Printed in U.S.A. Copyright © 2001 by The Endocrine Society Proteasomal Activation Mediates Down-Regulation of Inositol 1,4,5-Trisphosphate Receptor and Calcium Mobilization in Rat Pancreatic Islets*

Inositol 1,4,5-trisphosphate receptor (IP3R) protein levels in isolated rat pancreatic islets were investigated in response to carbachol (CCh) and sulfated cholecystokinin 26-33 amide stimulation. Within 2 h, CCh reduced IP3R-I protein levels by 22% and IP3R-II and -III levels to 65% or more below basal. Sulfated cholecystokinin 26-33 amide decreased the levels of IP3R-I, -II, and -III by 34%, 60%, and 66% below basal, respectively. The effect of CCh was concentration- and time-dependent, with a persistent decline in IP3R levels for up to 6 h after the onset of stimulation. CCh-pretreated islets also showed an inhibition of glucose-stimulated insulin secretion. Proteasome inhibition completely blocked the down-regulatory effects of CCh on IP3Rs and significantly increased the insulin secretory response to glucose stimulation in the presence of CCH: Islet stimulation by glucose, alpha-ketoisocaproic acid, and tolbutamide completely protected IP3Rs against the down-regulatory effects of CCH: 2-deoxyglucose and 3-O-methyl glucose failed to affect CCh-induced IP3R down-regulation. The protective effects of glucose on IP3R down-regulation were completely inhibited by the Ca(2+) channel-blocking agent nimodipine. Intracellular Ca(2+) ([Ca(2+)](i)) levels in Fura-2 (fluorescent Ca(2+) indicator)-loaded islets, in the absence of extracellular Ca(2+), increased in response to glucose stimulation; but in islets pretreated with CCh, glucose did not increase [Ca(2+)](i) above basal levels. However, in islets pretreated with CCh and the proteasomal inhibitor MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinyl-H), the glucose-stimulated increase in [Ca(2+)](i) was significantly higher than the change observed for glucose-stimulated [Ca(2+)](i) in the absence of MG-132. The results suggest that muscarinic receptor stimulation modulates IP3R protein levels in islets through a proteasomal activation pathway, and that down-regulation of IP3Rs has a profound effect on Ca(2+) mobilization in islets that may relate to insulin secretory responsiveness.

