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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 insulincontaining 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)P[sub 2]] 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)P[sub 2]-binding protein Ca[sup 2+]-dependent activator protein for secretion (CAPS) is present in β cells, and neutralization of the protein abolished both Ca[sup 2+]- and PI(4,5)P[sub 2]-induced exocytosis. We conclude that ADPinduced changes in PI 4-kinase activity, via generation of PI(4,5)P[sub 2], represents a metabolic sensor in the β cell by virtue of its capacity to regulate the release competence of the secretory granules. [ABSTRACT FROM AUTHOR]

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 β-cells by stimulation of Ca2+-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 A2 (cPLA2) inhibitors ACA and AACOCF3 and in cells treated with antisense oligonucleotide against cPLA2α. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF3, and cPLA2α antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA2 activity. We propose that cPLA2α plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting β-cells. [Copyright &y& Elsevier]

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 α- and β-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at ≤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 μM 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 μM). The stimulatory action of NNC77-0074 was dependent on protein kinase C activity. NNC77-0074 potently inhibited glucagon secretion from rat islets (EC50=11 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 α-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. [Copyright &y& Elsevier]

A number of potassium channels including members of the KCNQ family and the Ca2+ activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (∼50 μm2 macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of −5.0 ± 0.1 mmHg the increase amounted to 381 ± 146% (mean ±s.e.m., n= 6, P < 0.025). In contrast, in oocytes expressing the strongly volume-sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms. [ABSTRACT FROM AUTHOR]

The release of sPLA2 from single mouse pancreatic β-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of β-cells with agents that increased cytosolic free Ca2+ concentration ([Ca2+]i) induced a rapid release of sPLA2 to the extracellular medium. Exogenous sPLA2 strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA2 was mediated via inhibition of ATP-dependent K+ channels and an increase in [Ca2+]i. Measurements of cell capacitance in single β-cells revealed that sPLA2 did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA2 is operational in pancreatic β-cells and point to this enzyme as an autocrine regulator of insulin secretion. [Copyright &y& Elsevier]

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A[sub 2] (cPLA[sub 2]α) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca[sup 2+]-dependent exocytosis in single mouse pancreatic β-cells, cPLA[sub 2]α dose dependently (EC[sub 50] = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca[sup 2+] current or cytoplasmic Ca[sup 2+] levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPLA[sub 2]αstimulated exocytosis was antagonized by the specific cPLA[sub 2] inhibitor AACOCF[sub 3]. Ca[sup 2+]-evoked exocytosis was reduced by 40% in cells treated with AACOCF[sub 3] or an antisense oligonucleotide against cPLA[sub 2]α. The action of cPLA[sub 2]α was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl[sup -] uptake through ClC-3 Cl[sup -] channels. Accordingly, the stimulatory action of cPLA[sub 2]α was inhibited by the Cl[sup -] channel inhibitor DIDS and in cells pretreated with ClC-3 Cl[sup -] channel antisense oligonucleotides. We propose that cPLA[sub 2]α has an important role in controlling the rate of exocytosis in β-cells. This effect of cPLA[sub 2]α reflects an enhanced transgranular Cl[sup -] flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine. [ABSTRACT FROM AUTHOR]

The glycosphingolipid sulfatide is present in secretory granules and at the surface of pancreatic beta-cells, and antisulfatide antibodies (ASA; IgG1) are found in serum from the majority of patients with newly diagnosed type 1 diabetes. Here we demonstrate that sulfatide produced a glucose- and concentration-dependent inhibition of insulin release from isolated rat pancreatic islets. This inhibition of insulin secretion was due to activation of ATP-sensitive K(+)-(K(ATP)) channels in single rat beta-cells. No effect of sulfatide was observed on whole-cell Ca(2+)-channel activity or glucose-induced elevation of cytoplasmic Ca(2+) concentration. It is interesting that sulfatide stimulated Ca(2+)-dependent exocytosis determined by capacitance measurements and depolarized-induced insulin secretion from islets exposed to diazoxide and high external KCl. The monoclonal sulfatide antibody Sulph I as well as ASA-positive serum reduced glucose-induced insulin secretion by inhibition of Ca(2+)-dependent exocytosis. Our data suggest that sulfatide is important for the control of glucose-induced insulin secretion and that both an increase and a decrease in the sulfatide content have an impact on the secretory capacity of the individual beta-cells. [ABSTRACT FROM AUTHOR]

Examines the role of inositol (Ins) hexakisphosphate in improving dynamin-medicated endocytosis in the pancreatic cell. Molecular mechanism regulating the membrane homeostasis; Metabolic control of the Ins; Formation of the phosphatidylinositol biphosphate following activation of protein kinase C.