Relationship between Time of Activation of Phospholipase C-linked. Plasma Membrane Receptors and Reloading of Intracellular Ca2+. Stores in LAN-I Human ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 269, No. 27, Issue of July 8, pp. 18021-18027, 1994 Printed in U.S.A.
0 1994 by The American Society for' Biochemistry and Molecular Biology, Inc.
Relationship between Time of Activation of Phospholipase C-linked Plasma Membrane Receptors and Reloading of Intracellular Ca2+ Stores in LAN-I Human Neuroblastoma Cells* (Received forpublication, January 3, 1994, and in revised form, February 24, 1994) Alessandro Fatatis, Roberta Caporaso, Elodia Iannotti, Antonella Bassi, Gianfranco Di Renzo, and Lucio AnnunziatoS From the Section of Pharmacology, Department of Neuroscience, Schoolof Medicine, Federico ZZ University of Naples, 80131 Naples, Italy
The stimulation of plasma membrane receptors coupled to The effectof sequential stimulation of different inositol(1,4,5)-trisphosphate(IP,)-linked receptors on the phospholipase C via GTP-binding protein (G-protein) activates functioning of intracellular Ca2+stores was evaluated in the hydrolysis of poliphosphoinositides and leadsto the formasingle LAN-1 human neuroblastoma cells by means of tion of IP,,' which acts as a well known intracellular second fura-2microfluorimetry.Homologous restimulation both messenger (1).A fundamental phenomenon triggered by IP, in theabsence and in the presence of extracellular Ca2+ formation which follows phospholipase C activation is themowith endothelin-1 (ET-l),Lys-bradykinin (BK), and ATP bilization of Ca2+ fromintracellular compartments,most likely did not elicit an intracellular Ca2+increase, whereas a represented by a modified portion of the endoplasmic reticulum [CaBIielevation after carbachol (CCh) re-exposure was (ER) which possesses IP, receptors on its membrane(2). When obtained only in the presence of extracellular Ca2+. SinceIP, binds to its receptor, the ER IP,-sensitive Ca2+ channel thapsigargin and ionomycin, in the absence of extracelopens, and the stored Ca2+is released into the cytosol (1-3). The lular Ca", were still able to release Ca2+after ET-1, BK, is assured by the ER ATPrefilling of intracellular Ca2+ pools and ATP but not after CCh, it can be argued that inthe first case the stores were not completely depleted. This dependent Ca2+pump which is able to bring Ca2+ from the evidence was also confirmed the byfact that LAN-1cells, cytosol back into the stores (4,5 ) . Multiple simultaneous andor sequentialstimulations of sequentially exposed in different order to ET-1, BK, ATP, and upon extracellular Ca2+ removal, showed an increase plasma membrane receptors by different hormones, peptides, of [Ca2+li although progressively reduced in magnitude. or neurotransmitters may profoundly affect the intracellular By contrast, when CCh wasperfused as the first agonist, Ca2+ signaling.In fact, thedepletion and the subsequentrefillof an IP,it completely precluded any further Ca2+ mobilization by ing of intracellular Ca2+ stores due to the activation the other three agonists. In addition, the lack of poten- linked receptor might interfere with theCa2+transduction sigtiation of the Ca2+response when BK and ET-1 were su- nal which follows the activationof another IP,-linked receptor. perfused together and the potentiation of Ca2+ response Although this problem has been partially examined (6-9), the elicited by ET-1 after BK, when the plasma membrane effect of sequential stimulation of different receptors coupled Ca2+ efflux pathways were blockedby lanthanum during with IP, production on the functioning of intracellular Ca2+ the firstagonist exposure, indicated that LAN-1cells can stores still needst o be clarified. In order to study the relationrecycle cytoplasmic Ca2+ when exposed to ET-1, BK, ATP ship between multiple receptor stimulation and the responsivebut not when exposed to CCh. This inhibitory effect of ness of intracellular Ca2+stores, LAN-1 human neuroblastoma CCh (perfused for 90 s) on Ca2+ refilling was strictly de- cells appeared to be a suitable model to investigate this interpendent on the time of receptor occupancy since the