3-O-methyl glucose failed to affect CCh-induced IP3R down-regulation.The protective effects of glucose on IP3R down-regulation were completely inhibited by the Ca 2ϩ channel-blocking agent nimodipine.Intracellular Ca 2ϩ ([Ca 2ϩ ] i ) levels in Fura-2 (fluorescent Ca 2ϩ indicator)-loaded islets, in the absence of extracellular Ca 2ϩ , increased in response to glucose stimulation; but in islets pretreated with CCh, glucose did not increase [Ca 2ϩ ] i above basal levels.However, in islets pretreated with CCh and the proteasomal inhibitor MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinyl-H), the glucose-stimulated increase in [Ca 2ϩ ] i was significantly higher than the change observed for glucose-stimulated [Ca 2ϩ ] i in the absence of MG-132.The results suggest that muscarinic receptor stimulation modulates IP3R protein levels in islets through a proteasomal activation pathway, and that down-regulation of IP3Rs has a profound effect on Ca 2ϩ mobilization in islets that may relate to insulin secretory responsiveness.(Endocrinology 142: 1744 -1751, 2001) T HE INOSITOL 1,4,5-TRISPHOSPHATE (IP3) receptor (IP3R) is an integral ligand-activated Ca 2ϩ -selective channel found in many tissues (1,2), including the pancreatic islet ␤-cell (3).After receptor binding by the secondmessenger IP3, intracellular Ca 2ϩ ([Ca 2ϩ ] i ) is mobilized from intracellular stores.There are three mammalian IP3R isoforms (types I-III), all of which have been identified in the rat pancreatic islet and ␤-cell lines (3).Regulation of transcriptional activation of messenger RNA (mRNA) for IP3R-III has been reported to be an early response to glucose stimulation in islets, and IP3R-III levels were reported to increase shortly thereafter (4).In contrast, long-term glucose stimulation, both in vivo and in vitro, has also been associated with differential changes in the regulation of IP3R-II and -III transcriptional and posttranscriptional activity (4).IP3Rs have been implicated in the regulation of oscillations in [Ca 2ϩ ] i (5), and the different receptor subtypes may activate multiple IP3-sensitive Ca 2ϩ pools (6).It has been proposed that the selective expression in cells of IP3R subtypes with different affinities for IP3 determines the sensitivity of Ca 2ϩ mobilization to IP3 (7).Moreover, functional interactions between different IP3R isoforms predict that differential regulation of IP3R isoform levels will be reflected in functional changes in IP3-mediated Ca 2ϩ mobilization (8).
Insulin secretion is largely a Ca 2ϩ -dependent process, and restricted increases in [Ca 2ϩ ] i have been related to impairment of glucose-stimulated insulin release (9).Several lines of evidence point to IP3 playing an important role in insulin secretion.IP3 was shown to mobilize [Ca 2ϩ ] i in permeabilized insulin-secreting cells (10), and IP3 production correlated with Ca 2ϩ mobilization in intact cells (11).In the mouse anx7 (ϩ/Ϫ) phenotype, where there is a profound reduction in IP3R expression and Ca 2ϩ mobilization in islets, there is also defective insulin secretion (12).Numerous stimuli have been reported to activate the IP3 pathway, and receptor stimulation is commonly linked to phospholipase C activation, as with certain neurotransmitters, growth factors, and hormones (13).In addition, in islets, glucose stimulation acts through a Ca 2ϩ -dependent mechanism to activate phospholipase C (14,15), a phenomenon also reported for brain synaptosomes and iris smooth muscle, among other tissues (13).The generation of IP3 in cells not only activates the IP3Rs and mobilizes Ca 2ϩ but also leads to IP3R down-regulation (16).Previous reports have described the down-regulation of IP3R-I in response to muscarinic receptor activation by carbachol (CCh) (17); and of IP3R-I, II, and III in response to cholecystokinin (CCK), bombesin, and pituitary adenylylcyclase-activating peptide (18).The mechanisms participating in IP3R down-regulation include IP3 binding, increased Ca 2ϩ mobilization, and processes that mediate the activation of a ubiquitination pathway, targeting the IP3Rs for degradation by proteasomes.
The present study describes the effects of various stimuli on isolated rat pancreatic islet IP3R regulation.Characterization of IP3R down-regulation and the role of glucose metabolism in that process are described.In addition, the functional nature of the IP3R down-regulatory phenomenon is linked to altered [Ca 2ϩ ] i mobilization responses and insulin secretion in isolated islets.

Materials and Methods Materials
Collagenase type P was obtained from Roche Molecular Biochemicals (Indianapolis, IN).CMRL-1066 culture medium was purchased from Life Technologies, Inc. (Grand Island, NY).Affinity-purified antisera against the IP3R types I and II were a gift from Dr. R. Wojcikiewicz (SUNY Health Science Center at Syracuse, Syracuse, NY).Monoclonal antibody against IP3R-III was obtained from Transduction Laboratories, Inc. (Lexington, KY).Peroxidase-conjugated antirabbit and antimouse IgG secondary antibodies were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA).N-acetyl-Leu-Leu-norleucinol (ALLN) and carbobenzoxyl-leucinyl-leucinyl-leucinyl-H (MG-132) were from Calbiochem (San Diego, CA).CCK fragment 26 -33 amide sulfated, CCh, and BSA were obtained from Sigma (St. Louis, MO).Immobilon-P membrane was purchased from Millipore Corp. (Bedford, MA).Rat insulin for RIA standard was a gift from Eli Lilly & Co. (Indianapolis, IN).[ 125 I]Insulin (porcine) was obtained from NEN Life Science Products (Boston, MA).All other chemicals were reagent grade and were obtained from commercial sources.

