Muscarinic Receptor Activation Down-regulates the Type I Inositol 1,4,5-Trisphosphate Receptor by Accelerating Its Degradation*

Stimulation of SH-SY5Y human neuroblastoma cells with carbachol, a muscarinic agonist, down-regulates the type I inositol 1,4,5-trisphosphate (InsP,) receptor by > 90% with maximal and half-maximal effects after "6 h and "1 h, respectively. Examination of the mechanistic basis of this down-regulation revealed that carbachol increased the rate of type I InsP, receptor degradation (radiolabeled immunoprecipitable receptor was lost from cells with half-times of > 8 h and -1 h in the absence and presence of carbachol, respectively) and that the concentration of type I InsP, receptor mRNA, despite a transient decrease after 3 h, did not correlate with levels of the receptor. Only those muscarinic receptor subtypes coupled to stimulation of phosphoinositide hydrolysis were capable of causing type I InsPg receptor down-regulation. Ca2+ mobilization was pivotal to the mechanism of receptor down-regulation, since perturbation of Ca2+ ho- meostasis with either EGTA or thapsigargin blocked the ability of

lecular mass = 313 kDa) with putative membrane-spanning regions and a binding site for InsP3 close to the amino terminus ( 2 4 ) . Tetrameric complexes of this protein form channels that conduct Ca2+ in an InsP3-sensitive manner ( 5 , 6). Subsequent genetic analysis has led to the identification of slightly smaller variants of the type I InsP3 receptor, generated by alternative splicing (71, and the cloning and sequencing of types I1 and I11 InsP3 receptor genes (8,9) which encode proteins with > 60% homology to the type I receptor.
Analysis of the functional properties of the channels formed by complexed InsP3 receptors has revealed a number of acute regulatory features. In particular, the gating of Ca2+ by InsP3 is modulated by both intraluminal (10) and cytosolic (11) Ca2+, a n d an individual channel can exist in different rapidly interconverting conductance states (12). InsP3 receptors are also modulated over longer time scales. In SH-SY5Y human neuroblastoma cells, muscarinic receptor activation and consequent stimulation of PIPz hydrolysis (13, 14) cause type I InsP3 receptor down-regulation (14, E), whereas in human promyelocytic leukemic HL-60 cells, retinoic acid up-regulates the receptor (16). Both of these regulatory effects have functional correlates, since the Ca2+ mobilizing activity of InsP3 is suppressed in muscarinic agonist-pretreated SH-SY5Y cells (14) and is enhanced in retinoic acid-pretreated HL-60 cells (16).
We now describe analyses of the possible mechanisms by which the type I InsP3 receptor is down-regulated in SH-SY5Y cells and the signals that initiate this mechanism. We have also sought to determine whether other phosphoinositidase C-linked receptors are capable of down-regulating the type I InsP3 receptor and in which cell types this process can occur.

EXPERIMENTAL PROCEDURES
Cell Culture and Pretreatment-Cell monolayers were cultured routinely in 175-cm2 flasks; SH-SYBY cells in minimal essential medium with Earle's salts supplemented with 10% newborn calf serum and antibiotics (131, A10 cells in the same medium but with 5% newborn and 5% fetal calf serum, Chinese hamster ovary (CHO) cells in minimal essential medium-cr with 10% newborn calf serum and antibiotics, GH, cells in Ham's F-10 with 10% fetal calf serum and antibiotics, and Swiss 3T3 and rat H9c2 cells in Hepes-buffered Dulbecco's minimal essential medium with 10% fetal calf serum and antibiotics. Rat cerebellar granule cells were prepared and cultured as described (17). Following addition of agonists or inhibitors to culture medium, cells were maintained at 37 "C for the time of pretreatment required. Cells were then removed from flasks in 155 nm NaCl, 10 m M Hepes, 0.7 m M EDTA, pH 7.4 (HBSE); SH-SYBY, CHO, GH,, and A10 cells became detached from flasks following gentle agitation, whereas Swiss 3T3, granule, and H9c2 cells were removed mechanically. All cell types were then pelleted by centrifugation at 500 x g for 2 min, except for granule cells which were centrifuged at 1,000 x g for 3 min.
