Vol. 282, Issue 3, 1247-1252, 1997

Mechanisms of Bradykinin-Induced Insulin Secretion in Clonal beta Cell Line RINm5F¹

Chi Yang , Bumsup Lee, Ter-Hsin Chen and Walter H. Hsu

Department of Veterinary Physiology and Pharmacology (C.Y., B.L., T.C., W.H.H.), Iowa State University, Ames, Iowa, Department of Veterinary Medicine National Chung Hsing University, Taichung, Taiwan 40227 (C.Y.), and Department of Comparative Medicine (T.H., W.H.H.), Pig Research Institute of Taiwan, Chunan, Miaoli, Taiwan 35099, Republic of China

We investigated the mechanisms underlying bradykinin (BK)-induced rise in intracellular Ca⁺⁺ concentration [Ca⁺⁺]_i and insulin secretion using clonal beta cell line RINm5F. Incubation with a range of concentrations of BK increased in concentration-dependent manners both insulin secretion (BK of 10 nM to 10 µM) and [Ca⁺⁺]_i (BK of 100 nM to 100 µM). In Ca⁺⁺-containing medium, BK (1 µM) induced a biphasic [Ca⁺⁺]_i rise, which was characterized by a Ca⁺⁺ peak and a sustained Ca⁺⁺ phase. In the Ca⁺⁺-free medium, BK failed to increase insulin secretion and induced only a Ca⁺⁺ peak without the sustained Ca⁺⁺ phase. Thapsigargin (1 µM), an inhibitor of the Ca⁺⁺ pump in the endoplasmic reticulum, abolished the Ca⁺⁺ peak and the sustained phase. Nimodipine (1 µM), a voltage-dependent Ca⁺⁺ channel blocker, abolished the BK-induced sustained Ca⁺⁺ phase and inhibited BK-induced insulin release. The BK₁ receptor agonist des-Arg⁹-BK (1 µM) did not change either [Ca⁺⁺]_i or insulin secretion. Both the BK-induced insulin secretion and rise in [Ca⁺⁺]_i were inhibited by a selective BK₂ receptor antagonist, HOE 140 (3.3-100 nM), in concentration-dependent manners but were not by a BK₁ receptor antagonist des-Arg9,Leu⁸-BK (1 µM). Pretreatment with pertussis toxin (0.1 µg/ml) did not block the BK-induced insulin secretion or increase in [Ca⁺⁺]_i. U-73122 (4, 6 and 8 µM), a phospholipase C inhibitor, antagonized both the BK-induced insulin secretion and the increase in [Ca⁺⁺]_i in a concentration-dependent and parallel manner. BK increased intracellular concentrations of inositol-1,4,5-trisphosphate (IP₃). Neither (p-amylcinnamoyl)anthranilic acid (100 µM), a phospholipase A₂ inhibitor, nor N^G-nitro-L-arginine methylester (100 µM), a nitric oxide synthase inhibitor, inhibited these effects of BK. Taken together, these findings suggested that in beta cells, BK activates BK₂ receptors, which, in turn, activate a pertussis toxin-insensitive G protein. The G protein couples to phospholipase C, which promotes the formation of IP₃ and diacylglycerol. IP₃ releases [Ca⁺⁺]_i from the intracellular Ca⁺⁺ store, probably the endoplasmic reticulum, which triggers Ca⁺⁺ influx via voltage-dependent Ca⁺⁺ channels and thus increases insulin secretion.