Isolation and culture of rat islets
Pancreatic islets from adult male Sprague Dawley rats were isolated using collagenase, as described previously (19).All animal procedures were approved by the institutional animal care and use committee.Isolated islets were cultured overnight in CMRL-1066 medium containing 5.5 mm glucose, 9% FBS, penicillin (100 U/ml), and streptomycin (100 g/ml) in 5% CO 2 -95% air, at 35 C, as described previously (19).Cultured islets devoid of acinar tissue were placed into fresh CMRL-1066 medium or Krebs-Ringer bicarbonate (KRB) buffer containing 16 mm HEPES, 5.5 mm glucose, and 0.01% BSA (19) and were processed as described in the text.

Immunoblotting
Islets were cultured and incubated as described above for insulin release.After incubation, whole-cell islet homogenates were prepared for immunoblotting essentially as described previously (4).Equal amounts of protein per sample (15-30 g) for each experiment were separated on 5% SDS-PAGE gels and transferred to Immobilon-P membrane.Immunoblotting was carried out essentially as described previously (4), with IP3R-I, -II, and -III antibodies and peroxidase-conjugated secondary antibodies.Bound antibody was localized by chemiluminescence, and the density of each band was determined by densitometric scanning using Molecular Analyst software (Bio-Rad Laboratories, Inc.).

Insulin release
After an overnight culture, isolated islets were suspended in fresh CMRL-1066 and cultured for 2 h, in the absence or presence of CCh and MG-132, as indicated in the text.The islets were distributed at 10 islets/ sample, washed, and resuspended in KRB buffer (19) and were incubated at 37 C, under an atmosphere of 95% O 2 -5% CO 2 , in a shaking water bath.After 60 min, the KRB buffer was replaced with fresh buffer, and an aliquot of buffer was removed for determination of time-zero insulin levels.After a 60-min incubation, in the presence or absence of a stimulus as indicated in the text, an aliquot of buffer was removed for determination of insulin release.Insulin release values are minus timezero insulin values.Insulin levels were quantitated by RIA using rat insulin as standard.

Islet [Ca 2ϩ ] i measurements
Islets were cultured and incubated as described above for insulin release.For the measurement of [Ca 2ϩ ] i , islets were loaded with Fura-2/AM (1 m), for 30 min at room temperature, in KRB buffer (pH 7.4) containing (mm): NaCl (115), KCl (5), NaHCO 3 (24), CaCl 2 (2.5), MgCl 2 (1), HEPES (50), d-glucose (5.5), and 0.1% BSA.The islets were then washed and incubated for 1 h in KRB buffer, at room temperature, to allow hydrolysis of the ester.For Ca 2ϩ -free KRB buffer, CaCl 2 was omitted, and EGTA (0.1 mm) was added.A coverslip containing Fura-2-loaded cells was mounted in a cell chamber (Bioptechs FCS2; Bioptechs Inc., Butler, PA) on the stage of an inverted microscope.A ratio-based microscopic fluorescent spectrophotometer (Photon Technology International Inc., South Brunswick, NJ) was used to excite the dye at 340/380 nm alternatively at 2-Hz sampling rate.Fluorescence of Fura-2/AMloaded islets was measured at 35 C by perifusing KRB buffer at a flow rate of 0.34 ml/min.The vol of the fluid in the cell chamber was 0.1 ml.Excitation wavelengths were obtained using 340 nm and 380 nm excitation filters, and the emission spectrum was obtained using a 510-nm filter.Two image pairs were acquired every second.Changes of [Ca 2ϩ ] i , in one islet per coverslip, were recorded for 50 min per experiment.Basal [Ca 2ϩ ] i was recorded with 5.5 mm glucose for 10 min; and increases in [Ca 2ϩ ] i , after glucose stimulation (16.5 mm) in the absence or presence of Ca 2ϩ in KRB buffer, were recorded for 20 min.

