance of these nycthemeral variations in transcription remains unclear, they appear to be a hallmark of photoreceptor metabolism, because they have also been reported for a number of phototransduction markers in the retina (Brann and Cohen 1987; Bowes et al. 1988; Korenbrot and Fernald 1989; McGinnis et al. 1992; Pierce et al. 1993). One possibility is that a precisely timed transcription serves to provide newly synthesized proteins at the appropriate moment for the elaboration of photoreceptor outer segments. The diversity of transcripts encoding catalytic and regulatory subunits of PDE6 would argue for the expression of both rod and cone forms of the enzyme in the chicken pineal gland. The information obtained by cDNA cloning and by western blot analysis appear to agree on the presence of only one type of rod catalytic subunit in the pineal gland. However, cDNA sequencing identi®ed a b subunit, whereas the protein detected on western blot migrated like an a subunit. This was not due to interspecies differences in the size of PDE6 subunits, because the enzyme from chicken retina clearly showed two protein bands that migrated like the a and b subunits of bovine PDE6. It may be suggested that pineal-speci®c post-translational modi®cations of the b subunit could be responsible for the observed shift in electrophoretic mobility. It is then possible that the 97 kDa

band observed in the pineal contains both an a subunit that remained undetected in the cloning procedure and a shifted b subunit. Further studies would be required to settle this point and to fully elucidate the structure of chicken rod PDE6. It should also be noted that a single catalytic subunit should be suf®cient to provide enzyme activity, because a2 or b2 homodimers were previously shown to reconstitute PDE6 activity in a heterologous expression system (Piriev et al. 1993). The presence of both rod and cone PDE6 subunits in the chicken pineal is in keeping with a previous report indicating that rod and cone forms of cyclic GMPgated cation channel are expressed in this tissue (BoÈnigk et al. 1996). Several photopigments have also been identi®ed in the chicken pineal, including red cone opsin, green cone opsin and the pineal-speci®c pinopsin (Okano et al. 1994; Max et al. 1995). Although the presence of rhodopsin has not been speci®cally reported in the chicken pineal gland, this rod-speci®c photopigment has been detected by immunocytochemistry in the pineal of quail, a closely related species (Araki et al. 1992). We further clari®ed this point by cloning a 475-bp RT-PCR product from chicken pineal RNA, whose sequence was 99.5% identical to chicken rhodopsin (Voisin, unpublished results). So far, only transducin has been observed as being only in rod form in the chicken pineal (Kasahara et al. 2000). By analogy with the retina, it may be envisaged that rod and cone forms of the different phototransduction markers de®ne distinct subtypes of photoreceptors in the chicken pineal gland. In situ hydridization and immunocytochemical detection of chicken pineal photopigments would appear to support this view. Indeed, several studies agree on an extensive distribution of pinopsin-positive cells (Max et al. 1995; Okano et al. 1997), whereas red cone opsin was observed in only a small number of cells (Okano et al. 1997). Further studies are now required to describe the distribution of the different isoforms of PDE6 in the chicken pineal gland, because the isoform of widest distribution would be the most probable effector of the pinopsin-initiated phototransduction cascade. The present study provides the ®rst evidence that PDE6 may be the functional link between photosensitivity and inhibition of melatonin synthesis in the chicken pineal gland. This was achieved by showing that PDE5/6 inhibitors protect AA-NAT activity from inhibition by light without affecting the enzyme activity in darkness. Because AA-NAT activity and melatonin synthesis are strongly stimulated by cyclic AMP analogs, but not by cyclic GMP analogs, it appears likely that the inhibition of AA-NAT by light is consecutive to a drop in cyclic AMP levels (Deguchi 1979; Nikaido and Takahashi 1989; Zatz 1989). Several studies would support this view because they showed that cyclic AMP levels are decreased by light in chick pineal cells (Takahashi and Zatz 1982; Nikaido and Takahashi 1989). One possibility is that the drop in cyclic AMP levels results from the cyclic AMP-hydrolyzing activity of PDE6 (Miki