Immunoblotting-The pellet from a flask of control or pretreated cells was resuspended in 10 ml of ice-cold 10 m M Tris, 1 m M EGTA, 0.1 m~ phenylmethylsulfonyl fluoride, 1 nm dithiothreitol, 10 PM leupeptin, 10 PM pepstatin, pH 7.4 (homogenization buffer), was disrupted (Ultra 7963 l)pe I InsP3 Receptor Down-regulation Turrax homogenizer, 1 x 18 s) and was centrifuged (500 x g for 10 min a t 4 "C). This centrifugation step was omitted during preparation of granule cells. The supernatant was then centrifuged (38,000 x g for 10 min at 4 "C), and the pellet obtained was resuspended in homogenization buffer (3-10 mg of proteidml), aliquoted, and frozen at -20 "C until required. Samples of these microsomal preparations and a rat cerebellum preparation (15) were then subjected to electrophoresis (18), transferred to nitrocellulose, and incubated with monoclonal antibody 4 C l l (at a dilution of -1:8 of crude culture medium) or 18A10 (at a dilution of -1:5,000 of purified antibody) or polyclonal antibody CT1 (at a dilution of 1:1,000 of crude serum). The nitrocellulose was then incubated with peroxidase-conjugated goat anti-rat or rabbit antibodies (at a dilution of -1:500), ECL detection reagents (Amersham Corp.), and x-ray film. Immunoreactivity was quantified with a GS-670 densitometer (Bio-Rad).
Antibodies 4Cll and 18A10 were raised against the mouse type I InsPs receptor as described (2) and have epitopes between amino acids 679 and 727, and 2736 and 2747, respectively (2, 19). Antibody 4 C l l recognizes human type I and type 111 receptors, whereas 18A10 is type I-specific.2 Antibody CT1 was obtained by injecting a rabbit with a peptide corresponding to the carboxyl-terminal 19 amino acids of the rodent type I InsP, receptor (2,3). Antigen was prepared by coupling 3 mg of the peptide, which was synthesized with a n additional cysteine at its amino terminus, with 3 mg of maleimide-activated keyhole limpet hemocyanin (Pierce). Initially 200 pg of antigen in complete Freund's adjuvant was injected subcutaneously, followed 2 weeks later by subcutaneous injection of 200 pg in incomplete Freund's adjuvant. Boosts of 100 pg of antigen were then given intravenously a t 4-week intervals.
Recognition in immunoblots of a -275-kDa protein corresponding in size to the type I InsP3 receptor of rat cerebellum and SH-SY5Y cells, and the ability to immunoprecipitate a -275-kDa protein were optimal after the first boost. Preimmune serum at the same dilution did not react with any proteins. Immunoreactivity was blocked if 0.2 mg/ml of the peptide to which the antibody was raised was included in incubations, but not by peptides from the carboxyl termini of types I1 and I11 receptors. Interaction of 4C11, 18A10, and CT1 with the human receptor is to be expected, as the predicted amino acid sequence of the human type I InsP, receptor is very similar to that of the mouse.3 mRNAAnalysis-Three flasks of control or carbachol-pretreated SH-SY5Y cells were rinsed with and harvested in 25 ml of HBSE. Following centrifugation (500 x g for 2 min), RNA was extracted using acid guanidium thiocyanate-phenol-chloroform (20). mRNA was then isolated using oligo(dT)-coupled magnetic beads (Promega). For each treatment the quality of preparation was checked visually after electrophoresis of RNA and mRNA samples in ethidium bromide-containing agarose minigels. mRNA purity and concentration were then quantitated spectrophotometrically (18); A2,dAzso was always 2 2.0, and only minor differences in Azso of preparations from control and pretreated cells were detected. Samples of mRNA were then subjected to electrophoresis in formaldehyde, 1% agarose gels, blotted to nitrocellulose, and baked (18).
