Biomedical subjects

J W Leitner

Publications and source records attributed to J W Leitner.

At least 37 records · Page 2Linked to original sources

PubMed1995

Insulin inhibits nuclear phosphatase activity: requirement for the C-terminal domain of the insulin receptor.

Insulin's interaction with its receptor initiates a multitude of cellular effects on metabolism, growth, and differentiation. We recently described an insulin-mediated inhibition of nuclear protein phosphatase 2A (PP-2A), which is associated with an increase in phosphorylation of the transcription factor cAMP response element-binding protein. To clarify the role of nuclear PP-2A inhibition in the insulin signaling cascade, we examined the regulation of this phosphatase activity by insulin in Rat-1 fibroblasts overexpressing normal (HIRc) or mutant human insulin receptors (delta CT cells, deletion of a 43-amino acid C-terminal domain). The delta CT cells represent an excellent model of impaired metabolic and intact mitogenic action of insulin. Insulin inhibited nuclear PP-2A activity and enhanced cAMP response element-binding protein phosphorylation in HIRc cells, but not in delta CT cells. The delta CT cells exhibited normal ras activation and blunted mitogen-activating protein kinase phosphorylation and activation in response to insulin (16-fold in HIRc cells vs. 3-fold in delta CT cells), indicating that the mitogen-activating protein kinase pathway is important for the regulation of nuclear PP-2A activity by insulin. We conclude that insulin inhibits nuclear PP-2A activity, and that the carboxy-terminal domain of the insulin receptor is important for this effect.

Activating Transcription Factor 1↗

PubMed1994

The signaling pathway coupling epidermal growth factor receptors to activation of p21ras.

Epidermal growth factor (EGF) treatment causes autophosphorylation of the epidermal growth factor receptor (EGFR) leading to increased guanine nucleotide exchange factor (GEF; Sos) activity and enhanced formation of p21ras-GTP. The connection of the EGFR to p21ras activation can occur through binding of Grb2.Sos complexes to the EGFR or through the adaptor protein Shc via EGFR.Shc.Grb2.Sos multimeric complexes. Therefore, we investigated the importance of Shc in coupling the EGFR to activation of ras GEF (Sos). EGF treatment led to rapid tyrosine phosphorylation of Shc. Although phosphorylated EGFR can bind to both Shc and Grb2, the predominant linkage was observed between EGFR and Shc. Similarly, more Grb2 was associated with Shc than with EGFR after EGF stimulation. Immunoprecipitation of Shc from EGF-stimulated cells removed almost all EGFR-associated Grb2. Furthermore, immunodepletion of Shc proteins from membrane fractions of EGF-stimulated cells removed 93% of the ras GEF activity, whereas, precipitation of EGFR had only a small effect on ras GEF activity. These data indicate that coupling to Shc provides the major pathway linking activated EGFRs to Grb2.Sos and stimulation of the p21ras pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Shc is the predominant signaling molecule coupling insulin receptors to activation of guanine nucleotide releasing factor and p21ras-GTP formation.

Insulin stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and She in Rat1 fibroblasts overexpressing wild type insulin receptors. We investigated the relative role of IRS-1 and She in insulin activation of guanine nucleotide releasing factor (GNRF) and p21ras-GTP formation. The time course of insulin-stimulated tyrosine phosphorylation of IRS-1 was rapid, whereas Shc phosphorylation was relatively slow. Growth factor receptor bound protein-2 (Grb2) associated with IRS-1 rapidly and gradually dissociated after 5 min, whereas Grb2 association with Shc was slower and reached a maximum at 10 min after insulin stimulation. Thus, the kinetics of Grb2 association with IRS-1 and She corresponded closely to the time course of tyrosine phosphorylation of IRS-1 and Shc, respectively. Importantly, 3-13-fold more Grb2 was associated with Shc than with IRS-1. In addition, the kinetics of insulin-stimulated GNRF activity and p21ras-GTP formation corresponded more closely to the time course of Shc phosphorylation than to the kinetics of IRS-1 phosphorylation. Furthermore, immunoprecipitation of Shc proteins from cell lysates of insulin-stimulated cells removed 67% of the GNRF activity, whereas precipitation of IRS-1 had a negligible effect on GNRF activity. Thus, although both IRS-1 and Shc associate with Grb2, the current results indicate that Shc plays a more important role than IRS-1 in insulin stimulation of GNRF activity and subsequent p21ras-GTP formation.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Insulin and insulin-like growth factor-I signal transduction requires p21ras.