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et al. 1973). However, we observed in the present study that this activity was rather low, suggesting that indirect routes from PDE6 to AA-NAT should be taken into consideration. It may be hypothesized that `classical' phototransduction proceeds all the way to the hyperpolarization of the pineal cell membrane, thus causing the closure of voltage-gated Ca21 channels and the inhibition of a Ca21/calmodulindependent adenylate cyclase. Another possibility is that a drop in cyclic GMP, consecutive to the activation of PDE6 by light, may release the inhibition of a PDE3 (cyclic GMPinhibited PDE, see Lugnier and Komas 1993; Manganiello et al. 1995 for reviews), thus leading to a secondary drop in cyclic AMP levels. In favour of this hypothesis, we have observed that the peak of cyclic AMP-hydrolyzing activity that eluted immediately after PDE6 was indeed inhibited by cyclic GMP. A possible effect of light on glutamate release from chick pineal cells should also be considered, because recent studies have shown that an autocrine loop involving glutamate regulates melatonin synthesis in rat pinealocytes (Yamada et al. 1998; Moriyama et al. 1999). While the mechanism linking photosensitivity to melatonin synthesis in the chicken pineal is becoming clearer, other studies indicate that this question may extend to mammals. Indeed it was recently reported that the neonatal rat pineal gland explanted in organ culture responds to light with an inhibition of melatonin synthesis (Tosini et al. 2000). This response could be observed in a narrow developmental window and was obliterated by noradrenergic input to the pineal gland (Tosini et al. 2000). These recent ®ndings are in agreement with previous studies indicating that a large number of phototransduction markers can be detected by in situ hybridization in the neonatal rat pineal (Blackshaw and Snyder 1997), that PDE6 activity can be detected in this organ (Carcamo et al. 1995) and that norepinephrine decreases opsin immunoreactivity in cultured rat pinealocytes (Araki and Tokunaga 1990; Araki 1992). Therefore, elucidating the mechanism of pineal photosensitivity may not only improve our knowledge of pineal physiology in non mammals, but also our understanding of the development of pineal function in the mammalian embryo. Acknowledgements We wish to express our grateful thanks to Dr R. H. Cote for his kind gift of the 63F antibody against bovine catalytic PDE6. We also wish to thank Mrs F. Chevalier, Mrs N. Girard and Mr G. Epistolin for their technical assistance. This work was supported by the Fondation Langlois. FM was supported by a stipend from the ReÂgion Poitou-Charentes.

References Araki M. (1992) Cellular mechanism for norepinephrine suppression of pineal photoreceptor-like cell differentiation in rat pineal cultures. Dev. Biol. 149, 440±447.

