Protein Kinase C and Calcium Regulation of Adenylyl Cyclase in ...

Protein Kinase C and Calcium Regulation of Adenylyl Cyclase in Isolated Rat Pancreatic Islets Yingrao Tian and Suzanne G. Laychock

Rat islets express several isoforms of adenylyl cyclase (AC), and the regulation of AC activity in isolated islets by Ca2ⴙ and protein kinase C (PKC) was investigated. At basal 2.8 mmol/l glucose, the muscarinic receptor agonist carbamylcholine chloride (CCh) evoked a concentration-dependent increase in cAMP generation with a maximum increase at least 4.5-fold above control. In contrast, forskolin and glucagon-like peptide 1 fragment 7-36 amide increased cAMP accumulation 23-fold and almost 10-fold, respectively. Cholecystokinin 26-33 sulfated amide (CCK) also stimulated cAMP production by up to eightfold, as did the phorbol ester, phorbol 12,13-dibutyrate (PDBu). PDBu and CCh or CCK responses were not additive. The effects of phorbol ester, CCh, and CCK were inhibited by as much as 75% by the PKC inhibitors GF 109203X and Ro-32-0432 and after PKC downregulation. In the absence of extracellular Ca2ⴙ, PDBu-, CCh-, and CCK-induced cAMP production was inhibited by ⬃50% in each case. Chelation of intracellular Ca2ⴙ with 1,2-bis(o-amino-5-fluorophenoxy) ethane-N,N,Nⴕ,Nⴕ-tetraacetic acid tetraacetoxymethyl ester (BAPTA/AM) inhibited CCh- and CCK-stimulated cAMP generation by ⬃50% but did not inhibit the stimulatory effect of PDBu. Stringent Ca2ⴙ depletion by removal of extracellular Ca2ⴙ and inclusion of BAPTA/ AM allowed for increased cAMP production in response to CCh and CCK; PKC inhibitors and PKC downregulation prevented this stimulation. Glucose stimulation also increased islet cAMP production, but PDBu did not potentiate the glucose response. The results suggest that Ca2ⴙ influx, Ca2ⴙ mobilization, and PKC activation play important roles in the modulation of AC activity in pancreatic islets. Diabetes 50:2505–2513, 2001

From the Department of Pharmacology and Toxicology, the State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New York. Address correspondence and reprint requests to Dr. Suzanne G. Laychock, 102 Farber Hall, SUNY at Buffalo, School of Medicine, Buffalo, NY 14214. E-mail: [email protected]. Received for publication 26 March 2001 and accepted in revised form 1 August 2001. AC, adenylyl cyclase; ANOVA, analysis of variance; BAPTA/AM, 1,2-bis(oamino-5-fluorophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid tetraacetoxymethyl ester; BSA, bovine serum albumin; CCh, carbamylcholine chloride; CCK, cholecystokinin; GLP, glucagon-like peptide; IBMX, 3-isobutyl-1-methylxanthine; KRBH, Krebs-Ringer bicarbonate HEPES; ODG, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-1; PDBu, phorbol 12,13-dibutyrate; PDD, phorbol-12myristate-13-acetate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; RIA, radioimmunoassay; VDCC, voltage-dependent Ca2⫹ channels. DIABETES, VOL. 50, NOVEMBER 2001