We have investigated the role of cellular p21ras protein in insulin and insulin-like growth factor-I (IGF-I) signaling pathways. Insulin stimulation increased Ras-GTP formation in Rat-1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), far greater than in parental Rat-1 fibroblasts, indicating that competent insulin receptors mediate this response. Cellular microinjection of a dominant-negative mutant p21ras protein (N17 ras) or anti-p21ras monoclonal antibody (Y13-259) into HIRc-B cells reduced insulin- and IGF-I-stimulated DNA synthesis by 75-90%. Insulin-induced c-fos protein expression was also inhibited by 74%. Microinjection of oncogenic p21ras (T-24 ras) into HIRc-B cells activated the mitogenic pathway, and coinjection of N17 ras and T-24 ras showed that oncogenic p21ras rescued the cells from the N17 ras blockade. This later finding indicates that T-24 ras acts downstream of N17 ras. In conclusion, 1) microinjection of a dominant interferring ras mutant into quiescent cells abrogated subsequent insulin and IGF-I mitogenic signaling; 2) oncogenic ras protein rescued cells from the N17 ras blockade, indicating that T24 ras action is downstream of the site of N17 inhibition; and 3) p21ras is an intermediate signaling molecule in the insulin/IGF-I signal transduction pathway and is required for gene expression and DNA synthesis.

Animals↗

PubMed1994

Insulin and epidermal growth factor influence guanine nucleotide-releasing factor by distinct mechanisms.

Both insulin and epidermal growth factor (EGF) significantly increased the activation of p21Ras in Rat-1 fibroblasts transfected with human insulin receptors. As both growth factors have been shown to stimulate guanine nucleotide exchange on p21Ras in permeabilized cells, we examined their effects on total cellular guanine nucleotide exchange factor (GEF) activity in a direct assay, using the rate of dissociation of [3H]GDP from p21Ras as a measure of GEF activity. Insulin increased GEF activity in a time-dependent manner, whereas EGF had no effect on GEF activity at any time point studied. To assess whether EGF stimulates guanine nucleotide exchange by recruiting Grb-2-Sos complexes to the plasma membrane, we measured GEF activity in both the cytosol and the plasma membrane fractions of the insulin- and EGF-treated cells. Insulin increased GEF activity in both fractions (48 +/- 12% [3H]GDP released vs. 24 +/- 6% in control plasma membranes, and 65 +/- 13% vs. 13 +/- 4% in control cytosolic fractions), whereas EGF enhanced only the plasma membrane-associated activity (43 +/- 12% of [3H]GDP release in the plasma membrane fraction and 10 +/- 2% in the cytosol). Western blotting of the subcellular fractions with Grb-2 and Sos antibodies revealed translocation of these elements to the plasma membrane after stimulation of cells with either insulin or EGF. Thus, whereas insulin stimulates guanine nucleotide exchange on Ras by both translocating Grb-2-Sos complexes to the plasma membrane and increasing the GEF activity of Sos, EGF does so by causing a translocation of Grb-2-Sos without increasing GEF activity.

Animals↗

PubMed1993

Insulin activates p21Ras and guanine nucleotide releasing factor in cells expressing wild type and mutant insulin receptors.

Insulin stimulates the formation of p21RasGTP in Rat-1 fibroblasts overexpressing wild type (HIRc) or mutant (delta CT and Y/F2) insulin receptors. Maximal insulin effect was observed at 7 min in Y/F2 cells, at 10 min in delta CT cells, and at 15 min in HIRc cells. Mutant insulin receptors which display enhanced mitogenic signaling properties stimulated p21Ras.GTP formation to a greater extent than wild type receptors. The amount of p21Ras was not affected by insulin (Western blotting). Tyrosine kinase inhibitor, Lavendustin A (10 nM), completely prevented insulin-induced activation of p21Ras in all cell lines. Insulin did not lead to GAP phosphorylation, or a change in cellular GAP activity, but did result in marked stimulation of Ras guanine nucleotide releasing factor (GRF) activity (by 48% in HIRc, 71% in delta CT, and 120% in Y/F2 cells). These results indicated that p21Ras.GTP is an important signaling molecule in insulin's mitogenic pathway, but may not participate in metabolic signaling and that insulin's stimulatory effects on p21Ras.GTP formation are mediated through Ras GRF.

Animals↗

PubMed1988

Cyclosporin-induced inhibition of insulin release. Possible role of voltage-dependent calcium transport channels.