Statistical analysis
Values are the mean Ϯ se.Significant differences between treatment groups were determined by one-way ANOVA, with post hoc analysis, using Student's-Newman-Keuls multiple-comparison test.P Ͻ 0.05 was accepted as significant.

Regulation of IP3R levels
IP3R levels in isolated rat pancreatic islets were investigated in response to CCh and CCK stimulation.After a 2-h stimulation with CCh, IP3R-I, -II, and -III protein levels were significantly reduced (Fig. 1, A and B).IP3R-I was downregulated by 22% below basal levels, whereas IP3R-II and -III were reduced by more than 60%.After stimulating islets for 2 h with CCK, there were significant decreases in the levels of IP3R-I, -II, and -III similar to the effects observed in the presence of CCh (Fig. 1).The effect of CCh was concentration-and time-dependent (Fig. 2, A and B).A concentration of CCh as low as 10 Ϫ6 m caused a decrease of approximately 40% in IP3R-II levels, and higher concentrations of CCh evoked further decrements in IP3R-II levels (Fig. 2A).IP3R-II levels declined rapidly within 2 h after CCh stimulation, and levels remained depressed for up to 6 h after the onset of stimulation (Fig. 2B).Similar results were observed for IP3R-III responses to CCh stimulation (data not shown).

Effects of proteasomal inhibitors on IP3R levels and insulin secretion responses to CCh
IP3R responses to CCh were investigated in the presence of the proteasomal inhibitors MG-132 and ALLN.Islets were preincubated with MG-132 (50 m) or ALLN (50 m) for 1 h before the addition of CCh.Both MG-132 and ALLN completely blocked the down-regulatory effects of CCh on IP3R-II and -III (Fig. 3A).In a preliminary study, 50 m MG-132 was more effective than 10 m at reversing the effects of CCh (data not shown).In addition, IP3R-II in islets treated with CCh and MG-132 showed an increase in IP3R levels above basal (Fig. 3A).Neither MG-132 nor ALLN alone had an effect on IP3R levels (Fig. 3, A and B).
It has been previously reported that prolonged stimulation with CCh diminishes the glucose-induced insulin secretory responses of rat islets (20).In isolated islets in static incubation, a submaximal secretory concentration of glucose (10 mm) stimulated insulin secretion by 11-fold (Fig. 4).CChpretreated islets, stimulated with glucose (10 mm), demonstrated the inhibitory effect of the cholinergic agonist, with insulin secretion reduced approximately 65%, compared with glucose-stimulated control islets (Fig. 4).When islets were pretreated with MG-132 before CCh treatment, the secretory response to glucose stimulation was significantly higher than in similarly treated islets in the absence of the proteasomal inhibitor (Fig. 4).There was no significant difference (P Ͼ 0.05) between islet insulin secretion in the presence of glucose (10 mm) plus MG-132 vs. glucose (10 mm) plus CCh plus MG-132.Neither MG-132 nor CCh, alone or in combination, had any effect on basal insulin secretion (Fig. 4).

Modulatory events in IP3R down-regulation
In addition to cholinergic and CCK receptor-mediated increases in inositol phosphates, glucose also stimulates phospholipase C and increases IP3 levels in islets (21,22).However, when islets were stimulated with a maximal secretagogic concentration of glucose (20 mm), there was no change in IP3R-II or -III levels, compared with basal (Fig. 5,  A and B).When glucose-stimulated islets were challenged with CCh, there was significant protection of IP3R-II and-III against the down-regulatory effects of CCh (Fig. 5, A and B).Similarly, when islets were treated with ␣-ketoisocaproic acid (KIC), a leucine metabolite that increases mitochondrial ATP generation, IP3R-II and -III levels were completely protected against the down-regulatory effects of CCh (Fig. 5, A  and B).In contrast, 2-deoxyglucose (which is transported and phosphorylated, but not metabolized, by islet cells) and 3-Omethyl glucose (which is also not metabolized) failed to have any effect on CCh-induced IP3R down-regulation (Fig. 5, A  and B).
One of the major mechanisms mediating glucose effects on insulin secretion in ␤-cells is depolarization-dependent Ca 2ϩ influx through voltage-dependent Ca 2ϩ channels (VDCC).When CCh-pretreated islets were cultured with nimodipine, a VDCC blocker, the protective effects of glucose on IP3R down-regulation were completely inhibited (Fig. 5, A and B).However, nimodipine alone failed to have any significant effect on the CCh response (data not shown).In contrast, the sulfonylurea tolbutamide significantly increased IP3R-II and -III levels in the presence of CCh, compared with islets treated with CCh alone (Fig. 5A and B).