T

he ability of the second messenger cAMP, through protein kinase A (PKA) activation, to potentiate glucose-stimulated insulin secretion is widely documented (1,2). Mechanisms mediating cAMP action in ␤-cells include Ca2⫹ mobilization, Ca2⫹ influx, sequestration, and electrical activity (3–5). Recently, cAMP was demonstrated to induce insulin secretion independent of a glucose stimulus (6,7), and cAMPdependent PKA activation appeared to activate the secretory apparatus distal to changes in intracellular Ca2⫹ in ␤-cells (8 –10). In addition, both PKA-dependent and -independent actions of cAMP have been demonstrated to modulate insulin secretion through changes in release and refilling of the insulin secretory granule pool (10). In pancreatic islets, adenylyl cyclase (AC) generates cAMP from ATP in response to stimulatory GTP-binding (Gs) protein–mediated receptor and metabolic stimuli (1,6,7,11). The molecular cloning of mammalian AC has identified at least nine different isoforms (12,13). It was recently reported that rat islets contain AC types 2 (14) and 3–7 (15,16). Regulation of the heterogeneous AC isoforms is imposed by forskolin (stimulates all isoforms), Gs (stimulates all isoforms), Gi (inhibits types 1–3, 5, and 6), G␤␥ (stimulates types 2, 4, and 7, but inhibits 1 and 8), Ca2⫹/calmodulin (stimulates types 1, 3, and 8), Ca2⫹ (inhibits 5 and 6), protein kinase C (PKC) (stimulates types 2, 5, and 7), PKA (inhibits types 5 and 6), and calmodulin kinase (inhibits types 1 and 3) (13). Variations on this paradigm are reported. For instance, PKC can inhibit AC activity in response to certain agonists (17–19) or antagonize the inhibition of AC by Gi (20). PKC-␣ can increase type 2 AC activity but inhibit type 4 activity (21). PKC-⑀ and PKC-␮ have also been implicated in AC regulation (22). Glucose has been proposed to stimulate islet cAMP production through changes in ATP levels and Ca2⫹ mobilization/calmodulin activation, whereas glucagon and glucagon-like peptide (GLP) 1—among other stimulatory hormones—activate Gs-coupled receptors (7,23,24). PKC activation has been reported to affect islet AC by inhibiting the ability of ␣2-adrenoceptor stimulation to reduce cAMP generation and glucose-induced insulin secretion (25). That PKC response was associated with phosphorylation of Gi/Go family of pertussis toxin–sensitive G proteins and possibly the uncoupling of G proteins from the ␣2-adrenoceptor. Characterization of any direct effects of pancreatic islet PKC activation on AC regulation has not been previously reported. However, the ability of glucose and other secretagogues in islet ␤-cells to mobilize Ca2⫹ and stimu2505

ADENYLYL CYCLASE ACTIVITY IN ISLETS

1 h; and ODQ, 30 min. All islet samples were pretreated with the phosphodiesterase inhibitor IBMX (0.2 mmol/l), 20 min, before time-zero stimulation with agents described in the text. Islet cAMP levels were determined at time-zero and after up to 20 min of incubation. All treatments were performed in duplicate. The reaction was stopped by the addition of ice-cold 0.1 N HCl (final concentration), and samples were immediately frozen. After three freeze/thaw cycles, the samples were sonicated and neutralized with sodium acetate buffer (final concentration 25 mmol/l) before acetylation and radioimmunoassay (RIA), as described previously (7,27). The cAMP levels of islets at time-zero were subtracted from the later time values to determine cAMP production. Statistical analysis. Values are means ⫾ SE. Significant differences between treatment groups were determined by one-way analysis of variance (ANOVA) with post hoc analysis using Student-Newman-Keuls multiple-comparison test. Values of P ⱕ 0.05 were accepted as significant.

RESULTS

FIG. 1. CCh-stimulated cAMP production. Islets were incubated for 20 min in KRBH buffer containing 2.8 mmol/l glucose and IBMX (0.2 mmol/l) in the absence (C) or presence of CCh (1–500 ␮mol/l), as indicated. cAMP values were determined by RIA. Values are means ⴞ SE for the number of independent experiments shown at the base of each bar. *P < 0.001 vs. C, as determined by one-way ANOVA and multiple-comparison test.

late PKC activity suggests that certain isoforms of AC can be specifically modulated in these cells. The hypothesis to be tested in this study was that stimulation of islets by G-protein– coupled receptor agonists that regulate phospholipase C (PLC) activation, PKC stimulation, and Ca2⫹ mobilization have an effect on AC activity in pancreatic islets. The results show that both Ca2⫹ and PKC modulate AC activity in isolated islets.

Effects of CCh and CCK on islet AC activity. To determine AC activity in isolated rat pancreatic islets, cAMP levels were quantitated in the presence of a phosphodiesterase inhibitor, IBMX (0.2 mmol/l). Stimulation of muscarinic receptors with CCh increased islet cAMP levels in a concentration-dependent manner (Fig. 1). Maximal stimulation of AC occurred between 200 and 500 ␮mol/l CCh with a 4.5-fold increase in cAMP, although significant stimulation was observed at 10 ␮mol/l CCh with a 2.7-fold increase in cAMP (Fig. 1). CCh stimulation increased cAMP levels maximally within 5 min and levels remained significantly higher than control values after 20 min (Fig. 2). The presence of the muscarinic receptor antagonist atropine (50 ␮mol/l) did not significantly change basal cAMP levels compared with control values (data not shown) but inhibited the CCh (200 ␮mol/l)-induced increase in cAMP by 81 ⫾ 17% (P ⬍ 0.001), such that there was no significant change compared with control values. The specificity of the cAMP response was tested in