The exposure of normal pancreatic islets to cyclosporin-A (1 microgram/ml) for 24 hr resulted in significant inhibition of glucose-induced (16.7 mM) insulin release from 197 +/- 14 microU/10 islets/15 min (control) to 103 +/- 14 microU/10 islets/15 min (Cy-A-treated islets; P less than 0.001). Cy-A did not alter insulin release in the presence of non-stimulatory (1.7 mM) or submaximally effective glucose concentrations (9.2 mM). In parallel experiments, Cy-A reduced glucose-stimulated increases in cytosolic free calcium concentrations, [Ca2+]i (217 +/- 15 nM without and 137 +/- 3 nM with Cy-A in the presence of 16.7 mM glucose, P less than 0.01). To better define the site of Cy-A action, we studied its effect on insulin release and increases in [Ca2+]i induced by either K+ (50 mM), which promotes Ca2+ influx via voltage-dependent Ca2+ channels, or by forskolin (20 microM), dibutyryl cyclic AMP (1 mM) or arachidonic acid (49 microM), all of which stimulate mobilization of intracellular Ca2+ stores. Cy-A significantly inhibited K+-induced changes (203 +/- 13 nM without and 77 +/- 6 nM with Cy-A, respectively, P less than 0.001), but not those induced by forskolin, dibutyryl cyclic AMP or arachidonic acid. These observations suggest that Cy-A inhibits insulin release by interfering with Ca2+ influx via voltage-dependent calcium channels.

Animals↗

PubMed1988

Insulin and glucose modulate protein kinase C activity in rat adipocytes.

In the presence of 1 mM glucose, insulin (10 ng/ml) increases both catalytic and receptor-binding properties of adipocyte cytosolic protein kinase C (PKC). Preincubation of adipocytes with 10 mM glucose raises basal PKC catalytic activity and prevents further stimulation of this enzyme by insulin. The effect of hyperglycemia is likely to be mediated by direct conversion of glucose into diacylglycerol. Thus, an incorporation of 14C-glucose into diacylglycerol is enhanced 10-fold in the presence of 10 mM glucose. These observations indicate that, in normal adipocytes, both insulin and glucose activate PKC; hyperglycemia eliminates the ability of insulin to stimulate this enzyme, thereby interfering with insulin action.

Adipose Tissue↗

PubMed1988

Glyburide increases cytosolic-free calcium concentrations in normal rat pancreatic islet cells.

We have attempted to delineate the effect of glyburide on the regulation of cytosolic-free calcium concentrations, [Ca2+]i, in normal rat pancreatic islet cells. In the presence of extracellular calcium (1 mmol/L), glyburide increased [Ca2+]i from 70 nmol/L to 260 nmol/L in a dose-dependent manner. The maximal effect was seen at a concentration of 2 mumol/L with half-maximal stimulation observed at .25 mumol/L. The effect of glyburide (.25 mumol/L) was inhibited 90% by the calcium channel blocker, verapamil (30 mumol/L). At a maximally effective concentration of glyburide (2 mumol/L), the inhibitory effect of verapamil was only 17%. In the absence of extracellular calcium, glyburide increased [Ca2+]i from 55 nmol/L to 107 nmol/L, indicating its ability to mobilize intracellular calcium stores. These results correlated well with the ability of glyburide (2 mumol/L) to stimulate insulin secretion both in the presence (from 38 +/- 5 mumol/L/10 islets to 131 +/- 28 microU/10 islets) and in the absence (from 49 +/- 4 microU/10 islets to 93 +/- 7 microU/10 islets) of extracellular Ca2+. The present observations suggest that glyburide promotes calcium influx via voltage-dependent calcium channels and may mobilize intracellular calcium stores.

Animals↗

PubMed1987

Unmasking of arachidonate-induced insulin release by removal of extracellular calcium. Arachidonic acid mobilizes cellular calcium in rat islets of Langerhans.

Exogenous arachidonic acid does not stimulate insulin release in Ca++-containing medium, but a potent effect was unmasked by extracellular Ca++ depletion. This secretion met several criteria of exocytotic release. It did not require the oxygenation of arachidonate or its esterification into islet membranes, but was potentiated by the presence of 16.7 mM glucose such that 33 microM arachidonate could reverse the inhibitory effects of extracellular Ca++ removal on glucose-induced insulin secretion. Arachidonic acid alone stimulated a rise in intracellular Ca++ concentrations in dispersed islet cells (measured by the fura-2 technique) equal to that induced by 16.7 mM glucose in normal medium. Arachidonic acid may be a critical coupling signal in normal islets.