Prehybridization and hybridization in buffer containing 6 x SSPE, 2 x Denhardt's reagent, 0.1% sodium dodecyl sulfate, 100 pg/ml salmon sperm DNA, 50% formamide and the washing of membranes were performed as described (18). The probe used, a 1.2-kilobase fragment of human type I InsP3 receptor cDNA corresponding to residues 2993-4130 of the mouse sequence, was labeled with [ C X -~~P I~C T P using T7 DNA polymerase (Pharmacia LKB Biotechnology Inc.).
Immunoprecipitation of P5SIMethionine-labeled Receptor-Pilot experiments in which the immunoprecipitable type I InsP, receptor of SH-SY5Y cells was labeled with [35Slmethionine either in methioninefree medium without serum or normal SH-SY5Y cell culture medium yielded similar data.
For analysis of the rate of receptor degradation, subconfluent cells in 80-cm2 flasks were labeled in 12 ml of normal culture medium with 60 pCi of [36Slmethionine for 40 h. Monolayers were then washed with 15 ml of chase medium (normal culture medium supplemented with 5 nm methionine and 20 m Hepes, pH 7.5) and finally incubated with 10 ml of chase medium with or without carbachol. After 2-8 h the chase medium was removed, and the cells were harvested in 10 ml of HBSE and centrifuged at 500 x g for 2 min.
Cell suspensions were used when the effects of EGTA and thapsigargin on receptor degradation were examined. In these experiments subconfluent cells i n 175-cm2 flasks were labeled i n 20 ml of normal culture medium with 125 pCi of [35S]methionine for 40 h, monolayers were washed with 10 ml of chase medium, harvested in 10 ml of HBSE, and T. Furuichi and K. Mikoshiba, unpublished data. T. Furuichi and K. Mikoshiba, submitted for publication. centrifuged at 500 x g for 2 min. The pellet was then washed once in chase medium and finally resuspended in 2 ml of chase mediudflask of cells. After division into 1-ml aliquots, the cell suspensions were incubated for 6 h with stimuli or inhibitors at 37 "C with occasional shaking and finally were centrifuged (500 x g for 1 min).
Cell pellets from both monolayer and suspension experiments were then resuspended in 0.9 ml of ice-cold lysis buffer (50 nm Tris, 150 nm NaCl, 1% Triton X-100, 1 m~ EDTA, 2 pg/ml aprotinin, 10 p~ pepstatin and leupeptin, 0.2 m phenylmethylsulfonyl fluoride, pH 8.0) and transferred to 1.5-ml microcentrifuge tubes. After incubation on ice for 30 min, lysates were centrifuged (13,000 x g for 8 min a t 4 "C), and the supernatants were transferred to tubes containing 100 pl of 25% nonfat dried milk, 0.25% Tween 20, and 12 pl of crude CT1 antiserum. After 40 min on ice, 300 pl of protein A-Sepharose sluny (Pharmacia) was added, and after a further 30 min a t 4 "C, immune complexes were purified by washing the Sepharose beads five times with 1 ml of lysis buffer. After the addition of 30 pl of 2 x gel loading buffer (18) and 5 pl of 8 M urea, bound proteins were eluted by heating the beads for 3 min a t 100 "C. Proteins were then subjected to electrophoresis, and gels were fixed with 10% acetic acid, 50% methanol, impregnated with Amplify (Amersham), dried, and autoradiographed. Phosphorylation-Cells from 175-cmZ flasks were harvested in HBSE, centrifuged (500 x g for 2 rnin), washed twice i n phosphate-free Krebs-Hepes buffer (118 nm NaCl, 4.7 m~ KCl, 1.2 nm MgSO,, 1.3 nm CaC12, 10 nm glucose, 25 m~ NaHCO,, 10 m~ Hepes, pH 7.4), and finally resuspended in the same buffer (2 muflask of cells) containing 0.2 mCi/ml 32Pi. Cells were then aliquoted into 1-ml portions and incubated for 1 h at 37 "C. After the addition of stimuli and further incubation at 37 "C, cells were centrifuged (500 x g for 1 min) and resuspended in 0.9 ml of ice-cold lysis buffer supplemented with the phosphatase inhibitor p-nitrophenyl phosphate (7.6 nm). The type I InsP, receptor was then isolated as for [35Slmethionine-labeled cells, except that 18A10 (10 pl of crude culture medium) was the immunoprecipitating antibody.