ex- action. In fact, this clone is provided with multiple IP,-linked posure to CCh for a shorter period (15 s) produced the receptors, like M, and M, muscarinic subtypes (lo), and those same effect on Ca" refilling when ET-1, BK,and ATP were perfused, as firstagonist, for 90 s. Furthermore, the en- for ET-1(11),ATP, and BK (12). In addition, thesecells present tity of Ca2+refilling after 15 s ofBK receptor occupancy the peculiarity of the lack of caffeine and ryanodine-sensitive was similar to that observed after 90 s. This seemsto s u g internal Ca2+stores (101, thus the whole amount of Ca2+regest that the receptors for ET-1, BK, and ATP maintain leased into the cytoplasm upon stimulation of a poliphosphothe transductional mechanisms in an activated state for inositide hydrolysis-coupled receptor should derive only by the a time shorter than the time of receptor occupancy. This emptying of the IP,-dependent Ca2+stores. In the present work, LAN-1 clone was studied by fura-2 was confirmedby the fact that IP, levels during a 90-sBK exposure fell to prestimulated value within 30 s, whereas microfluorimetry and videoimaging to monitor changes of after CCh they reached a sustained plateau phase, after [Ca2+l,in a single cell. With this approach [Ca"], modifications the peak. in response to the stimulation of multiple IP,-linked receptors can be estimated in the same cell. In addition, most of the experiments with LAN-1 cells were performed in a system in * This work was supported by CNR Grant 92.02176.CT14 (to G. F. D.) which extracellular Ca2+ was omitted, since in this condition and 92.02455.CT04 (to L. A.) and by the CNR Target Project on Bio- the refilling of intracellular stores canbe exclusively provided technology and Bioinstrumentation. The costs of publication of this by the "new" Ca2+ released into thecytoplasm. These experiarticle were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent"in accordance mental approaches allowed us to verify if, in LAN-1 cells: (i) with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed: Section of Pharmacology, Dept.of Neuroscience, Schoolof Medicine, Federico I1 University The abbreviations usedare: IP,, inositol (1,4,5)-trisphosphate; ET-1, of Naples, Via Pansini 5, 80131 Naples, Italy. Tel.: 81-746-3318-3323; endothelin-1; BK, Lys-bradykinin; CCh, carbachol; [Ca2+l,,intracellular Fax: 81-746-3323. free calcium concentration; ER, endoplasmic reticulum.
18021
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Different IP,-linked Stimulation of
Ca2+released into thecytosol upon a first receptor stimulation can be reaccumulated back into the internal stores and then released again upon multiple sequential activation of the same or of different receptors;(ii) the time of receptor stimulation by one agonist is a factor that can affect the refilling process and therefore can regulate the further CaZ+ mobilization by a subsequent agonist action; (iii) the time of receptor occupancy by which the transducone agonist corresponds t o the time during tional mechanism is operating or conversely the transductional pathway turns off even if the agonist is still bound to its receptor.
Receptors ard buffer. After a brief centrifugation, 2 x lo6 cells were resuspended and incubated with Li' in presenceof 100 PM CCh or 1 p~ BK (10 pl) at room temperature (final volume 300 pli. Reactions were terminated adding 940 pl of a (1:2) chlorofodmethanol solution. Separation of [3Hlinositol phosphates was achieved through a 100-200 mesh size AG-1x8 anion-exchange resin(Bio-Rad) using ammonium formate solutions (4 ml) a t concentrations of 0.2,0.4, andl mM to elute[3H]inositol monophosphate, [3Hlinositol bisphosphate, and [3H]inositol trisphosphate, respectively. Finally, 1-ml aliquots of the threedifferent fractions were collected and radioactivity directly determined by liquid scintillation counting, using Ecolite (ICN, CA) as liquid scintillator. Materials-Drugs were obtained as follows: bradykinin, ATP, ionomycin, fura-2/AM were purchased from Calbiochem. Thapsigargin was purchased from LC Service (Woburn, MA). [3H]myo-inositol was purchased from Amersham Corp. All other drugs were purchased from Sigma.