RESEARCH DESIGN AND METHODS Materials. Adenosine 3⬘,5⬘-cyclic phosphoric acid 2⬘-O-succinyl [125I]iodotyrosine methyl ester was from New England Nuclear (Boston, MA). GLP-1 fragment 7-36 amide, cAMP, insulin-free bovine serum albumin (BSA), 3-isobutyl-1-methylxanthine (IBMX), carbamylcholine chloride (CCh), cholecystokinin fragment 26-33 sulfated amide (CCK), phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-didecanoate (PDD), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-1 (ODQ), forskolin and phorbol 12-myristate 13-acetate (PMA) were from Sigma Chemical (St. Louis, MO). Fetal bovine serum was from Atlanta Biologicals (Norcross, GA). Collagenase type P was from Roche Molecular Biochemicals (Indianapolis, IN). CMRL-1066 medium was from GIBCO/Life Technologies (Grand Island, NY). Bisindolylmaleimide (GF 109203X) and bisindolylmaleimide XI HCl (Ro-32-0432) were from Calbiochem-Novabiochem (San Diego, CA). 1,2-Bis(o-amino-5-fluorophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid tetraacetoxymethyl ester (BAPTA/AM) was from Molecular Probes (Eugene, OR). Antibody to cAMP was a gift from Dr. David L. Garbers (Howard Hughes Medical Institute, Dallas, TX). All other chemicals were reagent grade. Isolation and culture of rat islets. Pancreatic islets were isolated using the collagenase method from pancreases excised from decapitated male Harlan Sprague-Dawley rats (225–300 g). All animal procedures were approved by the Institutional Animal Care and Use Committee. Isolated islets were either used immediately as freshly isolated islets or were cultured for 20 h in CMRL-1066 culture medium containing 5.5 mmol/l glucose, 2 mmol/l glutamine, 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 ␮g/ml), as described previously (26). The islets were cultured in a humidified (95%) atmosphere of 5% CO2/95% air at 35°C. Levels of cAMP in freshly isolated versus cultured islets were not significantly different under basal or stimulated conditions. cAMP production. For each experiment, islets (10 islets/sample) were washed three times in a Krebs-Ringer bicarbonate HEPES (KRBH) buffer (pH 7.4) containing 2.8 mmol/l glucose (basal) and 0.01% BSA, as described previously (26). Both fresh and cultured islets were incubated in KRBH buffer for 30 – 60 min at 37°C in an atmosphere of 95% O2/5% CO2 in a shaking water bath. Then, the islets were preincubated at a final volume of 0.1 ml KRBH buffer with various agents: BAPTA/AM, 20 min; GF 109203X and Ro-32-0432, 2506

FIG. 2. Time course for CCh-stimulated cAMP generation. Islets were incubated in KRBH buffer containing 2.8 mmol/l glucose (C) and 0.2 mmol/l IBMX in the absence (F) or presence (Œ) of CCh (200 ␮mol/l). cAMP levels were determined by RIA at the times indicated. Values are means ⴞ SE for six independent experimental determinations. *P < 0.02, and **P < 0.001 vs. C, as determined by one-way ANOVA and multiple-comparison test. DIABETES, VOL. 50, NOVEMBER 2001

Y. TIAN AND S.G. LAYCHOCK

TABLE 1 Effects of forskolin, PKC inhibitors, and Ca2⫹-lowering agents on cAMP generation Treatments Basal GF 109203X Ro-32-0432 Forskolin Forskolin plus GF 109203X Forskolin plus Ro-32-0432 Forskolin plus BAPTA/AM Ca2⫹-free Basal Forskolin

FIG. 3. PDBu-, CCh-, and CCK-stimulated cAMP production. Islets were incubated for 20 min in KRBH buffer containing 2.8 mmol/l glucose (C), PDD (1 ␮mol/l), PDBu (1 ␮mol/l), CCh (500 ␮mol/l) (^), and CCK (400 nmol/l) ( ), as indicated, and cAMP levels were determined. Values are means ⴞ SE for the number of independent experiments shown at the base of each bar. *P < 0.001 vs. C, and other P values were determined by one-way ANOVA and multiple-comparison test.