Measurement of ZnsP3 M a s s X e l l s were subcultured into multiwell dishes (1.8 cm2/well) and after 24 h were incubated with agonists or inhibitors for various times in 1 ml of normal culture medium. Incubations were terminated by removal of media and the addition of 150 pl of 0.5 M trichloroaceteic acid and 40 pl of 10 m~ EDTA. Cell extracts were then transferred to 1.5-ml microcentrifuge tubes, 200 pl of 1:l freod octylamine was added, and following centrifugation (13,000 x g for 5 min), samples were assayed for InsP, mass as described (21).

Detection of !l'ype Z Imp3 Receptor Down-regulation-
Antibody 4 C l l recognizes a protein in rat cerebellum preparations with an apparent molecular mass -of 275 kDa (Fig. 1, lane 1 ), which corresponds to the 313-kDa product of the rodent type I InsP3 receptor gene (2,3). This antibody also recognizes a protein with the same mobility in SH-SYSY cells which corresponds in size to the human type I InsPs recepto? (Fig. 1,   lane 2). Pretreatment of SH-SY5Y cells in culture with carbachol for 6 h reduced immunoreactivity at -275 kDa by 92% (Fig. 1, lane 3 ) . This finding is in close agreement with our previous study on SH-SY5Y cells using antibody 18A10 (15), in which carbachol induced an -90% fall in type I InsP3 receptor immunoreactivity by 6 h (half-maximal effect at -1 h) which was maintained for up to 24 h. As 4 C l l and 18A10 recognize amino-terminal and carboxyl-terminal regions of the type I InsP, receptor, respectively, these data show that the cellular concentration of the entire type I InsP3 receptor polypeptide is reduced. Analysis of o p e I Imp3 Receptor mRNA Concentration-The effects of muscarinic stimulation on type I InsP3 receptor mRNA concentration were examined. Fig. 2A shows that a probe derived from human type I InsP3 receptor cDNA hybridized with a -10-kilobase mRNA species from control cells (lane 1 ) and that the concentration of this species was the same after treatment with carbachol for 20 h (lane 2). The concentration of this species, which is the appropriate size for the precursor of InsP3 receptor polypeptides, was, however, reduced by shorter incubations with carbachol, decreasing sharply after 1 h to a nadir of 38 2 15% of control levels a t 3 h (Fig. 2B 1. Despite this decrease, these data reveal no correlation between mRNA levels and the kinetics of changes in type I InsP3 receptor concentration (15).
Analysis of Receptor Degradation-We next examined the possibility that degradation of the type I InsP3 receptor was accelerated by carbachol. SH-SY5Y cells were labeled for 40 h with [35S]methionine and the rate at which radioactivity was lost from immunoprecipitable type I InsP3 receptor during subsequent incubations in medium containing 5 m M nonradioactive methionine was examined. Fig. 3A shows that the halftime of loss of radioactivity (tin) in unstimulated cells was > 8 h, whereas in the presence of carbachol, tIl2 was < 2 h. These data indicate that an increase in the rate of type I InsP3 receptor degradation accounts for its down-regulation. This effect was specific, as carbachol did not alter the overall 35S content of the array of proteins detected after electrophoresis of crude cell lysates4 Effects of Perturbation of Ca2+ Homeostasis on Receptor Down-regulation and Degradation-As Ca2+ mobilization is pivotal to the intracellular signaling initiated by phosphoinositidase C-linked muscarinic receptors (23, 24) the effects of perturbing Ca2+ homeostasis were examined. We have shown previously that reducing extracellular Ca2+ concentration to -200 m with EGTA blocks the ability of carbachol to cause type I InsP3 receptor down-regulation (15). Fig. 3B shows that this results from a reversal of the ability of carbachol to accelerate receptor degradation; carbachol accelerates degradation in cells suspended in normal medium (lanes 1 and 2) but not in cells incubated with EGTA (lanes 3 and 4 ).