EXPERIMENTAL PROCEDURES Cell Culture--LAN-1 humanneuroblastoma cell line(generously supplied by Dr. Massarelli, Centre de Neurochimie-Cronenbourg, Strasbourg-Cedex, France)wascultured as a monolayer in polystyrene RESULTS dishes and maintained in Dulbecco's modified Eagle's medium (ICN Effects of Different ZP,-linked Agonists on [Ca2+],-The expoLaboratories, Milan, Italy) containing 10% heat-inactivated fetal calf sure of LAN-1 cells to increasing concentrations of ET-1, BK, serum (Hyclone, UT), 100 IU of penicillirdml, 100 pgof streptomycidml. Cells were grown in a humidified incubator at 37 "C in a 5%CO, ATP, and CCh, in the absence of extracellular Ca2+, caused a atmosphere and were fed twice weekly. All experiments were performed dose-related increase of [Ca2+lias shown in Fig. 1. The lowest with cells from passages 80-86. concentration of each agonist elicitingthe maximal increase of IntracellularCa2+ Measurements-LAN-1 cells weregrown on [Ca2+l,(ET-1, 100 I"; BK, 1PM; ATP, 50 p ~ and ; CCh, 100 25-mm clean no. 1 glass coverslips, previously coated with collagen (VitrogenR") and poly-L-lysine (30 pg/ml), and transferred in a 35-mm was used in all the following experiments. Among the four Petri plastic dish (Falcon). The cells were loaded with 5 p~ fura-2 agonists, CCh induced the highest elevation of [Ca2+],. acetoxymethylester (Calbiochem,San Diego, CAI using a balanced salt The percentage of LAN-1 cells responsive t o ET-1, BK, ATP, solution (standard buffer)of the following composition: 159 mM NaC1, 5 and CCh were 43% (60n),52% (60n),78%(5111) and 70% (44x11, mM KCl, 1.2 mM MgSO,, 10 mM HEPES, 10 mM glucose, 1.5 mM CaCl,, respectively. This percentage was calculated by considering and 0.2 mM BSA, (osmolality 320mOsmkg), pH adjusted to7.4, with 1 only the cells showing at leasta 2-fold increase of basal [Ca2+l, M Tris. The cells were incubated with fura-2 for 45 min at room temof the four perature t o avoid probe compartmentalization and then washed for 5 when exposed t o each single agonist. When each IP,-linked agonists was superfused in the absence of extracelmin before starting the experiment. Coverslips were mounted on a coverslip chamber (Medical System lular Ca2+,a rapid elevation of[Ca"], occurred (Fig. 2). The Co., Greenvale, N Y ) for fluorescence measurements.All measurements time interval required by the cells t o restore the basal [Ca"], were made at 26 "C. Cells were continuously superfused using a periafter a 90-s stimulation with each agonist (peak duration)was staltic pump (Gilson, France)(flow rate of 300 pl/min), and the perfu148 2 8, 63 f 3, 86 2 4, and 76 2 10 s for CCh, BK, ET-1, and sion medium was directly perfusedon the cells under observation by a ATP, respectively. microtube that was positioned with a macromanipulator (Narishige, Another feature which emerged from the experimentshown Japan). Theremoval of experimental solutionsfrom the coverslip chamber (300-pl volume) wasachieved using a microaspirator (Medical Sys- in Fig. 2 was the difference in the pattern of Ca2+ responseto tem Co., Greenvale, N Y ) connected to a vacuum pump and the entire CCh and t o the other threeagonists when the cation was presvolume of the chamber turned over in less than 50s. A two-way valve ent in the extracellularspace. In fact, muscarinic receptor ac(Thomson, Springfield,VA) controlled the flow from a separate injection tivation was characterized by a sustained plateau phase of loop which wasusedtoperfuse cells withdifferentexperimental [Ca2+li,whereas thisresponse was absent duringET-1, BK, and solutions. Ca2+-free experiments were performed using a nominally Ca2+-free ATP exposure. However, these latter three agonists showed a standard buffer containing 0.2 mM EGTA. Before starting the experi- peak duration which was longer than thatobtained in absence ment, the cells were perfused with this buffer for 5 min in order t o of extracellular Ca2+ (Fig. 2). Furthermore, among the four completely remove Ca2+from the extracellular space. agonists studied, CCh was the only one which displayed alower The cytoplasmic fluorescence appeared uniform throughout the cells. peak in the absence of extracellular Ca2+.When