islets in two ways. In the absence of IBMX, CCh failed to significantly increase cAMP levels (3.3 ⫾ 0.3 fmol cAMP/10 islets) compared with basal values (1.3 ⫾ 1.3 fmol cAMP/ 10 islets) after a 20-min stimulation. Moreover, in the presence of a specific guanylyl cyclase inhibitor, ODQ (10 ␮mol/l), islet cAMP generation in response to CCh (727 ⫾ 124 fmol cAMP/10 islets) was not significantly different (P ⬎ 0.05) from levels with CCh treatment alone (629 ⫾ 103 fmol cAMP/10 islets). Thus, guanosine 3⬘,5⬘-cyclic monophosphate did not contribute to the perceived effects of CCh on cAMP generation. CCK is an intestinal hormone that stimulates receptors on ␤-cells and regulates insulin secretion (28). Similar to CCh, CCK (400 nmol/l) increased cAMP levels 4.1-fold compared with control values (Fig. 3). Effects of phorbol ester on cAMP generation. The effects of the PKC-activating phorbol ester, PDBu, on islet cAMP levels were determined. PDBu increased cAMP levels 6.2-fold compared with control (Fig. 3), whereas PDD, a biologically inactive phorbol ester, did not significantly affect cAMP generation. The combination of CCh and PDBu did not potentiate cAMP accumulation compared with PDBu alone, although a significant increase in cAMP generation was observed compared with CCh (Fig. 3). A combined stimulus with PDBu and CCK increased cAMP levels as much as 6.3-fold, but this was not different from the PDBu response alone (Fig. 3). Thus, neither CCh nor CCK effects were additive with PDBu. The relative efficacy of PDBu was compared with the direct-acting AC activator forskolin and the GLP that activates receptor-mediated GTP-binding protein G␣s and AC. Forskolin (10 ␮mol/l) increased islet cAMP levels up to 58-fold (Table 1), and GLP (0.1 ␮mol/l) increased cAMP levels almost 10-fold (2,694 ⫾ 827 fmol cAMP/10 islets) DIABETES, VOL. 50, NOVEMBER 2001

cAMP levels (fmol/10 islets)

n

144 ⫾ 133 157 ⫾ 53 149 ⫾ 88 8,400 ⫾ 288* 7,520 ⫾ 1,153* 7,300 ⫾ 663* 6,373 ⫾ 1,051*†

5 4 4 5 5 5 3

155 ⫾ 47 8,625 ⫾ 1,261*

4 3

Islets were incubated in KRBH buffer containing IBMX (0.2 mmol/l) in the absence (basal) or presence of forskolin (10 ␮mol/l), GF 109203X (0.5 ␮mol/l), Ro-32-0432 (0.2 ␮mol/l), or BAPTA/AM (10 ␮mol/l) for 20 min. Islets were also incubated in calcium-free buffer with 100 ␮mol/l EGTA and the absence or presence of the agents indicated. Values are means ⫾ SE for the number of independent determinations indicated (n). Significant differences were determined by one-way ANOVA and Student-Newman-Keuls multiplecomparison test. *P ⬍ 0.001 vs. basal; †P ⬍ 0.02 vs. forskolin in the presence of extracellular Ca2⫹.

(P ⬍ 0.001) after a 20-min incubation. Thus, PDBu did not maximally activate islet AC compared with these stimuli. Glucose is the primary stimulus for insulin secretion in islets and has been previously reported to increase cAMP levels in islets (2). In the present study, glucose (20 mmol/l) stimulation increased cAMP levels 2.7-fold. When glucose (20 mmol/l) was combined with PDBu (1 ␮mol/l), there was a 6.7-fold increase in cAMP (Fig. 4). However, the glucose and PDBu responses were not additive and

FIG. 4. PDBu- and glucose-stimulated cAMP production. Islets were incubated for 20 min in KRBH buffer containing 2.8 mmol/l glucose (C) in the absence or presence of PDBu (1 ␮mol/l) or incubated in 20 mmol/l glucose (G) in the absence or presence of PDBu (1 ␮mol/l), and cAMP values were determined. Values are means ⴞ SE for the number of independent experiments shown at the base of each bar. *P < 0.001 vs. C, and other P values were determined by one-way ANOVA and multiple-comparison test. 2507