Animals↗

PubMed1987

Cytosolic free-calcium concentrations in normal pancreatic islet cells. Effect of secretagogues and somatostatin.

We have assessed the effect of somatostatin on glucose-, potassium-, forskolin-, and dibutyryl cAMP-induced changes in cytosolic free [Ca2+] in normal rat pancreatic islet cells with the new Ca2+ indicator fura 2. The cytosolic free [Ca2+] in islet cells incubated with nonstimulatory concentrations of glucose (30 mg/dl) ranged from 54 to 64 nM. In the presence of extracellular Ca2+ (1 mM), glucose (300 mg/dl) rapidly increased the cytosolic free [Ca2+] to a level of 90-110 nM. In the absence of extracellular Ca2+, glucose failed to increase the cytosolic free [Ca2+], which remained at a level of 55-60 nM. Somatostatin inhibited glucose-induced increases in cytosolic free [Ca2+] in a dose-dependent manner (maximal inhibition was 34%). Half-maximal inhibition was observed at 10(-9) M somatostatin, which correlated well with somatostatin binding to islet cells (Kd = 2.6 X 10(-10) M). Potassium (50 mM) rapidly increased the cytosolic free [Ca2+] to 110-120 nM, and its effect was not influenced by the presence of somatostatin. Forskolin (20 microM) and dibutyryl cAMP (1 mM) rapidly increased cytosolic free Ca2+ both in the presence and absence of extracellular Ca2+. More than 80% of the overall increase in cytosolic free-Ca2+ levels could be accounted for by the mobilization of intracellular Ca2+ stores. Somatostatin effectively blocked the forskolin effect (32% inhibition) but not the dibutyryl cAMP-induced effect. Somatostatin appears to inhibit secretagogue-induced increases in cytosolic free [Ca2+] by interfering with cAMP production and probably with Ca2+ transport across the cell membrane.

Animals↗

PubMed1986

Feedback inhibition of insulin on insulin secretion in isolated pancreatic islets.

We have examined the effect of exogenous insulin on secretion vesicle margination and secretion vesicle lysis in isolated perifused rat pancreatic islets. Recruitment of somatostatin (SRIF) receptors to the plasma membrane was used as a marker of secretion vesicle margination, whereas insulin release reflected the process of secretion vesicle lysis. A newly designed perifusion protocol allows one to interrupt intermittently either secretion vesicle margination or secretion vesicle lysis. Islets were initially perifused with glucose (30, 100, 165, 200, or 300 mg/dl) in the presence of sodium isethionate. Sodium isethionate inhibits secretion vesicle lysis, but not the recruitment of SRIF receptors. Thus, the margination of secretion vesicles to the surface membrane continued without their lysis. Sodium isethionate was then removed, and islets were challenged with 400 microM isobutylmethylxanthine (IBMX). In the islets perifused with high glucose concentrations, IBMX lysed a greater number of vesicles and caused enhanced release of insulin. The presence of exogenous insulin during the initial phase of secretion vesicle margination did not affect subsequent IBMX-induced insulin secretion from the islets perifused with low glucose concentrations (30 or even 100 mg/dl). When the glucose concentration was increased to 165, 200, or 300 mg/dl, insulin significantly diminished IBMX-induced insulin release. In separate experiments, increasing concentrations of insulin (50, 100, and 200 microU/ml) reduced glucose-induced recruitment of SRIF receptors in a dose-dependent manner. Our observations strongly suggest the existence of a well balanced relationship between ambient glucose and insulin concentrations in terms of their positive and negative feedback actions on insulin release. Their influences seem to be exerted at the level of secretion vesicle margination at the plasma membrane.

1-Methyl-3-isobutylxanthine↗

PubMed1986

Insulin secretion in aging: studies with sequential gating of secretion vesicle margination and lysis.