LAN-1 cells Fura-2 fluorescence was imaged with an inverted Nikon Diaphot microscope using a Nikon 40d1.3 NA Fluor DL objective lens. The cells were subjected t o an homologous restimulation withET-1, BK, were illuminated witha 100-watt Xenon lamp (Osram, Germany) with and ATP, after a washing period of 90 s, the second exposure quartz collector lenses. Ashutter anda filter wheel containing the two was unable to reproduce a [Ca2+li elevation, regardlessof the different interference filters (340 and 380 nm) were controlled by a presence or the absence of extracellular Ca'' (Fig. 2). The only computer. Emitted light was passed through a400-nm dichroic mirror, exception was representedby CCh which, only in thepresence filtered a t 510 nm, and collected by a CCD camera connected with a of extracellular Ca2+, was able t o reinduce a second peak of light intensifier (Applied-Imaging Ltd., Dukesway Gateshead, United Kingdom). Images of as many as 30 cellslfield were digitized and aver- [Ca2+l,.The failure of ET-1, BK, and ATP to induce a second aged inan image-processor (Applied-Imaging Ltd.) connected t o a com- homologous elevation of [Ca2+],did not seem to be due to a puter equipped with Tardis software. Eachcell in the image was inde- complete depletion of the internal Ca2+pools. In fact, when the pendently analyzed for each time point in the captured sequence. All sesquiterpene lactone thapsigargin, an inhibitor of the ER individual cell [Ca2+],traces shown are representative responsesfor a Ca2+-ATPase(14), was added after ET-1 and BK in theabsence given field of cells, which were observed in at least three separate of extracellular Ca2+,it was still able to elicit a significant experiments. Intracellular calcium concentration was determined as previously [Ca2+],increase (Fig. 3). By contrast, when thapsigargin was superfused after CCh, only a negligible release from intraceldescribed (10). Measurement of PPt nrnouer-Measurement of PP, turnover was lular Ca2+stores wasdetected. The same resultsobtained with performed as described (13) with some modifications. In brief, LAN-1 thapsigargin were observed with theCa2+-ionophoreionomycin cells were ailowed to prelabel for 2 days in Ham's F-10 medium, 10% (Fig. 3). fetal calf serum containing 3 pCi/ml [3Hlmyo-inositol (370-740 GBs/ Time Course of ZP3Production upon M , l M , and BK Receptor mmol, 10-20 Wmmol). In preliminary experiments, it was determined Stimulation-IP, levels were determined in LAN-1 cells after that a 36-h labeling period was sufficient for the lipids to attain an 100 PM CCh and 1 PM BK, which represents the group of the isotopic equilibrium of labeling. Cells were washed with a standard buffer containing 10mM myo-inositol and then detached using a standagonists that did not show the homologous restimulation, at
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ATP [pMI FIG.1. Concentration-dependent effects of ET-1, BK, ATP, and CCh on [Ca2+l,in fura-2-loadedL A N - 1 cells, in the absence of extracellular Ca2+.Data are the mean of three to five separate determinations. Vertical burs represent S.E. Lanthanum Superfusion during the First Agonist Exposure different time intervals. CCh and BK induced an early peak phase of different magnitude which occurred at about 10 s. At Potentiated Intracellular Ca2+Release Evoked by the Second IP, levels in BK-treatedcells fell Agonist Exposure-Fig. 8 shows that when the trivalentcation the endof the peak phase, the to a steady state slightly higher than the base line, whereas lanthanum (La3+), a potent inhibitor of the plasmamembrane upon CCh treatment the IP, values reached a sustained pla- Ca'' pump (15, 161, was superfused to LAN-1 cells before (5 min) and during BK exposure (90 s), the following addition of teau (Fig. 4). Effect of Sequential Superfusion, in Different Order, of ET-1, 100 n~ ET-1, after a 5-min washing period in theabsence of the BK, and ATP on [Ca"J in LAN-1Cells-After a first stimula- lanthanide, evoked a second [Ca"], peak which was of the same tion with ET-1, BK, and ATP, LAN-1 cells were sequentially magnitude of that elicited by BK (397 2 22 nM versus 349 2 27 exposed to the other two