were not significantly higher than cAMP levels with PDBu stimulation alone. A role for PKC stimulation in islet cAMP generation was confirmed with the specific PKC inhibitors GF 109203X and Ro-32-0432. The presence of GF 109203X and Ro-320432 resulted in an ⬃60% decrease in PDBu-induced cAMP levels (Fig. 5A). Similarly, both GF 109203X and Ro-320432 decreased CCh-stimulated cAMP levels by ⬃50% (Fig. 5B). GF 109203X also inhibited CCK-stimulated cAMP generation by ⬃30%, but Ro-32-0432 completely inhibited the CCK response (Fig. 5B). Neither GF 109203X nor Ro32-0432 affected basal cAMP levels or forskolin-stimulated cAMP levels (Table 1). A role for Ca2ⴙ in the effects of CCh, CCK, and phorbol ester. To determine whether Ca2⫹ mediated the activation of AC, islets were treated with an intracellular Ca2⫹ chelator, BAPTA/AM (29). In BAPTA/AM-treated islets, the cAMP responses to CCh and CCK were inhibited by 50 and 45%, respectively, although there was no significant effect on basal cAMP levels (Fig. 6). BAPTA/AM also significantly reduced forskolin-stimulated cAMP production by ⬃25% (Table 1). In contrast, PDBu responses were not significantly affected by the presence of BAPTA/AM (Fig. 6). Additional experiments were performed in the absence of extracellular Ca2⫹ and the presence of EGTA (100 ␮mol/l) to chelate residual Ca2⫹. Under Ca2⫹-free conditions, cAMP generation in response to PDBu, CCh, and CCK was significantly inhibited by ⬃50% for each agent (Fig. 7). Forskolinstimulated cAMP production, however, was not significantly affected by Ca2⫹-free conditions (Table 1). AC activation in the absence of extracellular Ca2⫹ and the presence of EGTA and BAPTA/AM was determined. Even with this stringent Ca2⫹ depletion, CCh significantly increased cAMP levels by 11-fold compared with the effects of CCh in the absence of BAPTA/AM (Fig. 8A). Likewise, with Ca2⫹ depletion, CCK significantly increased cAMP levels by 5.4-fold compared with effects in the absence of BAPTA/AM (Fig. 8B). To determine whether PKC played a role in AC activation under stringent Ca2⫹depleted conditions, PKC inhibitors were included in the islet treatment. In the presence of CCh plus GF 109203X or Ro-32-0342, cAMP levels were decreased by 75 and 51%, respectively, compared with Ca2⫹-depleted islets in the absence of inhibitors (Fig. 8A). The PKC inhibitors also decreased CCK-induced cAMP production to a similar extent in Ca2⫹-depleted islets (Fig. 8B). Effects of PKC downregulation on AC activity. To determine whether PKC downregulation modulated the cAMP response to CCh or CCK, islets were cultured in the presence of the phorbol ester PMA (500 nmol/l) for 20 h to induce PKC downregulation (30). In PMA-pretreated islets, PMA, CCh, and CCK each failed to increase cAMP production above basal levels, whereas each of the stimuli significantly increased cAMP production in untreated islets (Fig. 9A). Moreover, islets that were pretreated with PMA showed a complete inhibition in AC activity in response to PMA, CCh, and CCK compared with similarly stimulated paired control islets (Fig. 9A, shaded versus open bars). Under stringent Ca2⫹ depletion in the absence of extracellular Ca2⫹ and in the presence of EGTA and BAPTA/AM, PMA-pretreated islet cAMP production in 2508

FIG. 5. PKC-mediated cAMP production. Islets were incubated in KRBH buffer in the absence (C) or presence of PDBu (1 ␮mol/l) (A), CCh (200 ␮mol/l) (B), CCK (400 nmol/l) (C), and GF 109203X (GF) (0.5 ␮mol/l) or Ro-32-0432 (Ro) (0.2 ␮mol/l), as indicated. Values are means ⴞ SE. *P < 0.01 vs. C values for each group, and other P values were determined by one-way ANOVA and multiple-comparison test.

response to PMA, CCh, and CCK was significantly inhibited by 41, 59, and 72%, respectively, compared with similarly stimulated control islets that were not downregulated (Fig. 9B). In comparison with basal values, PMA but not CCh or CCK evoked a small stimulation of cAMP DIABETES, VOL. 50, NOVEMBER 2001


FIG. 6. Intracellular Ca2ⴙ-dependent cAMP production. Islets were incubated for 20 min in KRBH buffer in the absence or presence of BAPTA/AM (10 ␮mol/l), PDBu (1 ␮mol/l) (_), CCh (200 ␮mol/l) (^), or CCK (400 nmol/l) ( ), as indicated, and cAMP production was determined. Values are means ⴞ SE for the number of independent experiments shown at the base of each bar. *P < 0.001 vs. basal values for control islets, and other P values were determined by one-way ANOVA and multiple-comparison test.

