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 NG-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.
Footnotes
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Send reprint requests to: Dr. Walter H. Hsu, Department of Biomedical Sciences, Iowa State University, Ames, IA 50011.
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↵1 This work was partially supported by National Science Council, ROC(NSC86-2313-B-005-113-T).
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↵2 C. Yang, B. Lee, T.-H. Chen and W. H. Hsu, unpublished obervations.
- Abbreviations:
- ACA
- (p-amylcinnamoyl)anthranilic acid
- AM
- acetoxymethyl ester
- BK
- bradykinin
- DAG
- diacylglycerol
- ER
- endoplasmic reticulum
- HOE 140
- IP3, inositol-1,4,5-trisphosphate
- KRB
- Krebs-Ringer bicarbonate solution
- L-NAME
- NG-nitro-l-arginine methylester
- PIP2
- phosphatidylinositol-4,5-bisphosphate
- PTX
- pertussis toxin
- PLA2
- phospholipase A2
- PLC
- phospholipase C
- TG
- thapsigargin
- VDCC
- voltage-dependent Ca++ channel
- Received September 3, 1996.
- Accepted May 12, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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