agonists, and the second and third n~ respectively, n = 8). By contrast, when La3+ was added to agonist, regardless of the orderof superfusion, were still able to LAN-1 cells after the refilling process was finished and the elicit an increase of [Ca"],, although progressively reduced in [Ca"], had returned to the base line, the peak elicited by the magnitude (Fig. 5). second agonist ET-1 was lower than that evoked byBK, as Whatever the sequence of the three agonists tested, the already observed in Fig. 4B in the absence of inhibition of mean percentage reduction of the second peak, when compared plasma membrane CaZ+ pump(391 2 26 nM versus 181 ? 18 nM, with the first one, was 42.5% (218 2 13 nM versus 379 2 10 nM; respectively, n = 9). n = 60) and that of the third compared with the second was Different Time of Cytoplasmic Ca" Removal upon CCh and 54.6% (99 6 nM uersus 218 2 13 nM; n = 60) (Fig. 5). BK Exposure-The main mechanisms involved in thedecline of CCh Completely Precluded Any Further Intracellular Ca2+ the [Ca2+lifrom a n elevated level to thebase lineare theplasma Mobilization by the Other Three Agonists-The [Ca"], elevation membrane Ca'' pump, the Na2+/Ca2+ exchanger,and the ER induced in LAN-1 cells by the muscarinic agonist CCh, as first Ca'+-ATPase (5, 17, 18). When all the efflux mechanisms are stimulus, completely precluded the Ca'+ releasing effect of working, the time required to bringdown the [Ca2+lifrom the ET-1, BK, and ATP (Fig. 6). This confirmed that after the stimu-arbitrary level of 200 nM to the baseline was twice longer for lation of muscarinic receptors the internal Ca2+ stores were CCh (105 2 5 s ) than that for BK (42 2 2 s). When the ER completely depleted, as already observed in the experiments Ca2+-ATPasewas blocked by thapsigargin and the efflux pathshown in Fig. 3. ways working were the plasma membrane Ca2+ pumpand the Comparison of Simultaneous Versus Single Superfusion with Na+-Ca2+ exchanger, no significant difference between CCh and ET1 and BK-In order to verify whether the reduced Ca'' BK was observed (150 2 10 versus 176 2 17 s). response elicited by the second agonist was due to a partial Effect of Different Time of Receptor Occupancy by CCh and releasing action of the first agonist on the Ca2+ stores, BK and BK on Intracellular Ca2+Refilling-& shown in Figs. 6 and 2, ET-1 were added simultaneously to the superfusion medium. a single pulse of CCh for 90 s was able to inhibit any further The two peptides evoked an elevation of [Ca'+], which was com- release of internal Ca2+by the other three agonists or by the pletely comparable to thatelicited by the singly superfused BK homologous restimulation. However, when the time of CCh and ET-1 (Fig. 7). exposure wasprogressively reduced from 90 to 60,30, and15 s,
Stimulation of Different IP,-linked
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a recovery ofCa'' response to the following stimulation with BK was observed (Fig. 9). Particularly, the magnitude of the [Ca"], increase due to the second stimulation was inversely related to the timeof CCh exposure. In addition, the duration of the [Ca2+l,peak induced by CCh was directly related t o the time exposure to themuscarinic agonist(Table I). Interestingly, when BK was perfused for only 15 s, the magnitude of [Ca2+], increase and the durationof the peak were equivalent to that obtained after 90 s of exposure (Table I and Figs. 5B and 9). Furthermore, the entityof Ca2+ refillingafter 15 s of BK receptor occupancy was similar t o that observed after 90 s since the following ET-1 superfusion, in a Ca'+-free medium, elicited a [Ca"], increase which was of the same magnitude either after 15 or 90 s of BK receptor occupancy.
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FIG.2. Effect of homologous re-exposure with BK, ET-1, ATP, and CCh on [Ca"],. The chosen concentrations for each of the four agonists were the lowest which elicited the maximal Ca2' response, in the absence ( l e f t )or presence (right) of extracellular Ca". Duces are from an individual cell in a field of cells which was stimulated twice consecutively for 90 s and are representative of twotofour other experiments.