The effects of thapsigargin were also examined. By inhibiting endoplasmic reticulum ATPases (25), thapsigargin releases intracellular Ca2+ stores including those normally moblized by InsP3 (24-26) and in some cells, including SH-SY5Y cells,5 stimulates Ca2+ entry via "capacitative refilling" (1,261. These tion (24-26). Intriguingly, thapsigargin alone did not alter InsP3 receptor immunoreactivity, but blocked the ability of carbachol to cause down-regulation (Fig. 4). Again, this blockade results from the fact that in the presence of thapsigargin, carbachol does not accelerate degradation of the type I InsPB receptor (Fig. 3B, lanes 5 and 6). Despite its effects on Ca2+ mobilization, thapsigargin does not on its own stimulate InsP3 formation (Table I) or alter the ability of carbachol to activate PIP2 hydrolysis persistently; the intracellular InsP3 concentration was maintained at an elevated level 12 h after initiation of muscarinic stimulation in the absence or presence of thapsigargin (Table I). Together, these data suggest that for carbachol to accelerate InsP3 receptor degradation, functional InsP3-sensitive Ca2+ stores must be present and that discharge of these stores via routes other than that involving the InsP3 receptor is not sufficient to accelerate degradation.
As the Ca2+-dependent neutral protease calpain has been shown recently to cleave the cerebellar InsP3 receptor (27) we examined the effect of the membrane-permeable calpain inhibitor EST (28) on carbachol-induced down-regulation. At 50 pg/ml this inhibitor did not affect the ability of carbachol to reduce type I InsP3 receptor imm~noreactivity.~ At this concentration, EST is capable of inhibiting calpain activity in Ca2+ ionophore-stimulated intact platelets totally (28). Qpe Z InsP, Receptor Phosphorylation-As the type I InsP3 receptor has been shown to be a substrate for several second messenger-dependent protein kinases in cell-free systems (291, we examined whether phosphorylation might be involved in initiating its degradation. Fig. 5 shows that in intact SH-SY5Y cells the type I InsP3 receptor is a phosphoprotein (lane 11, but that carbachol does not enhance phosphorylation (lanes 2 and 3), either at times corresponding to the initial rapid burst of PIPz hydrolysis and Ca2+ mobilization or during the subsequent lower but sustained phase of the response (13,241. Phosphorylation was not activated by forskolin (lane 4 ) which, in these cells, produces a large increase in intracellular CAMP concentration (30). the type I InsP, receptor was immunoprecipitated using antibody CT1 and isolated in 6% polyacrylamide gels. The position of the type I InsP, receptor is indicated by arrows. Autoradiographs shown are representative experiments, and data shown graphically are the means of values from two independent experiments. Panel A, labeled cells were incubated with chase medium in the absence (-) or presence (+) of 1 mM carbachol for 0 h (lanes 1 and 2 ) , 2 h (lanes 3 and 4 ) , 4h (lanes 5 and 6), 6 h (lanes 7 and 8), or 8 h (lanes 9 and 10). The graph depicts the loss of radioactivity from the type I InsP, receptor expressed as a percentage of control values (lanes 1 and 2). Panel B , labeled cells were incubated for 6 h in suspension with chase medium in the absence (-) or presence (+) of 1 m~ carbachol with vehicle (lanes 1 and 2), 3.5 m~ EGTA (lanes 3 and 4 ) , or 2 p~ thapsigargin (lanes 5 and 6). Thegraph depicts the loss of radioactivity from the type I InsP, receptor expressed a s a percentage of control values (lane 1 ). 1 2 3 4 -+ -+ Fig. 1 except that antibody 18A10 was used. Cells were incubated for 18 h with either vehicle (0.1% dimethyl sulfoxide, lanes 1 and 2 ) or 2 p~ thapsigargin (lanes 3 and 4 ) in the absence (-) or presence (+) of 1 mM carbachol. The position of the type I InsP, receptor is indicated by the arrow.

