Imidazoline NNC77-0074 stimulates insulin secretion and inhibits glucagon release by control of Ca 2+-dependent exocytosis in pancreatic α- and β-cells

Abstract

We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1 H-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 (EC 50) 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 Ca 2+ 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 (EC 50=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 (EC 50=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 Ca 2+-dependent exocytosis in single rat α-cells (EC 50=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 Ca 2+-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.