A new sequential gating perifusion technique was employed to investigate secretion vesicle margination and granule lysis in islets isolated from 2- and 18-month-old Fischer 344 rats. The technique is based on sequential perifusion (periods A, B, and C) of isolated islets with glucose (30, 165, or 300 mg/dl) in the presence of sodium isethionate, an inhibitor of granule lysis, followed thereafter by trifluoperazine, an inhibitor of secretion vesicle margination, and glucose (300 mg/dl) or isobutylmethylxanthine (IBMX; 400 microM). When glucose was employed during period A to marginate secretion vesicles to the plasma membrane, subsequent glucose- and IBMX-induced insulin release (period C) was depressed in islets from 18-month-old rats [maximal increase above the basal rate of release (delta max), 9 +/- 2 nU/micron X min] compared to that in the 2-month-old animals (delta max, -19 +/- 3 nU/micron X min). With glyburide (400 microM) used to induce secretion vesicle margination, glucose- and IBMX-induced insulin release was the same in young and old animals (delta max, 14 +/- 3 and 15 +/- 3 nU/micron X min, respectively). Insulin release was then studied as a function of secretion vesicle margination at the plasma membrane by measuring somatostatin (SRIF) receptor recruitment. The islets from older animals must be stimulated with 300 mg/dl glucose to attain the same level of SRIF binding as in islets isolated from younger animals stimulated with 150-165 mg/dl glucose. Insulin release per unit SRIF binding was identical in young and old animals (65 and 69 nU/liter fmol SRIF binding), indicating normal lysis of marginated secretion granules. These studies implicate glucose-induced secretion vesicle margination as the site of impairment in age-related insulin release.

1-Methyl-3-isobutylxanthine↗

PubMed1985

Control of secretion vesicle margination and lysis by glucose, IBMX, and glyburide.

We have attempted to define the effect of glucose, glyburide, and isobutylmethylxanthine (IBMX) upon secretion vesicle margination to the plasma membrane and granule lysis. Margination of secretion vesicles during exocytosis was assessed by measuring the recruitment of somatostatin (SRIF) receptors. Secretion vesicle lysis was studied by measuring insulin release into the incubation media. Our observations suggest that glucose directly affects both secretion vesicle margination and lysis. Both events are rapidly influenced by this secretagogue (within 10 min of incubation). Trifluoperazine (TFP) and removal of Ca2+ from the incubation media significantly reduced glucose-induced margination of secretion vesicles and their lysis (P less than 0.001). IBMX primarily influences the lysis of secretion vesicles and not their margination. IBMX caused a rapid increase in insulin secretion within 10 min without recruitment of SRIF receptors. Neither TFP nor the absence of extracellular Ca2+ affected IBMX-induced insulin release. Late enhancement in margination of secretion vesicles (30 min of incubation with IBMX) is probably related to the initial event of secretion vesicle lysis and is independent of extracellular calcium. Glyburide exerts its action in a manner similar to glucose, affecting both the lysis of secretion vesicles and their margination. However, the action of glyburide is independent of extracellular calcium and partially dependent upon calmodulin.

1-Methyl-3-isobutylxanthine↗

PubMed1985

A unique control mechanism in the regulation of insulin secretion. Secretagogue-induced somatostatin receptor recruitment.

In this study, we have correlated the translocation of somatostatin (SRIF) receptors from the cell interior to the plasma membrane with the ability of SRIF to inhibit insulin release. Islets were perifused with glucose (30, 100, 165, 200, or 300 mg/dl) in the presence of sodium isethionate. Sodium isethionate inhibits insulin release, but not the recruitment of SRIF receptors. Thus, the recruitment of SRIF receptors to the surface membrane continued without the lysis of secretion vesicles. SRIF binding rose from 3.75 +/- 0.16 to 6.46 +/- 0.28 fmol/10 islets as glucose concentration increased. Sodium isethionate was then removed, islets perifused with low glucose (30 mg/dl), and challenged with 400 microM isobutylmethylxanthine (IBMX) with or without SRIF (5 micrograms/ml). In the islets perifused with high glucose concentration, IBMX lysed a greater number of vesicles and caused enhanced release of insulin. The greater the number of secretion vesicles marginated to the plasma membrane by glucose, the greater the response to IBMX. Colchicine (1 mM) prevented secretion vesicle migration and this potentiation effect of higher concentrations of glucose was eliminated. In experiments with IBMX and SRIF, the degree of inhibition of IBMX-induced insulin release by SRIF was proportional to the magnitude of SRIF binding to these islets. SRIF inhibited insulin release by 20 microU/100 islets initially perifused with low glucose (30 mg/dl) and by 875 microU/100 islets perifused with high glucose (300 mg/dl). The maximal effect of SRIF was observed when its binding reached a level of 5.4 fmol/10 islets. We conclude that inhibition of insulin release by SRIF is proportional to the SRIF receptor concentration, and that translocation of SRIF receptors during exocytosis plays an important role in paracrine regulation of insulin secretion by rendering the islets more sensitive to SRIF.

1-Methyl-3-isobutylxanthine↗

PubMed1985

The nature of insulin secretory defect in aging rats.