Receptor Down-regulation in Other Cell
Types-To characterize further type I InsP3 receptor down-regulation, the effects of a range of agonists a t phosphoinositidase C-linked receptors were examined in a variety of cell types. In SH-SYSY cells, bradykinin as well as muscarinic receptors are coupled to phosphoinositidase C activation, although bradykinin produces a much smaller and more transient response than carbachol: and af'ter incubation with 20 VM bradykinin for 12 h, the intracellular InsP3 concentration was no different from control valu e~.~ Fig. 6A (lanes 2-6) shows that although carbachol caused down-regulation with a half-maximal effect a t -0.1 p~, brady-G . B. Willars and S. R. Nahorski, manuscript in preparation.

TABLE I Effects of thapsigargin on carbachol-induced increases
in InsP,? concentration SH-SY5Y cells in multiwell dishes were incubated in normal culture medium in the absence or presence of 1 m~ carbachol with either vehicle (0.1% dimethyl sulfoxide) or 2 p~ thapsigargin. After 12 h a t 37 "C InsP3 mass was assessed. Data shown are mean t SE of sextuplicate incubations. kinin was ineffective (lane 7). Similar effects of carbachol were seen in rat cerebellar granule cells (Fig. 6B 1, in which carbachol also persistently enhances InsPs formation (17). As SH-SYSY and granule cells express multiple muscarinic receptors subtypes (17,31) we sought to determine which ones initiate down-regulation. Of the five subtypes known, m l , m3, and m5 are coupled preferentially to activation of phosphoinositidase C , and m2 and m4 to inhibition of adenylate cyclase and activation of K+ channels (23). Thus, we examined the effects of carbachol in CHO cells transfected with human m l , m2, and m3 receptor cDNA (23). In these cells, phosphoinositi-  lanes 1 and 2 ) , CHO-m2 cells (lanes 3 and 4 ) and CHO-m3 cells (lanes 5 and 6 ) were incubated for 18 h with either vehicle (lanes 1,3, and 5 ) or 1 mM carbachol (lanes   2, 4, and 6). Panel D, GH3  dase C-linked receptor activation elevates InsP3 concentration for hour-long periods (32), whereas m2 receptors do not stimulate InsP3 f~r m a t i o n .~ Fig. 6C shows that carbachol causes type I InsP3 receptor down-regulation in those cells expressing m l and m3 receptors, but not in those expressing m2 receptors. Fig. 7 provides the functional correlate of this finding, since InsP3-induced 45Ca2+ mobilization was suppressed only in those cells expressing m l and m3 receptors. Thus, it can be concluded that only the phosphoinositidase C-linked muscarinic receptors are capable of causing InsP3 receptor downregulation. Type I InsP3 receptor immunoreactivity was also detected in GH3 rat pituitary cells (Fig. 6D 1, in A10 rat smooth muscle cells (Fig. 6E), in Swiss mouse 3T3 fibroblasts (Fig. 6F), and in rat H9c2 cardiac muscle cells (Fig. 6G). However, none of the agonists tested altered the level of immunoreactivity. This may be because the agonists used did not stimulate phosphoinositide hydrolysis persistently. For example, thyrotropin-releasing hormone produces a robust increase in phosphoinositide hydrolysis and InsP, formation within seconds of its addition to GH3 cells (331, but when measured after 12 h of incubation, InsP3 mass in cells stimulated with 2 J~M thyrotropin-releasing hormone was the same as that in control cells (25.2 2 0.8 and 26.1 2 0.7 pmoVmg protein, respectively; mean 2 S.E. of sextuplicate incubations).