Effects of proteasomal inhibition on Ca 2ϩ mobilization
To determine the effects of CCh on Ca 2ϩ mobilization responses, islets were loaded with the fluorescent Ca 2ϩ in- dicator Fura-2/AM.Perifusion of the islets with Ca 2ϩ -free KRB medium containing 16.5 mm glucose resulted in a small, but significant, increase in peak [Ca 2ϩ ] i above basal (Fig. 6A and Table 1).In islets pretreated with CCh, a subsequent stimulation with glucose did not significantly increase [Ca 2ϩ ] i above basal (Fig. 6B and Table 1).However, in CChand MG-132-pretreated islets, the glucose-stimulated increase in [Ca 2ϩ ] i , to 29 Ϯ 5% above basal, was significantly higher (P Ͻ 0.01) than the response observed for glucosestimulated [Ca 2ϩ ] i in the absence of MG-132 (3 Ϯ 5%) (Fig. 6C and Table 1).
In another series of experiments, islets were perifused with Ca 2ϩ -containing medium to determine whether the CCh response could be quantitated under physiological conditions.Glucose and KCl stimulation evoked significant increases in peak [Ca 2ϩ ] i , which were higher than those observed in the absence of extracellular Ca 2ϩ (Fig. 7A and Table 1).When islets were pretreated with CCh for 2 h, the glucose (16.5 mm)-stimulated increase in peak [Ca 2ϩ ] i , to 28 Ϯ 3% above basal, was significantly lower than that observed for control islets (58 Ϯ 3% above basal), whereas KCl-stimulated peak [Ca 2ϩ ] i reached 90 Ϯ 15% above basal and was not significantly different from the response in control islets (Fig. 7B and Table 1).When islets were pretreated with both CCh and MG-132, the glucose (16.5 mm)-stimulated peak [Ca 2ϩ ] i of 51 Ϯ 6% above basal was significantly (P Ͻ 0.05) higher than in the absence of MG-132 and not significantly different from the glucose response in control islets (Fig. 7C and Table 1); the KCl-stimulated peak [Ca 2ϩ ] i (81 Ϯ 13% above basal) in these treated islets was not significantly different from the response in control islets (Table 1).When islets were treated only with MG-132 (50 m), the [Ca 2ϩ ] i response to glucose or KCl was not different from untreated islets (data not shown).