Araki M. and Tokunaga F. (1990) Norepinephrine suppresses both photoreceptor and neuron-like properties expressed by cultured rat pineal glands. Cell Differ. Dev. 31, 129±135. Araki M., Fukada Y., Shichida Y., Yoshizawa T. and Tokunaga F. (1992) Differentiation of both rod and cone types of photoreceptors in the in vivo and in vitro developing pineal glands of the quail. Brain Res. Dev. Brain Res. 65, 85±92. Baehr W., Devlin M. J. and Applebury M. L. (1979) Isolation and characterization of cGMP phosphodiesterase from bovine rod outer segments. J. Biol. Chem. 254, 11669±11677. Bernard M., Klein D. C. and Zatz M. (1997) Chick pineal clock regulates serotonin N-acetyltransferase mRNA rhythm in culture. Proc. Natl Acad. Sci. USA 94, 304±309. Binkley S. A., Riebman J. B. and Reilly K. B. (1978) The pineal gland: a biological clock in vitro. Science 202, 1198±1120. Blackshaw S. and Snyder S. H. (1997) Developmental expression pattern of phototransduction components in mammalian pineal implies a light-sensing function. J. Neurosci. 17, 8074±8082. BoÈnigk W., Muller F., Middendorff R., Weyand I. and Kaupp U. B. (1996) Two alternatively spliced forms of the cGMP-gated channel a-subunit from cone photoreceptor are expressed in the chick pineal organ. J. Neurosci. 16, 7458±7468. Bothwell A., Yancopoulos G. D. and Alt F. W. (1990) RNA preparation using LiCl-urea, in Methods for Cloning and Analysis of Eukaryotic Genes, pp. 15±17. Jones and Bartlett, Boston. Bowes C., Van Veen T. and Farber D. B. (1988) Opsin, G-protein and 48-kDa protein in normal and rd mouse retinas: developmental expression of mRNAs and proteins and light/dark cycling of mRNAs. Exp. Eye Res. 47, 369±390. Brann M. R. and Cohen L. V. (1987) Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina. Science 235, 585±587. Carcamo B., Hurwitz M. Y., Craft C. M. and Hurwitz R. L. (1995) The mammalian pineal expresses the cone but not the rod cyclic GMP phosphodiesterase. J. Neurochem. 65, 1085±1092. Deguchi T. (1979) Role of adenosine 3 0 ,5 0 -monophosphate in the regulation of circadian oscillation of serotonin N-acetyltransferase activity in cultured chicken pineal gland. J. Neurochem. 33, 45±51. Deguchi T. (1981) Rhodopsin-like photosensitivity of isolated chicken pineal gland. Nature 290, 706±707. Delfau M. H., Kerckaert J. P., Collyn D. M., Fenaux P., Lai J. L., Jouet J. P. and Grandchamp B. (1990) Detection of minimal residual disease in chronic myeloid leukemia patients after bone marrow transplantation by polymerase chain reaction. Leukemia 4, 1±5. Deterre P., Bigay J., Forquet F., Robert M. and Chabre M. (1988) cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits. Proc. Natl Acad. Sci. USA 85, 2424±2428. Donis-Keller H. (1979) Site speci®c enzymatic cleavage of RNA. Nucleic. Acids. Res. 7, 179±192. Dryer S. E. and Henderson D. (1991) A cyclic GMP-activated channel in dissociated cells of the chick pineal gland. Nature 353, 756±758. Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high speci®c activity. Anal. Biochem. 132, 6±13. Gillespie P. G. and Beavo J. A. (1988) Characterization of a bovine cone photoreceptor phosphodiesterase puri®ed by cyclic GMPsepharose chromatography. J. Biol. Chem. 263, 8133±8141. Grechez-Cassiau A., Bernard M., Ladjali K., Rodriguez I. R. and Voisin P. (1998) Structural analysis of the chicken hydroxyindole-Omethyltransferase gene. Eur. J. Biochem. 258, 44±52. Kasahara T., Okano T., Yoshikawa T., Yamazaki K. and Fukada Y. (2000) Rod-type transducin alpha-subunit mediates a

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 88±99

Chicken pineal PDE6 99

phototransduction pathway in the chicken pineal gland. J. Neurochem. 75, 217±224. Klein D. C., Auerbach D. A., Namboodiri M. A. A. and Wheler G. H. T. (1981) Indole metabolism in the mammalian pineal gland, in The Pineal Gland, Vol. I: Anatomy and Biochemistry (Reiter R. J., ed.), pp. 199±227. CRC Press, Boca Raton, Florida. Korenbrot J. I. and Fernald R. D. (1989) Circadian rhythm and light regulate opsin mRNA in rod photoreceptors. Nature 337, 454±457. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Lugnier C. and Komas N. (1993) Modulation of vascular cyclic nucleotide phosphodiesterases by cyclic GMP: role in vasodilatation. Eur. Heart J. 14, 141±148. Lugnier C., Muller B., Le Bec A., Beaudry C. and Rousseau E. R. D. (1993) Characterization of indolidan- and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions. J. Pharmacol. Exp. Ther. 265, 1142±1151. Manganiello V. C., Murata T., Taira M., Belfrage P. and Degerman E. (1995) Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Arch. Biochem. Biophys. 322, 1±13. McGinnis J. F., Whelan J. P. and Donoso L. A. (1992) Transient, cyclic changes in mouse visual cell gene products during the light-dark cycle. J. Neurosci. Res. 31, 584±590. Max M., McKinnon P. J., Seidenman K. J., Barrett R. K., Applebury M. L., Takahashi J. S. and Margolskee R. F. (1995) Pineal opsin: a nonvisual opsin expressed in chick pineal. Science 267, 1502±1506. Miki N., Keirns J. J., Marcus F. R., Freeman J. and Bitensky M. W. (1973) Regulation of cyclic nucleotide concentrations in photoreceptors: an ATP-dependent stimulation of cyclic nucleotide phosphodiesterase by light. Proc. Natl Acad. Sci. USA 70, 3820±3824. Moriyama Y., Yamada H., Hayashi M. and Yatsushiro S. (1999) Intrinsic glutaminergic system negatively regulates melatonin synthesis in mammalian pineal gland. Adv. Exp. Med. Biol. 460, 83±90. Nikaido S. S. and Takahashi J. S. (1989) Twenty-four hour oscillation of cAMP in chick pineal cells: role of cAMP in the acute and circadian regulation of melatonin production. Neuron 3, 609±619. Okano T., Yoshizawa T. and Fukada Y. (1994) Pinopsin is a chicken pineal photoreceptive molecule. Nature 372, 94±97. Okano T., Takanaka Y., Nakamura A., Hirunagi K., Adachi A., Ebihara S. and Fukada Y. (1997) Immunocytochemical identi®cation of pinopsin in pineal glands of chicken and pigeon. Brain Res. Mol. Brain Res. 50, 190±196. Pierce M. E., Sheshberadaran H., Zhang Z., Fox L. E., Applebury M. L. and Takahashi J. S. (1993) Circadian regulation of iodopsin gene expression in embryonic photoreceptors in retinal cell culture. Neuron 10, 579±584. Piriev N. I., Yamashita C., Samuel G. and Farber D. B. (1993) Rod