DISCUSSION
The main conclusion to be drawn from the data presented is that the type I InsP3 receptor of SH-SY5Y cells is down-regu-

Type I InsP3
Receptor Down-regulation lated during muscarinic stimulation because it is degraded more rapidly than in resting cells. The tllz of the receptor in carbachol-stimulated cells was -1 h, whereas in resting cells the tI1, was > 8 h. This latter value immediately rules out a reduction in the rate of receptor synthesis as the sole cause of down-regulation, since even if type I InsP, receptor production was blocked totally upon addition of carbachol, receptor concentration would only fall with a half-time of > 8 h. Rather, the increase in the rate of receptor degradation is alone sufficient to reduce rapidly the cellular concentration of the type I InsP, receptor. Indeed, tlIz in carbachol-stimulated cells concurs well with the time a t which down-regulation of the type I InsP, receptor is half-maximal (15).
Despite the evidence against a role for regulation of receptor synthesis as the cause of down-regulation, type I InsP3 receptor mRNA levels did fall after 3 h of stimulation but then returned to basal levels. Such complex changes during muscarinic stimulation are common t o other mRNA species, for example, m2 and m3 muscarinic receptor mRNA in cerebellar granule (34) and SH-SY5Y (35) cells, and suggest that reduced type I InsPs receptor synthesis may contribute toward down-regulation at certain times. However, verification of this suggestion will require that the rates of mRNA translation are measured directly.
The studies with CHO cells revealed that only phosphoinositidase C-linked muscarinic receptor subtypes caused downregulation, Although stimulation of these receptors initiates a cascade of intracellular signaling (23), the primary response to receptor activation appears to be confined to the breakdown of PIP, and the formation of the second messengers InsP, and 1,2-diacylglycerol. InsP, releases intracellular Ca2+ stores via InsP3 receptors and also seems to be involved in causing entry of Ca2+ into the cell, either directly by interacting with plasma membrane InsP, receptors (1) or indirectly via capacitative refilling of discharged intracellular stores (1,26). In contrast, 1,2-diacylglycerol activates protein kinases C (36). Although protein kinase C activation does not initiate type I InsP, receptor down-regulation (151, the data presented here indicate that both InsP3 and Ca2+ stores have roles to play in this process. Initial evidence for this came from the finding that acceleration of receptor degradation was blocked by chelation of extracellular Ca2+ with EGTA. This manipulation inhibits Ca2+ influx and also eventually depletes intracellular Ca2+ stores. However, as Ca2+ derived from both stores and entry acts as a feedback activator of phosphoinositide hydrolysis during muscarinic stimulation of SH-SY5Y cells (24), depletion of extracellular Ca2+ not only suppresses Ca2+ mobilization, but also markedly inhibits PIP, hydrolysis. Thus, treatment with EGTA does not discriminate between Ca2+ mobilization or InsP3 as the signal that initiates acceleration of receptor degradation.
More revealing are the results obtained with thapsigargin. Without significantly raising InsP3 levels, thapsigargin persistently discharges intracellular Ca2+ stores, including those normally released by InsP3 (24,26), and also, because of capacitative refilling, causes Ca2+ entry (26). However, thapsigargin alone did not accelerate the degradation of or cause downregulation of the type I InsP3 receptor. These data together with the observation that that persistent depolarization of SH-SY5Y cells with K+ does not cause down-regulation (15) show that accelerated degradation is not simply a consequence of chronic release of intracellular Ca2+ stores, Ca2+ entry, or elevation of cytosolic Ca2+ concentration. Most significantly though, thapsigargin blocked the ability of carbachol to cause down-regulation without inhibiting its ability to elevate InsP, concentration. Thus, persistent activation of PIP, hydrolysis alone is not sufficient to cause down-regulation. Furthermore, as the InsP3 formed in the presence of thapsigargin will be unable to enhance the passage of Ca2+ through the channels formed by tetrameric InsP, receptors, because the gradient driving this movement will have been discharged, it appears that functional intracellular Ca2+ stores are required for downregulation to occur. This linkage of Ca2+ stores with the mechanism of type I InsP, receptor down-regulation is given credence by the observation that half-maximal inhibition of type I InsP3 receptor immunoreactivity was seen at -0.1 p~ carbachol. This value is very close to that which gives half-maximal Ca2+ mobilization during prolonged incubations of SH-SY5Y cell monolayers with carbachol (0.3 p~) .~ Thus, we propose that the signal that initiates acceleration of type I InsP, receptor degradation is the efflux of Ca2+ from intracellular stores via the InsP3 receptor itself. Perhaps the conformational changes that appear to accompany type I InsP3 receptor activation (5) and the flux of Ca2+ (12) expose the type I InsP3 receptor to a degradative pathway.
