Endocannabinoid Inhibition of Ion Channels of Pancreatic Beta Cells

Abstract

The secretion of insulin from pancreatic beta cells is regulated by many factors, chiefly the plasma glucose concentration. Glucose initiates a chain of events that raises the ATP:ADP ratio within the cell. When the K(ATP) channel is inhibited by this increased ratio, the cell depolarizes, calcium action potentials ensue, and the raised cytosolic calcium concentration leads to exocytosis of insulin secretory granules. Preliminary evidence from our lab indicates that endocannabinoids are negative regulators of insulin secretion, and we previously demonstrated that the endocannabinoid 2-arachidonylglycerol (2-AG) inhibits sodium, delayed rectifier potassium, and high voltage-activated (mostly L-type) calcium channels at low micromolar concentrations in the insulinoma cell line R7T1.In the R7T1 cell line, inside-out patches (+60 mV) displayed unitary currents of about 4.2 pA and various multiples, which were identified as K(ATP) channels by being nearly completely inhibited by 1 mM ATP. 2-AG inhibited the K(ATP) channel with an IC50 of 1 uM. This block was irreversible, even in the presence of lipid free bovine serum albumin. To test whether the block by 2-AG were mediated by the CB1 receptor, the CB1 antagonist AM-251 (10 uM) was added to the pipette solution. Though a >95% antagonism was predicted, no effect of the AM-251 was observed, suggesting that the CB1 receptor did not mediate the 2-AG blockade. This block was evident in current clamped (perforated patch) primary mouse beta cells, which showed a small (2.5 mV median) but consistent depolarization in the presence of 10 uM 2-AG. Moreover, the depolarization induced by 10 mM glucose was blunted by 10 uM 2-AG, evidence that the ion channel blockade seen in the insulinoma cells can apply to primary beta cells.