In the present study, CCh evoked a concentrationdependent stimulation of cAMP production in islets in the presence of a low substimulatory concentration of glucose (2.8 mmol/l). The specificity of the muscarinic receptor response was demonstrated by the inhibitory effect of atropine. Concentrations of acetylcholine (10⫺6 ⫺ 10⫺3 mol/l) similar to those for CCh in the present study also increased intracellular Ca2⫹ in rat islet ␤-cells at a low concentration of glucose (4.4 mmol/l) (46), suggesting that intracellular Ca2⫹ levels modulated CCh-induced AC activity. Moreover, CCK, a gut hormone that stimulates PLC activation (47) and elevates ␤-cell Ca2⫹ levels (46), also stimulated AC activity in rat islets. Glucose promotes insulin secretion by increasing Ca2⫹ influx (33) and Ca2⫹ mobilization (2,37). The present study confirms previous reports that glucose stimulates AC activity (1,27). Thus, CCh, CCK, and glucose increase cellular Ca2⫹ levels that together with calmodulin could modulate activity of AC type 3 (Fig. 10). Several PKC isoforms are expressed in rat islets (48–50), including the conventional Ca2⫹-dependent/diacylglycerolactivated isoforms ␣ and ␤, the novel Ca2⫹-independent/ diacylglycerol-activated isoforms ␦ and ⑀ (48), and the atypical Ca2⫹-independent/diacylglycerol-insensitive isoforms ␨ and ␫ (50). PKC isoform sensitivity to phorbol esters parallels their diacylglycerol sensitivity (51). Since glucose (52–54), CCh (52,55), and CCK (56) increase islet and/or ␤-cell PKC activation, the effects of a PKC-activating phorbol ester were investigated for effects on AC activity. PDBu and PMA stimulated islet cAMP generation, whereas a biologically inactive phorbol ester, PDD, did not. However, no potentiation or additivity was evident

production in downregulated islets (Fig. 9B). In contrast, PMA, CCh, and CCK significantly stimulated cAMP production by 400 –500% above basal levels under Ca2⫹depleted conditions in control islets (Fig. 9B). DISCUSSION

AC production of cAMP in ␤-cells has been attributed to glucose-evoking changes in ATP levels and Ca2⫹ mobilization/calmodulin activation (31–34). Indeed, Ca2⫹-stimulated AC activity has been reported for plasma membrane preparations of rat insulinoma cells (35). Stimulation of muscarinic receptors by CCh activates PLC with the generation of inositol 1,4,5-trisphosphate that mobilizes Ca2⫹ in rat islets (36,37) and diacylglycerol that stimulates PKC activation, while voltage-dependent Ca2⫹ channels (VDCC) (32,38,39) and store-operated channels (40) contribute to Ca2⫹ influx. Yet, there is controversy as to whether muscarinic receptor stimulation regulates islet AC activity. An early report described rat islet AC in homogenates as being sensitive to acetylcholine (0.1 mmol/l) (41), but later studies reported that muscarinic receptor stimulation by acetylcholine or CCh in intact rat islets did not affect cAMP production (32,42,43). However, these latter studies were performed in the absence of a phosphodiesterase inhibitor that is required to detect changes in islet cAMP levels. A lack of effect of acetylcholine on ob/ob mouse islet cAMP levels has also been reported (44); however, ob/ob mouse islets may be deficient in AC isoforms (45). DIABETES, VOL. 50, NOVEMBER 2001

FIG. 7. Extracellular Ca2ⴙ-dependent cAMP production. Islets were incubated in KRBH buffer containing 2.8 mmol/l glucose for 1 h. Then, the Ca2ⴙ-free islets were washed three times in Ca2ⴙ-free KRBH buffer containing 100 ␮mol/l EGTA. cAMP production was determined after a 20-min incubation in Ca2ⴙ-containing (ⴙ) or Ca2ⴙ-free (-) buffer, as indicated, in absence (C) (ⵧ)or presence of PDBu (1 ␮mol/l) (^), CCh (200 ␮mol/l) ( ), or CCK (400 nmol/l) (_). Values are means ⴞ SE for the number of independent experiments shown at the base of each bar; Ca2ⴙ-depleted C, n ⴝ 7. *P < 0.001 vs. Ca2ⴙ-containing C; ␺ P < 0.01 vs. Ca2ⴙ-depleted C; and other P values were determined by one-way ANOVA and multiple-comparison test. 2509


FIG. 8. cAMP production in Ca2ⴙ-depleted islets. Islets were incubated in KRBH buffer containing 2.8 mmol/l glucose for 1 h. Then, the islets were washed three times in Ca2ⴙ-free KRBH buffer containing 100 ␮mol/l EGTA, and cAMP production was determined after a 20-min incubation in this buffer. Islets were incubated in the absence (C) or presence of CCh (200 ␮mol/l) (A) or CCK (400 nmol/l) (B), and BAPTA/AM (BAP) (10 ␮mol/l), GF 109203X (GF) (0.5 ␮mol/l), and Ro-32-0432 (Ro) (0.2 ␮mol/l), as indicated. Values are means ⴞ SE for the number of independent experiments shown at the base of each bar; in A, C was n ⴝ 11. *P < 0.05 vs. C, and other P values were determined by one-way ANOVA and multiple-comparison test.