Regarding the nature of this pathway, it has been shown recently that calpain cleaves the type I InsP3 receptor into fragments of -130 and -95 kDa (27). However, cleavage by calpain was not responsible for the down-regulation seen in SH-SY5Y cells, as EST did not block this process, and immunoreactive fragments did not appear as type I InsP3 receptor levels declined. Neither does phosphorylation of the type I InsP3 receptor seem to play a role in its down-regulation. Although the receptor is clearly a phosphoprotein in resting SH-SY5Y cells, the extent of its phosphorylation was unaltered by carbachol. It is noteworthy that forskolin also had no effect on the extent of phosphorylation. Thus, at least in SH-SY5Y cells, activation of the two major signaling pathways used by hormone and neurotransmitter receptors does not regulate type I InsP, receptor phosphorylation. This contrasts with data obtained from cell-free systems (29).
Investigation of whether or not down-regulation occurred in other cell types revealed that muscarinic receptor stimulation was effective in reducing immunoreactivity in CHO cells expressing phosphoinositidase C-linked muscarinic receptors and also in cerebellar granule cells. The latter finding is particularly significant as it shows that down-regulation is not just a feature of cell lines, but can also occur in primary cultures. In each of these cell types, muscarinic receptor activation stimulates PIP, hydrolysis persistently (14,17,32), and it is likely that other receptors that share this characteristic (37, 38) will be those with the capacity to cause InsP3 receptor down-regulation. It will also be interesting to ascertain how persistent phosphoinositidase C activation relates t o the observation that GTP-binding proteins that couple receptors to phosphoinositidase C in CHO-ml cells are down-regulated during chronic stimulation with carbachol (39). In contrast to muscarinic receptors, however, many phosphoinositidase C-linked receptors are subject to rapid and severe desensitization (37, 38). The transience of the responses generated by such receptors may explain why, for example, bradykinin did not cause type I InsP3 receptor down-regulation in SH-SY5Y cells and why in the other cell types examined, none of the agonists tested was effective.
As yet we have only examined type I InsP3 receptor downregulation. This is the predominant type in the central nervous system (40) and in SH-SY5Y cells appears to mediate the majority of Ca2+ store mobilization. This can be concluded because down-regulation of the type I InsP, receptor by -90% suppressed markedly InsP3 action; maximal Ca2+ mobilization was reduced by half, and the potency of InsP3 was reduced 3-fold (14). Similarly, in CHO cells, type I InsP, receptor down-regulation reduced the maximal effect of InsP3 by one-third and its potency 2-3-fold. Nevertheless, significant amounts of types I1 and I11 receptor are expressed in brain (40) and in other tissues Type I InsP3 Receptor Down-regulation 7969 and cell lines (8, 9), and it will be fascinating to see whether these too are down-regulated.
In conclusion, this study has shown that persistent muscarinic receptor stimulation causes severe down-regulation of the type I InsP, receptor in several cell types. Acceleration of type I InsP, receptor degradation provides the mechanistic basis of this regulation, and sustained InsPs generation and Ca2+ release from intracellular stores appear to be essential in the initiation of this process.