Receptors DISCUSSION
The resultsof the present study demonstrated thatLAN-1 in cells the reloading of intracellular Ca'' stores which follows the activation of IPS-linked receptors can be different if the transductional pathway was activated by ET-1, BK, and ATP or by CCh. Furthermore, the CaZ+ responseelicited by the stimulation of plasma membrane receptors for ET-1, BK, and ATP displayed a rapid inactivation either in the presence or in the absence of extracellular Ca'' since the homologous restimulation did not occur. This phenomenon was not observed upon CCh stimulation of M1/MS receptors, since in the presence of extracellular Ca'' a second and equivalent[Ca"], increase was obtained after homologous restimulation (Fig. 2). Onthe other hand, since the lack of homologous restimulation with BK, ET-1, and ATP was observed also in the presence of extracellular Ca", the lack of the response can be attributed to an impairment of the receptor-effector system rather than to a complete depletion of intracellular Ca2+pools. In fact, thapsigargin and ionomycin were able to induce a significant Ca2+ release into thecytoplasm when addedafter ET-1, BK, and ATP (Fig. 3). It is interesting to underline that the Ca2+ response induced by the three agonists which did not show the homologous restimulation wascharacterized, in thepresence of extracellular Ca", by a rapid peak followed bya slow return t o basal Ca'' levels, without anevident and sustained plateau phase. In addition, the magnitude of the peak phase induced by these three agonists was not reduced by the removal of extracellular Ca". On the contrary, M,/M, receptor stimulation produced a sustained plateau phase lasting until receptor the wasoccupied by the agonist and showed a peak phase which was significantly reduced by extracellular Ca2+removal (Fig. 2). These results indicated that, when muscarinic receptorsare activated in LAN-1 cells, there is an earlyopening of plasma membrane Ca'' "refilling" channels (19) that remain open as long as the receptor is occupied. On the contrary, the occupation of the receptors for BK, ET-1, and ATP did not appear to induce a rapid andprolonged opening of these plasma membrane channels, the opening of which seems to be regulated by the Ca2+ content of the intracellular pools (19-21). Therefore, from the data obtained upon BK, ET-1, and ATP exposure, it can be hypothesized that LAN-1 cells need to refill the intracellular Ca" stores to a lesser extent thanwhen exposed to the muscarinic agonist. This hypothesis is supported by the experiments inwhich, after theexposure to BK, ET-1,and ATP, in the absence of extracellular Ca'+, LAN-1 cells still contained a sig[Ca'+]i nM
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FIG.6.Effect on [Ca*'l,by ET-1 (100MI), BK (1 J~M), and ATP (50
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H FIG.5. Effect on [Caa+Ii by sequential superfusion, in different 1 min orders, of ET-1 (100 m),BK (1 p ~ ) and , ATP (50 p"). Each agonist FIG.7. Comparison ofthe [Ca*+], increase obtainedby ET-1 (100 was superfused for 90 s. Duces are from an individual cell in a field of MI) and BK (1 p ~ alone ) and when perfused together for 90 s. cells and are representative of two other experiments. Duces are from an individual cell in a field of cells and are represent-
nificant amount of Ca2+ in the intracellular stores, whereas after CCh stimulation Ca2+pools seemed t o be almost totally depleted (Fig. 3). This evidence is also confirmed by the fact that BK, ET-1, and ATP, perfused in sequence, were able to induce a repeated release of Ca2+(Fig. 5). This could be explained by different mechanisms: 1)the first agonist partially depleted the internal Ca2+ stores and following the agonist exposure progressively mobilized the remaining amountof Ca2+; 2 ) the first agonist induced a complete depletion of intracellular Ca2+pools, and the release observed after the other agonists was due toa refilling of the Ca2+stores through a recycling of the Ca2+ released by the previous agonist. The first hypothesis seems unlikely for when BK and ET-1 were perfused together there was a lack of potentiation of Ca2+response (Fig. 7). The second hypothesis seemsto bethe most likely one.In fact, when the plasma membraneCa2+efflux pathways wereblocked with lanthanum, added during the exposure of L A N - 1 cells to the first agonist, the second heterologous stimulation, evoked a [Ca2+l,increase which was comparable to thatproduced by the first agonist, instead of eliciting a reduced Ca2+ response(Fig. 8). Theseresultsdemonstrated that LAN-1 cells upon BK, ET-1, and ATP receptor stimulation are able to recycle Ca2+ from the cytoplasm to the internal stores and that progresthe sive reduction of the Ca2+ response following heterologous stimulation was due to the loss of Ca2+through the plasma membrane efflux pathways. The refilling of the internal stores occurring after BK, ET-1, and ATP receptor activation was not
ative of three other experiments.