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among CCh, CCK, glucose, and PDBu in terms of AC activation. The results suggest that PDBu evoked a maximal PKC activation response—a mechanism shared by CCh, CCK, and glucose. However, the PKC-mediated AC activation was not maximal in terms of the islet cell capacity to generate cAMP. Both forskolin and GLP evoked larger increases in cAMP production than did PDBu, CCh, or CCK, suggesting that not all AC isoforms were maximally activated by the latter agents alone or in combination (Fig. 10). Forskolin is characterized as directly activating all AC isoforms, whereas GLP is receptor mediated and activates all isoforms through Gs regulation (13). It is probable that PKC stimulated AC types 2, 5, and 7 in islets (Fig. 10). PKC mediation of the effects of PDBu on AC was confirmed by two specific PKC inhibitors; GF 109203X inhibits PKC-␣, -␤I, -␤II, -␥, -␦, and -⑀ (57), whereas Ro-32-0432 inhibits PKC-␣, -␤1, and -⑀ (58). At the concentrations used in this study, the inhibitors are specific for PKC (59,60). GF 109203X may inhibit PKA (Ki ⫽ 2 ␮mol/l) and has been reported to affect intracellular Ca2⫹ stores at a concentration 10 times higher than that used in the present study (61). In islets, both PKC inhibitors reduced PDBu-, CCh-, and CCK-stimulated cAMP production. CCh-stimulated cAMP levels were decreased by half in the presence of either inhibitor, suggesting that PKC activation was partly responsible for mediating CCh-stimulated cAMP production. Similarly, GF 109203X partially inhibited CCK-stimulated cAMP production. However, Ro-32-0342 completely prevented CCK-stimulated increases in cAMP. The reason for the latter effect is not clear. Ro-32-0342 may be more potent at the possibly lower stimulation caused by nanomoles of CCK versus micromoles of CCh. However, the high concentration of CCh may induce other effects in the ␤-cell, such as changes in ion flux (38) that potentially affect AC activity and are not mediated by PKC. Neither GF 109203X nor Ro-32-0342 affected AC activity under basal conditions. Further evidence that PKC mediates the activity of AC in islets came from experiments in which PMA pretreatment was used to downregulate PKC. In PMA-pretreated cells, neither PMA, CCh, nor CCK evoked a change in AC activity, as compared with the robust increase in cAMP observed with these agents in the absence of PKC downregulation. Thus, these data support the hypothesis that PKC activation mediates cholinergic and CCK stimulation of AC activation and cAMP generation in rat islets. The role of extracellular Ca2⫹ on AC activation was studied by incubating islets in a Ca2⫹-free KRBH buffer with EGTA to chelate Ca2⫹. PDBu-, CCh-, and CCK-stimulated cAMP generation was significantly inhibited under Ca2⫹-free conditions, suggesting that Ca2⫹ influx plays an important role in AC activation. CCh promotes Ca2⫹ influx through VDCC (39) and by capacitative Ca2⫹ entry through store-operated channels (40,62). CCK-induced cytoplasmic Ca2⫹ mobilization in rat islet cells (63) may mediate effects on AC activation. However, the present results also suggest that Ca2⫹ influx—perhaps through capacitative Ca2⫹ entry—is a component of islet CCK effects. The dependence of AC activity on intracellular Ca2⫹ was studied by inclusion of an intracellular Ca2⫹ chelator, BAPTA/AM. The presence of BAPTA/AM decreased CChDIABETES, VOL. 50, NOVEMBER 2001


ly, glucose augments insulin release when PKA and PKC are activated simultaneously in the absence of changes in intracellular Ca2⫹ with BAPTA (29). The question of whether AC activity would be further affected by depletion of both extracellular and intracellular Ca2⫹ was investigated. Unexpectedly, stringent Ca2⫹ depletion resulted in increased CCh- and CCK-stimulated cAMP production compared with responses in Ca2⫹-free buffer alone. PKC may play a role in this stimulatory response, since the increase in cAMP levels was reversed by the inclusion of PKC inhibitors. Similarly, PKC downregulation also markedly reduced AC responses to CCh and CCK under Ca2⫹-depleted conditions. There was a small AC stimulation above basal by PMA in PKC-downregulated Ca2⫹-depleted islets that may be due to residual PKC activity. The relationship between PKC activity and Ca2⫹ depletion under these conditions is not entirely understood. It is likely that some islet PKC isoforms are active under stringent Ca2⫹-depleted conditions (48,50), such as novel PKC-␦ and PKC-⑀. Ca2⫹-independent novel PKCs have been implicated in upregulation of AC activity in macrophages (22), and AC activation in erythroid progenitor cells (64). Alternatively, PKC can phosphorylate ␤␥ subunits and increase activation of type 2 AC (65). In islets, PKC isoform effects on AC may become apparent in the absence of Ca2⫹-supported enzyme activity. CCh or CCK stimulation under stringent Ca2⫹ depletion also suggests that a Ca2⫹-related AC inhibition was relieved. Whether or not there is a Ca2⫹-mediated inhibitory component (66,67) to islet AC regulation is not known. However, if AC activity were relieved from an endogenous inhibitory effect of Ca2⫹, it is possible that PKC effects might become more pronounced, as in the present study. In conclusion, this is the first study to characterize a role