Discussion
IP3 production is increased in pancreatic islets and other tissues in response to receptor agonists acting through a G q protein-coupled receptor-activating phosphoinositidase C (1).Both CCh and CCK activate receptors coupled to the production of IP3.Between 0.1 mm and 1 mm CCh has been reported to evoke maximal IP3 production in rat islets (21), and 0.5 mm CCh was selected for use in the present studies.Among the CCh-responsive muscarinic receptor subtypes, only m1, m3, and m5 stimulate IP3 production, and m1 and m3 (but not m2) receptor subtypes have been shown to down-regulate IP3Rs (17).In islets and ␤-cell lines, m1 and m3 receptor subtypes are functionally expressed (23)(24)(25).The binding of IP3 to IP3Rs activates the receptor ion channel; and Ca 2ϩ stored in the endoplasmic reticulum, and perhaps in other subcellular organelles, enters the cytoplasm.It has been reported that the binding of IP3 to its receptors and mobilization of Ca 2ϩ are required for initiating events that lead to down-regulation in the expression level of IP3R subtypes specifically (17, 26 -30).In the rat pancreatic islet and insulinoma cell lines, IP3R isoforms I, II, and III have been  M) for 1 h, followed by an additional 2 h of culture in the absence or presence of CCh (0.5 mM).Then, the islets were washed, and incubated in the absence or presence of glucose (10 mM), as indicated.Insulin release was determined after 1 h.Insulin release values are the mean Ϯ SE for seven or eight independent determinations.Significant differences between groups were determined by one-way ANOVA and multiple-comparison test.*, P Ͻ 0.001 vs. basal.
identified and characterized for regulation in response to short-term and long-term glucose stimulation (3,4).In islets, the regulation of IP3R-I, of -II, and of -III are independent.Whereas IP3R-I showed little response to glucose, short-term exposure to a glucose stimulus increased the mRNA and protein levels for IP3R-III, and long-term glucose stimulation increased IP3R-II mRNA and protein but decreased IP3R-III mRNA and protein levels.
The present study represents another aspect of IP3R regulation in pancreatic islets (that of down-regulation by phosphoinositidase activating agents).Both CCh and CCK increased the down-regulation of islet IP3Rs.Both CCh and CCK down-regulated each of the three subtypes of IP3R, but IP3R-I to a lesser degree than IP3R-II and -III.CCK has been reported to be more potent than CCh at down-regulating each of the IP3R subtypes in AR4 -2J cells, a response correlated with persistent IP3 generation (18).However, CCh (1 mm) also caused ubiquitination, and presumably down-regulation by proteasomal mechanisms, of IP3R-I and -III in the ␤-cell line INS-1 (that, unlike rat islets, does not express IP3R-II) (18).Interestingly, the time course of CCh-induced IP3R-II and -III down-regulation in the present study showed an initial rapid decline in receptor levels, which remained at reduced levels over several hours.This suggests that some persistent stimulation event targets the IP3Rs for degradation and that new receptor synthesis does not rapidly reverse the effect.
Proteasomal inhibition by MG-132 or ALLN prevented the down-regulatory response to CCh on IP3Rs in islets.ALLN, a peptide inhibitor of calpain and other cysteine proteases (31) as well as the proteasome (32)(33)(34), has been previously demonstrated to inhibit IP3R down-regulation (30,35).It has been proposed that calpain is activated by local increases in Ca 2ϩ mobilization in cells and can specifically degrade IP3Rs (30).Previous studies have shown that ligand binding of IP3Rs and Ca 2ϩ mobilization mediates the ubiquitination of IP3Rs and their targeting for degradation by the proteasomal pathway (18).Protein kinase C (PKC) activation does not mediate this process.The degradation of IP3R-I is expected in conjunction with IP3R-II and -III down-regulation, because it has been reported that the type II and III receptors, primarily in association with type I, are susceptible to ubiquitination.
Other cellular stimuli were also investigated for effects on IP3R levels.In a previous study, we reported that short-term glucose stimulation resulted in an increase in IP3R-III protein levels in freshly isolated islets (4).In the present study, islets were cultured overnight, before glucose stimulation, to allow all adherent acinar tissue to dissociate from the islets, and it was apparent that there was no significant change in IP3R-III levels in these cultured islets in response to glucose.Thus, overnight culture of islets seems to alter glucose responsivity of the IP3R pathway.However, there was an interaction between CCh and glucose in these islets, because the reduction in IP3R-II and -III levels in response to CCh was largely inhibited by the presence of a glucose stimulus.KIC evoked a protective response similar to that of glucose.Two glucose analogs, 2-deoxyglucose and 3-O-methyl glucose, which are not metabolized to produce energy in islets, failed to affect the down-regulatory response to CCh, suggesting that increased carbohydrate metabolism, in some way, protects the IP3R from proteasomal degradation.One of the major mechanisms mediating glucose and KIC effects on the ␤-cell is stimulation of the citric acid cycle, increasing ATP levels in the ␤-cell and closing ATP-sensitive K ϩ channels, thus inducing depolarization of the