observed with M,/M, receptor stimulation. In fact, after CCh supefision a complete inhibition of the Ca2+ refilling occurred, as shownby the absenceof any [Ca2+liincrease upon stimulation with the other three agonists (Fig. 6) or upon muscarinic homologous restimulation (Fig. 2). The idea thatMJM, receptor activation impairs Ca2+ refillingwas further supported by the fact that either thapsigargin or ionomycin were unable to release a significant amount of Ca2+from intracellular stores after CCh exposure (Fig. 3). Another evidence in favor of the idea thatM,/M, receptor stimulation blocks the refilling of Ca2+ into the internal storesderives from the experiments inwhich the ER Ca2+-ATPasewas blocked by thapsigargin and theonly [Ca2+l,lowering mechanisms were the plasma membrane CaZ+ pump and the Na+-Ca2+ exchanger. In fact, in this condition the difference in the time of Ca2+ removal between CCh and BK which was observed when both [Ca2+l,lowering mechanisms were operating, disappeared. This suggests that CCh impairs the Ca2+ refilling process.In addition, the inhibitoryeffect exerted by MIDI, receptor stimulation on theCa2+ refilling mechanisms seems to be strictly dependent on the time of receptor occupancy. In fact, the progressive reduction in the time of M,/M, receptor occupancy from 90 s (the time required to inhibit therefilling) to 15 s, produced a progressive increase of [Ca2+l,elicited by the following addition of BK (Fig. 9). Interestingly, when M,/M, receptors wereexposed to the agonist for
Stimulation of Different IP,-linked Receptors
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FIG.8. Effect of different timesof lanthanum superfusion on the [Caa+Ii increase evoked by the second agonist exposure.BK (1p ~ and ) ET-1 (100 nM) were superfused for 90 s, and lanthanum (2 mM) was superfused for 5 min before and during BK exposure (A) or 5 min before and duringET-1 exposure (B). Duces arefrom a n individual cell in a field of cells and are representative of two other experiments.
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the shortest time (15 s) the [Ca2+],increase evoked by the following BK exposure was of the same extent as thatobserved when the first agonist was different from CCh (205 2 19 nM uersus 218 2 13 nM, respectively; seealso Table I) and the second agonist was ET-1, BK, or ATP. A similar observation on the effect of short periods of muscarinic receptor occupancy on the following homologous stimulation was already made by 400 Negulescu and Machen (22). In addition, the peak duration induced by 15 and 30 s of CCh exposure was similar to that obtained with 90-s exposure t o ET-1, BK, and ATP (Table I). Since the 15-s muscarinicreceptor occupancy produced the same effect on Ca2+refilling observed when BK, ET-1, and ATP BK ET-1 occupied their receptors for 90 s, it canbe hypothesized that the 1 receptors for these three agonists maintain the transductional CaZ+ free mechanisms in an activated state for a time shorter than the I min time of receptor occupancy. This hypothesis was supported by FIG.9. Effect of the duration of the exposure to the first agothe resultsof the experiment in which BK, when superfusedfor nist onthe [Caa+Ii increase inducedby the secondagonist. CCh, 15 s, elicited the same [Ca2+],increase, peak duration, and BK, and ET-1 were perfused at the concentration of 100 VM, 1p f , and intracellular Ca2+ refilling as observed after a 90-s exposure 100 nM, respectively. CCh exposure lasted 15 (A), 30 (B), and 60 ( C ! s, (Fig. 9). The different effect on the refilling process exerted by while the exposure to BK as firstagonist lasted for 15 s (D). CCh action washing CCh and BK, ET-1, and ATP when superfused for 90 s may was rapidly interrupted by the addition of 4 p~ atropine in the depend on the different level and timecourse of IP, production buffer. BK and ET-1 when used as second agonist were perfused for 90 evoked by these IP,-linked receptors. In fact, the timecourse of s. Duces are from an individual cell in a field of cells and are representative of two other experiments. CCh-induced IP, formation was characterized by an early peak (10 s ) followed by a sustained and progressively increasing TABLE I plateau phase. By contrast, BK, after the early peak phase, produced a much lower plateau phase. The sustained and el[Ca''] peak induced Condition Duration of peak by the'second agonist evated level of IP, elicited by CCh may leave the ER IP,-sensitive CaZ+ channel in an prolonged open state allowing a conS tinuous and significant Ca2+leakage from the stores. In this CCh 100 PM 90 s 148 -c 8 CCh 100 PM 60 s 123 -c 6 way the intracellularreloading of Ca2+stores exertedby the ER 102 f 2 CCh 100 PM 30 s 86 -c 4 185 f 22 Ca2+-ATPasevanished (23, 24). By contrast, since after BK CCh 100 pf 15 s 49 205 f2 -c 19 ER stimulation the IP, levels returned to basal values, the IP,-sensitive Ca2+ channels close, and the intracellular stores BKlW90s 218 63 -c 3 -c 13 may reload Ca". Therefore, the results of the present study BK 1 PM 15 s 58 f 2 210 -c 27 seem to suggest that the difference in theIP, levels before and 76 f 10 ATP 50 p~ 90 s after 60 s of receptor stimulation iscritical for the occurrence of ET-1 100 nM 90 s 86 -c 4 the refilling process. This hypothesis seems tobe supported by the finding shown in Fig. 9 in which CCh did not inhibit the A possible explanation for the fact that the transductional refilling process if removed from the MJM, receptors before 60 s. On the contrary, when CCh was removed after 60 s from mechanisms of BK, ET-1, and ATP receptors inactivate even if muscarinic receptors the intracellular Ca2+stores can not be the receptor is occupied could be the development of the phenomenon of receptordesensitization, which has previously refilled. In fact,even if IP, levels fall and the ERIP,-sensitive released into the cytoplasm is already ex- been described for these receptors (25-28) and that in this case channel closes, ea2+ membrane plasma truded by the efflux pathways. amears to be raDid and comdete. By contrast, M,/M, receptor
IDI
-
1.