We have attempted to define the nature of insulin secretory defect(s) in aged animals. In these studies, pancreatic islets were isolated from 2- and 18-mo-old Fischer 344 rats. Margination of secretion vesicles during exocytosis was assessed by measuring the recruitment of somatostatin (SRIF) receptors to the surface membrane. Section vesicle lysis was studied by measuring insulin release into the incubation media. Submaximal and maximal glucose-induced insulin secretion was significantly greater in islets isolated from younger rats (P less than 0.01). SRIF receptor recruitment was stimulated by glucose in both younger and older Fischer 344 rats. However, an increase in SRIF receptor recruitment was reduced in islets isolated from older animals (from 2.14 +/- 0.4 to 4.6 +/- 0.4 fmol/10 islets) (P less than 0.01) as compared with islets from younger animals (from 2.6 +/- 0.2 to 6.2 +/- 0.4 fmol/10 islets). When secretion vesicle lysis was inhibited by the presence of sodium isethionate in the incubation media, glucose (300 mg/dl) failed to stimulate secretion vesicle margination to the plasma membrane. In contrast, glyburide (0.6 micrograms/ml) continued to stimulate directly secretion vesicle margination in islets from aged animals (from 2.1 +/- 0.3 to 6.0 +/- 0.3 fmol/10 islets). We conclude that glucose-induced margination of secretion vesicles at the plasma membrane is impaired by the aging process. This impairment results in lower submaximal and maximal insulin secretory response to glucose. The fact that glyburide is capable of stimulating secretion vesicle margination suggests that glucose signal recognition and/or stimulus-secretion coupling may be the locus of impairment in the process of insulin secretion in older animals.

1-Methyl-3-isobutylxanthine↗

PubMed1985

Localization of somatostatin receptors in secretion vesicles in anterior pituitary cells and pancreatic islets.

Secretion vesicles in anterior pituitary cells and pancreatic islets appear to translocate somatostatin receptors from the cell interior to the plasma membrane. In this study we attempted to localize somatostatin receptors on either the cytoplasmic or the intraluminal surface of the secretion vesicles. 125I-somatostatin binding was determined in intact secretion vesicles and vesicles disrupted either by sonication or solubilization. The binding of 125I-somatostatin was identical in intact and disrupted vesicles, indicating cytoplasmic orientation of somatostatin receptors. Pronase treatment of intact secretion vesicles removed approximately 90% of specific somatostatin binding. Sonication of pronase treated secretion vesicles did not reveal latent somatostatin binding sites. Gold-conjugated somatostatin binds to isolated secretion vesicles confirming the presence of somatostatin binding sites on the outer surface of these vesicles. We conclude that somatostatin binding sites are located on the cytoplasmic surface of secretion vesicles isolated from anterior pituitary cells and pancreatic islets.

Animals↗

PubMed1984

Calmodulin and cyclic AMP. Possible different sites of action of these two regulatory agents in exocytotic hormone release.

It may now be possible to identify certain intracellular events that impact specifically on secretion-granule fusion to the plasma membrane or on granule lysis. Secretion vesicles in isolated rat islets appear to translocate somatostatin (SRIF) receptors from the Golgi apparatus to the plasma membrane. We have proposed that secretion granule fusion to the plasma membrane can be determined by measuring recruitment of SRIF receptors to the surface membrane. Granule lysis can be assessed by measuring insulin release. To activate cyclic AMP (cAMP)-dependent pathways, we employed isobutylmethylxanthine (IBMX, 400 microM), glucagon (10 microM), and forskolin (20 microM), a diterpene activator of adenylate cyclase. These agents evoked rapid release of insulin (from 0.41 +/- 0.02 to 1.88 +/- 0.02; 0.41 +/- 0.02 to 1.93 +/- 0.08; and 0.41 +/- 0.02 to 1.66 +/- 0.03 microU/islet/min, respectively, P less than 0.001). There was no concomitant recruitment of SRIF receptors. Somatostatin (10 micrograms/ml), which inhibits cAMP-stimulated protein phosphorylation, suppresses insulin release evoked by IBMX, glucagon, or forskolin (inhibition: 80, 75, or 82%, respectively). In contrast, trifluoperazine (10 microM), an inhibitor of calmodulin, did not suppress insulin release induced through cAMP-dependent pathways. Trifluoperazine suppresses glucose-induced insulin release and the recruitment of SRIF receptors to the surface membrane, suggesting the possible role of calmodulin in promoting secretion-granule fusion with the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