cell and opening VDCC for Ca 2ϩ influx (36).The protective effect of glucose on CCh-induced down-regulation of IP3Rs was completely overcome with the blockade of VDCC by nimodipine, providing evidence for the importance of Ca 2ϩ influx mediating the protective effects of glucose and presumably KIC on IP3Rs.Further support for this hypothesis comes from the islet response to tolbutamide.Sulfonylureas bind to and block ATP-sensitive K ϩ -channels, depolarize the ␤-cell, and promote Ca 2ϩ influx through VDCC.The protective response to tolbutamide, in preventing the down-regulation of IP3Rs in the presence of CCh, is likely to also be mediated by increased [Ca 2ϩ ] i .It is not known how increased [Ca 2ϩ ] i antagonizes the CChinduced IP3R down-regulatory effects, but increased [Ca 2ϩ ] i might increase calmodulin binding to IP3R-I, which report-  Islets were precultured with or without MG-132 (50 M) for 1 h, and then CCh (0.5 mM) was added for an additional 2 h.Islets were loaded with Fura-2/AM, washed, and perifused with KRB buffer in the absence of added Ca 2ϩ and the presence of EGTA (100 M) (Ca 2ϩ -free) or in the presence of 2.4 mM Ca 2ϩ (Ca 2ϩ -containing).Islets were stimulated with glucose (16.5 mM) and KCl (35 mM) as indicated.Values (the fluorescence ratio of 380/390 nm) are means Ϯ SE for three or four independent experiments.The basal fluorescence ratios in the Ca 2ϩ -free perifusion group were control (0.89 Ϯ 0.10), CCh-pretreated (0.83 Ϯ 0.03), and CCh-, MG-132-pretreated (1.01 Ϯ 0.15), in the Ca 2ϩ -containing perifusion group, basal ratios were control (0.86 Ϯ 0.04), CCh-pretreated (0.91 Ϯ 0.05), and CCh-, MG-132-pretreated (0.86 Ϯ 0.04).Significant differences were determined by one-way ANOVA and multiple-comparison test.a, P Ͻ 0.01 vs. basal; b, P Ͻ 0.05 vs. control for the same treatment group.
edly inhibits IP3-induced Ca 2ϩ mobilization (37).Because IP3-induced Ca 2ϩ mobilization from endoplasmic reticulum stores is required for IP3R down-regulation (17), a Ca 2ϩsuppressed Ca 2ϩ release phenomenon may antagonize IP3R down-regulation in ␤-cells.It has also been reported that blockade of L-type Ca 2ϩ channels in A7r5 cells induced IP3R-I down-regulation (29), suggesting that, in these cells too, Ca 2ϩ influx inhibits IP3R degradation.Alternatively, increased Ca 2ϩ -influx may alter phosphorylation of IP3R by various protein kinases (38 -40) and alter IP3R activity and down-regulation.
Because CCh has been reported to desensitize the islet to further stimulation by glucose or other agonists (20,41), the role of proteasomal activation in this process was investigated.Pretreatment of islets with CCh for 2 h markedly diminished the subsequent insulin secretory response to glucose.However, when MG-132 was present during the CCh challenge, a subsequent stimulation by glucose showed that insulin release was maintained close to control levels, suggesting that a proteasome activation event mediated, in part, the effects of CCh on islets.CCh, in addition to increasing IP3 levels as a result of phosphoinositidase C stimulation, also increases diacylglycerol levels and activates PKC.It is not likely that PKC modulated IP3R levels, because it has been previously reported that activation of PKC with phorbol ester does not down-regulate IP3R levels (27,28).
The effect of CCh on IP3R levels was also investigated for correlation with Ca 2ϩ mobilization in islet cells.Muscarinic receptor types 1 and 3, expressed in pancreatic ␤-cells, are functionally linked to phosphoinositidase C that is associated with stimulation of insulin secretion (25).Moreover, acetylcholine, in the absence of extracellular Ca 2ϩ , has been reported to trigger an increase in [Ca 2ϩ ] i mobilization (42).In the present study, the effects of CCh pretreatment on glucose-induced Ca 2ϩ mobilization were investigated to determine whether down-regulation of IP3Rs correlated with changes in [Ca 2ϩ ] i .In the absence of extracellular Ca 2ϩ , it was observed that glucose stimulation increased islet [Ca 2ϩ ] i but that pretreatment with CCh completely prevented glucose stimulation.The inclusion of the proteasomal inhibitor MG-132, during the CCh pretreatment of islets, protected the glucose response, suggesting that it was not depletion of [Ca 2ϩ ] i pools that mitigated glucose stimulation.Under more physiological conditions, with islets in the presence of extracellular Ca 2ϩ , glucose-induced elevations in [Ca 2ϩ ] i were also inhibited by pretreatment with CCh, and again the inhibition of the proteasomal pathway augmented the response to glucose stimulation.These data suggest that there is a correlation between inhibition of the IP3R down-regulation and [Ca 2ϩ ] i mobilization in response to glucose.We have not ruled out the possibility that other proteins may also be down-regulated by the proteasomal pathway during CCh pretreatment and may affect the islet response to glucose.
In summary, the well-known reduction in glucose-stimulated insulin secretion by pretreatment with CCh has been correlated to down-regulation of the IP3Rs and to diminished glucose-responsive islet cell Ca 2ϩ mobilization.Proteasomal degradation of IP3Rs seems to mediate the muscarinic receptor-induced down-regulatory phenomenon in islets.These data support the conclusion that IP3R regulation plays a role in long-term effects of muscarinic receptor and CCK-receptor stimulation in ␤-cells.

