Marianne Høy

Somatostatin inhibits exocytosis in rat pancreatic α-cells by Gi2-dependent activation of calcineurin and depriming of secretory granules

1 Measurements of cell capacitance were used to investigate the molecular mechanisms by which somatostatin inhibits Ca2+‐induced exocytosis in single rat glucagon‐secreting pancreatic α‐cells. 2 Somatostatin decreased the exocytotic responses elicited by voltage‐clamp depolarisations by 80 % in the presence of cyclic AMP‐elevating agents such as isoprenaline and forskolin. Inhibition was time dependent and half‐maximal within 22 s. 3 The inhibitory action of somatostatin was concentration dependent with an IC50 of 68 nm and prevented by pretreatment of the cells with pertussis toxin. The latter effect was mimicked by intracellular dialysis with specific antibodies to Gi1/2 and by antisense oligonucleotides against G proteins of the subtype Gi2. 4 Somatostatin lacked inhibitory action when applied in the absence of forskolin or in the presence of the L‐type Ca2+ channel blocker nifedipine. The size of the ω‐conotoxin‐sensitive and forskolin‐independent component of exocytosis was limited to 60 fF. By contrast, somatostatin abolished L‐type Ca2+ channel‐dependent exocytosis in α‐cells exposed to forskolin. The magnitude of the latter pool amounted to 230 fF. 5 The inhibitory effect of somatostatin on exocytosis was mediated by activation of the serine/threonine protein phosphatase calcineurin and was prevented by pretreatment with cyclosporin A and deltamethrin or intracellularly applied calcineurin autoinhibitory peptide. Experiments using the stable ATP analogue AMP‐PCP indicate that somatostatin acts by depriming of granules. 6 We propose that somatostatin receptors associate with L‐type Ca2+ channels and couple to Gi2 proteins leading to a localised activation of calcineurin and depriming of secretory granules situated close to the L‐type Ca2+ channels.

Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells.

Insulin secretion is controlled by the β cell′s metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P2-binding protein Ca2+-dependent activator protein for secretion (CAPS) is present in β cells, and neutralization of the protein abolished both Ca2+- and PI(4,5)P2-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P2, represents a metabolic sensor in the β cell by virtue of its capacity to regulate the release competence of the secretory granules.

CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells

1 Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+‐induced exocytosis in single insulin‐secreting mouse pancreatic B‐cells. 2 ACh (250 μM) increased exocytotic responses elicited by voltage‐clamp depolarizations 2.3‐fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (τ) for the recovery of [Ca2+]i following a voltage‐clamp depolarization increased 5‐fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 μM) for much longer periods in the presence than in the absence of the agonist. 3 The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in τ for the refilling of the RRP from 31 to 19 s. 4 Collectively, the effects of ACh on the RRP and τ result in a > 10‐fold stimulation of the rate at which granules are supplied for release.

Increase in cellular glutamate levels stimulates exocytosis in pancreatic β‐cells

Glutamate has been implicated as an intracellular messenger in the regulation of insulin secretion in response to glucose. Here we demonstrate by measurements of cell capacitance in rat pancreatic β‐cells that glutamate (1 mM) enhanced Ca2+‐dependent exocytosis. Glutamate (1 mM) also stimulated insulin secretion from permeabilized rat β‐cells. The effect was dose‐dependent (half‐maximum at 5.1 mM) and maximal at 10 mM glutamate. Glutamate‐induced exocytosis was stronger in rat β‐cells and clonal INS‐1E cells compared to β‐cells isolated from mice and in parental INS‐1 cells, which correlated with the expressed levels of glutamate dehydrogenase. Glutamate‐induced exocytosis was inhibited by the protonophores FCCP and SF6847, by the vacuolar‐type H+‐ATPase inhibitor bafilomycin A1 and by the glutamate transport inhibitor Evans Blue. Our data provide evidence that exocytosis in β‐cells can be modulated by physiological increases in cellular glutamate levels. The results suggest that stimulation of exocytosis is associated with accumulation of glutamate in the secretory granules, a process that is dependent on the transgranular proton gradient.

Rapid Association of Protein Kinase C-ϵ with Insulin Granules Is Essential for Insulin Exocytosis

Glucose-dependent exocytosis of insulin requires activation of protein kinase C (PKC). However, because of the great variety of isoforms and their ubiquitous distribution within the β-cell, it is difficult to predict the importance of a particular isoform and its mode of action. Previous data revealed that two PKC isoforms (α and ϵ) translocate to membranes in response to glucose (Zaitzev, S. V., Efendic, S., Arkhammar, P., Bertorello, A. M., and Berggren, P. O. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9712–9716). Using confocal microscopy, we have now established that in response to glucose, PKC-ϵ but not PKC-α associates with insulin granules and that green fluorescent protein-tagged PKC-ϵ changes its distribution within the cell periphery upon stimulation of β-cells with glucose. Definite evidence of PKC-ϵ requirement during insulin granule exocytosis was obtained by using a dominant negative mutant of this isoform. The presence of this mutant abolished glucose-induced insulin secretion, whereas transient expression of the wild-type PKC-ϵ led to a significant increase in insulin exocytosis. These results suggest that association of PKC-ϵ with insulin granule membranes represents an important component of the secretory network because it is essential for insulin exocytosis in response to glucose.

Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells

1 Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+‐dependent exocytosis in isolated glucagon‐secreting rat pancreatic A‐cells. 2 When applied extracellularly, tolbutamide stimulated depolarization‐evoked exocytosis 4.2‐fold without affecting the whole‐cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration‐dependent increase in cell capacitance. Half‐maximal stimulation was observed at 33 μm and the maximum stimulation corresponded to a 3.4‐fold enhancement of exocytosis. 3 The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mm) and quinine (10 μm). A similar stimulation was elicited by 5‐hydroxydecanoate (5‐HD; 10 μm), an inhibitor of mitochondrial ATP‐sensitive K+ (KATP) channels. 4 Tolbutamide‐stimulated, but not TEA‐induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. 5 Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide‐ and Ca2+‐evoked exocytosis. Furthermore, tolbutamide‐ and Ca2+‐induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V‐type) H+‐ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide. 6 We propose that during granular maturation, a granular V‐type H+‐ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown mechanisms, possibly involving granular alkalinization.

Scinderin-derived actin-binding peptides inhibit Ca2+- and GTPγS-dependent exocytosis in mouse pancreatic β-cells

Abstract Using capacitance measurements, we have explored the effects of three different scinderin actin-binding peptides (Sc77–89; Sc138–146; Sc511–523) on Ca2+- and GTPγS-induced exocytosis in single mouse pancreatic β-cells. Sc77–89 (10 μM) reduced exocytosis by 43% in whole-cell experiments in which secretion was triggered by intracellular dialysis with a Ca2+-EGTA buffer with a free Ca2+ concentration of 2 μM. A more pronounced reduction of the rate of exocytosis was observed with Sc138–146 (72%) but not with Sc511–523 (39%). Sc138–146 also reduced depolarisation-induced exocytosis by 61% without affecting the whole-cell Ca2+ current. When exocytosis was triggered by infusion of GTPγS, all scinderin-binding peptides reduced exocytosis by 59–75%. These data suggest that scinderin might be important for controlling cortical actin network dynamics in mouse pancreatic β-cells and that scinderin-induced cortical filamentous actin disassembly is required for insulin secretion.

Selectivity of prandial glucose regulators: nateglinide, but not repaglinide, accelerates exocytosis in rat pancreatic A-cells

The effects of the two prandial glucose regulators, repaglinide and nateglinide, on ATP-sensitive K(+) (K(ATP)) channel activity, membrane potential and exocytosis in single rat pancreatic A-cells were investigated using the patch-clamp technique. K(ATP) channel activity was reversibly blocked by repaglinide (K(d)=22 nM) and nateglinide (K(d)=410 nM) and this was associated with membrane depolarisation and initiation of electrical activity. The effect of repaglinide and nateglinide on stimulation of glucagon secretion by direct interference with the exocytotic machinery was investigated by the use of capacitance measurements. Nateglinide, but not repaglinide, at concentrations similar to those required to block K(ATP) channels potentiated Ca(2+)-evoked exocytosis 3-fold. In alphaTC1-9 glucagonoma cells addition of nateglinide, but not repaglinide, was associated with stimulation of glucagon secretion. These results indicate that the fast-acting insulin secretagogue nateglinide is glucagonotropic primarily by stimulating Ca(2+)-dependent exocytosis.

Imidazoline NNC77-0074 stimulates insulin secretion and inhibits glucagon release by control of Ca2+-dependent exocytosis in pancreatic α- and β-cells

We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)pyridine (NNC77-0074) on stimulus-secretion coupling in isolated pancreatic alpha- and beta-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at less than or equal to 2.5 mM glucose and maximal stimulation occurred at 10-15 mM glucose. NNC77-0074 produced a concentration-dependent stimulation of insulin secretion. Half-maximal (EC50) stimulation was observed at 24 muM and at maximally stimulatory concentrations insulin release was doubled. The stimulatory action of NNC77-0074 on insulin secretion was not associated with membrane depolarisation or a change in the activity of ATP-sensitive K+ channels. Using capacitance measurements, we found that NNC77-0074 stimulated depolarisation-induced exocytosis 2.6-fold without affecting the whole-cell Ca2+ current when applied via the extracellular medium. The concentration dependence of the stimulatory action was determined by intracellular application of NNC77-0074 through the recording pipette. NNC77-0074 stimulated exocytosis half-maximal at 44 nM and at maximally stimulatory concentrations the rate of exocytosis was increased twofold. NNC77-0074 stimulated depolarised-induced insulin secretion from islets exposed to diazoxide and high external KCl (EC50 = 0.45 muM). The stimulatory action of NNC77-0074 was dependent on protein kinase C activity. NNC77-0074 potently inhibited glucagon secretion from rat islets (EC50 = I I nM). This was not associated with a change in spontaneous electrical activity and ATP-sensitive K channel activity but resulted from a reduction of the rate of Ca2+-dependent exocytosis in single rat alpha-cells (EC50=9 nM). Inhibition of exocytosis by NNC77-0074 was pertussis toxin-sensitive and mediated by activation of the protein phosphatase calcineurin. In rat somatotrophs, PC12 cells and mouse cortical neurons NNC77-0074 did not stimulate Ca2+-evoked exocytosis, whereas the other imidazoline compounds phentolamine and efaroxan produced 2.5-fold stimulation of exocytosis. Our data suggest that the imidazoline compound NNC77-0074 constitutes a novel class of antidiabetic compounds that stimulates glucose-dependent insulin release while inhibiting glucagon secretion. These actions are exclusively exerted by modulation of exocytosis of the insulin- and glucagon-containing granules

Imidazoline NNC77-0074 stimulates Ca2+-evoked exocytosis in INS-1E cells by a phospholipase A2-dependent mechanism.

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic beta-cells by stimulation of Ca(2+)-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A(2) (cPLA(2)) inhibitors ACA and AACOCF(3) and in cells treated with antisense oligonucleotide against cPLA(2)alpha. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF(3), and cPLA(2)alpha antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA(2) activity. We propose that cPLA(2)alpha plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting beta-cells.