FIG. 9. PKC downregulation and AC activity in islets. Islets were cultured for 20 h in CMRL-1066 medium containing 5.5 mmol/l glucose and the absence (o) or presence (䡺) of PMA (500 nmol/l). A: Islets were washed and incubated in KRBH buffer at 2.8 mmol/l glucose in the absence (basal) or presence of PMA (500 nmol/l), CCh (200 ␮mol/l), or CCK (400 nmol/l) for 20 min for determination of cAMP levels. B: Ca2ⴙ-depleted islets were washed three times in Ca2ⴙ-free KRBH buffer containing 100 ␮mol/l EGTA. cAMP levels were determined in islets incubated for 20 min in Ca2ⴙ-free buffer containing BAPTA/AM (10 ␮mol/l) in the absence (basal) or presence of PMA (500 nmol/l), CCh (200 ␮mol/l), or CCK (400 nmol/l). Values are means ⴞ SE for three or four independent experiments. *P < 0.001, and ŒP < 0.05 vs. similarly treated basal values, and other P values were determined by one-way ANOVA and multiple-comparison test.

and CCK-stimulated cAMP levels by ⬃50%, suggesting that intracellular Ca2⫹ was partially responsible for the receptor agonist stimulatory effects. However, BAPTA/AM did not affect the response to PDBu, suggesting that phorbol ester and PKC effects on AC activation were not dependent on increases in intracellular Ca2⫹ levels. InterestingDIABETES, VOL. 50, NOVEMBER 2001

FIG. 10. Model of AC regulation in the ␤-cell. Stimuli such as CCh, CCK, and glucose stimulate activation of PLC that induces hydrolysis of phosphatidylinositol bisphosphate (PIP2) to produce inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates receptors on the endoplasmic reticulum to release sequestered Ca2ⴙ; this, in turn, can induce Ca2ⴙ influx through store-operated channels (SOC). Depolarization of the cell also increases Ca2ⴙ influx through VDCC. The increase in intracellular Ca2ⴙ activates calmodulin (CaM) modulated AC type 3 (AC3) to produce cAMP. DAG production activates PKC isoforms that phosphorylate and stimulate AC types 2, 5, and 7 to form cAMP. Phorbol ester directly activates PKC to stimulate AC activity. Forskolin and G-protein coupled receptor stimuli (GPCR) can activate each of the known AC isoforms. Stimulatory effects are indicated by a ‘ⴙ’. 2511


for PKC in AC activation in islets. A model for AC regulation in the ␤-cell (Fig. 10) shows CCh, CCK, and glucose stimulation activating PLC, with a resulting increase in inositol 1,4,5-trisphosphate–mediated Ca2⫹ mobilization and Ca2⫹ influx through either VDCC or store-operated channels. Ca2⫹/calmodulin activates AC type 3. Both intracellular Ca2⫹ mobilization and extracellular Ca2⫹ influx are important components of the islet AC response. The alternative diacylglycerol-mediated pathway, like phorbol esters, activates PKC, which then potentially activates AC types 2, 5, and 7. Forskolin and G-protein coupled receptor stimuli can stimulate each of the cyclase isozymes. A previous report characterized glucose stimulation of AC in ␤-cells as dependent on glucagon secreted from ␣-cells (68). In the present study, effects of CCh, CCK, or PDBu on glucagon secretion and stimulation of G-protein– coupled receptors (Fig. 10) cannot be ruled out as a mechanism contributing to the changes in cAMP production in islets. Moreover, the cAMP levels observed could be generated in any or all of the heterogeneous cell types that compose the pancreatic islet. However, the results imply that cholinergic innervation and gastrointestinal CCK may play an important role in the regulation of cAMP production, as well as insulin secretion, in islets of Langerhans. ACKNOWLEDGMENTS

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