FIG. 1 .
FIG. 1. IP3R isoform regulation in rat isolated islets.Islets were cultured, in the absence (basal) or presence of CCh (0.5 mM) or CCK (0.1 M), for 2 h.A, Islet homogenates were analyzed by Western blotting for IP3R-I, -II, or -III, as indicated.Equal amounts of protein (15 g) were loaded per lane.B, Western blot densitometric analyses of IP3R isoforms expressed as percent of basal values.Values are the mean Ϯ SE for 3-11 independent determinations.Significant differences from basal were determined by one-way ANOVA and multiplecomparison test for each type.ࡗ, P Ͻ 0.05; *, P Ͻ 0.02; OE, P Ͻ 0.001 vs. basal.

FIG. 3 .
FIG. 3. Effects of MG-132 and ALLN on CCh-induced changes in IP3R-II (A) and IP3R-III (B) protein expression.Rat islets cultured at 5.5 mM glucose were preincubated in the absence (basal) or presence of MG-132 (50 M) and ALLN (50 M) for 1 h, before incubation for an additional 2 h in the absence or presence CCh (0.5 mM), as indicated.Values are the mean Ϯ SE for three independent determinations.Significant differences between groups were determined by one-way ANOVA and a multiple-comparison test.*, P Ͻ 0.01 vs. basal.

FIG. 7 .
FIG. 7. Effects of CCh on glucose-induced Ca 2ϩ mobilization in Ca 2ϩ -containing buffer.Islets were cultured in the absence (A and B) or presence of MG-132 (50 M) (C) for 1 h, before culture in the absence (A) or presence of CCh (0.5 mM) (B and C) for 2 h.Islets were loaded with Fura-2/AM and perifused at 35 C in KRB buffer containing 2.5 mM Ca 2ϩ .Islets were perifused in KRB buffer containing 2.5 mM Ca 2ϩ and stimulated with glucose (16.5 mM), followed by KCl (35 mM), as indicated by the horizontal bars.The glucose stimulations are representative of four independent determinations.

TABLE 1 .
Effect of CCh pretreatment and MG-132 on peak [Ca 2ϩ ] i in isolated rat islets