I
.
Stimulation of IP,-linked Receptors Different
18027
7. Chau, L. Y., Lin, T. A,, Chang, W. T., Chen, C. H., Shue, M. J., Hsu, Y. S., Hu, occupation results only in a partial desensitization, as shown C. Y., Tsai, W. H., and Sun, G. Y. (1993)J. Neurochem. 60, 454460 by the fact that IP, levels remained higher than after the BK 8. Ito, S., Mochizuki-Oda, N., Hori, K., Ozaki,K., Miyakawa, A,, and Negishi, M. receptor stimulation (Fig. 4). A mechanismthat canaccount for (1991)J. Neurochem. 56, 531-540 9. Yorek, M. A,, Davidson, E. P., Dunlap, J. A,, and Stefani,M. R. (1993)Biochim. the different pattern of desensitization observed with BK, ET-1, Biophys. Acta 1177, 215-220 ATP, and CCh could be the phosphorylation process of the re- 10. Fatatis, A,, Bassi, A,, Monsurrb, M. R., Sorrentino, G., Mita, G. D., Di Renzo, G. F., and Annunziato, L. (1992)J . Neurochem. 69, 1-9 ceptor. In fact, it has already been demonstrated thatphospholipase C/IP,-linked receptors undergo a phosphorylation step at 11. Fatatis, A,, Bassi, A,, Mita, G. D., Di Renzo, G. F., and Annunziato, L. (1992) Funct. Neurol. 7, 239-242 the level of the third intracellular loop, which is the primary 12. Fatatis, A,, Bassi, A,, Di Renzo, G. F., and Annunziato,L. (1992)22ndAnnual Meeting of the Society for Neuroscience, Oct. 25-30, 1992, Anheim, CA site of the coupling to G-proteins (29). Generally, the phospho(Abstr. 260.3) rylated form is believed to represent the desensitized state of 13. Berridge, M. J.,Dawson, R. M. C., Downe, C. P., Heslop, J.P., and Imine, R. F. the receptor (30).Therefore, a different degreeof phosphoryla(1983)Biochem J. 212,473482 tion of M1/M3 and BK, ET-1, and ATP receptors may be respon- 14. Thastrup, O., Cullen, P. J., Drobak, B. IC, Hanley, M. K., and Dawson, A. l? (1990)Proc. Natl. Acad. Sci. U. S. A. 87, 2466-2470 sible for the difference in the receptor functioning. 15. Penniston, J. T. (1983) in Calciumand Cell Function (Wai, Y. C., ed) pp. In conclusion, the receptors that displayed a rapid and com99-149, Academic Press, New York plete desensitization have a more marked fall of IP, levels, and 16. Kwan, C. Y., Takemura, H., Obie, J.F., Thastrup, O., and Putney, J. W. (1990) Am. J. Physiol. C1006-C1015 the refilling process can occur. In this way the heterologous 17. Reeves J. P. (1990) in Intracellular Calcium Regulation (Bronner, F., ed.) pp. restimulation of Ca2+mobilization can occur. On the contrary, 305-347, Wiley-Liss, New York 18. Carafoli, E. (1992) J. Biol. C k m . 267, 2115-2118 the M1/M3 receptors that showed only a partial desensitization 19. Putney, J. W. (1986) Cell Calcium 7, 1-12 have a less marked fall of IP, levels, of which the sustained and 20. Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W. J. (1989)J. Biol. prolonged elevation is responsible for the inhibition of Ca2+ Chem. 264, 12266-12271 21. Putney, J. W. J. (1990) Cell Calcium 11, 611-624 refilling and for the lack of heterologous restimulation.
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