Abstract

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 betacells, 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. IP₃releases [Ca⁺⁺]_i from the intracellular Ca⁺⁺ store, probably the endoplasmic reticulum, which triggers Ca⁺⁺ influxvia voltage-dependent Ca⁺⁺ channels and thus increases insulin secretion.