photoreceptor cGMP-phosphodiesterase: analysis of alpha and beta subunits expressed in human kidney cells. Proc. Natl Acad. Sci. USA 90, 9340±9344. Reiter R. J. (1980) The pineal and its hormones in the control of reproduction in mammals. Endocr. Rev. 1, 109±131. Robertson L. M. and Takahashi J. S. (1988) Circadian clock in cell culture: II. In vitro photic entrainment of melatonin oscillation from dissociated chick pineal cells. J. Neurosci. 8, 22±30. Semple-Rowland S. L. and Green D. A. (1994) Molecular characterization of the alpha 0 -subunit of cone photoreceptor cGMP phosphodiesterase in normal and rd chicken. Exp. Eye Res. 59, 365±372. Stoclet J. C., Keravis T., Komas N. and Lugnier C. (1995) Cyclic nucleotide phosphodiesterases as therapeutic targets in cardiovascular diseases. Exp. Opin. Invest. Drugs 4, 1081±1100. Takahashi J. S. and Zatz M. (1982) Photic regulation of cyclic nucleotide levels and N-acetyltransferase activity in the cultured avian pineal. Soc. Neurosci. 8, 546. Takanaka Y., Okano T., Iigo M. and Fukada Y. (1998) Light-dependent expression of pinopsin gene in chicken pineal gland. J. Neurochem. 70, 908±913. Tosini G., Doyle S., Geusz M. and Menaker M. (2000) Induction of photosensitivity in neonatal rat pineal gland. Proc. Natl Acad. Sci. USA 97, 11540±11544. Van Veen T., Ostholm T., Gierschik P., Spiegel A., Somers R., Korf H. W. and Klein D. C. (1986) alpha-Transducin immunoreactivity in retinae and sensory pineal organs of adult vertebrates. Proc. Natl Acad. Sci. USA 83, 912±916. Virmaux N., Urban P. F. and Waehnheldt T. V. (1971) Proteins of bovine retinal outer segments electrophoresis on polyacylamide gels in the presence of sodium dodecyl sulfate. FEBS Lett. 12, 325±328. Voisin P., Van Camp G., Pontoire C. and Collin J. P. (1993) Prostaglandins stimulate serotonin acetylation in chick pineal cells: involvement of cyclic AMP-dependent and calcium/ calmodulin-dependent mechanisms. J. Neurochem. 60, 666±670. Yamada H., Yatsushiro S., Ishio S., Hayashi M., Nishi T., Yamamoto A., Futai M., Yamaguchi A. and Moriyama Y. (1998) Metabotropic glutamate receptors negatively regulate melatonin synthesis in rat pinealocytes. J. Neurosci. 18, 2056±2062. Yar®tz S. and Hurley J. B. (1994) Transduction mechanisms of vertebrate and invertebrate photoreceptors. J. Biol. Chem. 269, 14329±14332. Zatz M. (1989) Relationship between light, calcium in¯ux and cAMP in the acute regulation of melatonin production by cultured chick pineal cells. Brain Res. 477, 14±18. Zatz M., Mullen D. A. and Moskal J. R. (1988) Photoendocrine transduction in cultured chick pineal cells: effects of light, dark, and potassium on the melatonin rhythm. Brain Res. 438, 199±215. Zimmerman N. H. and Menaker M. (1979) The pineal gland: a pacemaker within the circadian system of the house sparrow. Proc. Natl Acad. Sci. USA 76, 999±1003.

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