Bradykinin induces elevations of cytosolic calcium through mobilisation of intracellular and extracellular pools in bovine aortic endothelial cells

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Abstract
1 In the presence of 1.8 mm extracellular calcium, bradykinin (0.3 nm–100 nm) induced a biphasic elevation of intracellular calcium ([Ca2+]i) in bovine aortic endothelial cells, consisting of an initial, large transient component followed by a lower sustained component. 2 When endothelial cells were bathed in nominally calcium-free solution containing 0.5 mm EGTA, bradykinin induced only a transient elevation of [Ca2+]i: the magnitude of this was significantly smaller than that obtained in the presence of extracellular calcium and the sustained phase was abolished. In the continued presence of bradykinin, re-addition of extracellular calcium to achieve a level of around 1.8 mm resulted in the induction of a biphasic elevation of [Ca2+]i consisting of a large initial component followed by a lower sustained component. 3 In the presence of 1.8 mm extracellular calcium, caffeine (5 mm) induced a small elevation of [Ca2+]i. When endothelial cells were bathed in nominally calcium-free solution containing 0.5 mm EGTA, the caffeine-induced elevation of [Ca2+]i was almost completely abolished. 4 In the presence of 1.8 mm extracellular calcium, treatment of endothelial cells with the calcium influx blocker, nickel chloride (4 mm), had no effect on resting [Ca2+]i or on the magnitude of the bradykinin-induced initial transient elevation of [Ca2+]i but abolished the sustained component. 5 In the presence of 1 mm extracellular calcium, treatment with the calcium chelator EGTA (2 mm; 1 min) had no effect on resting [Ca2+]i but the magnitude of the bradykinin-induced initial transient elevation of [Ca2+]i was significantly reduced. Increasing the exposure time or concentration of EGTA resulted in no further reduction in the magnitude of the bradykinin-induced transient component. 6 Treatment of endothelial cells with the putative inhibitor of intracellular calcium release, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, 0.1 mm) increased resting [Ca2+]i slightly but had no effect on the magnitude of the bradykinin-stimulated elevation of [Ca2+]i. 7 These findings suggest that, in bovine aortic endothelial cells, the bradykinin-induced initial transient elevation of [Ca2+]i is completely dependent upon release of calcium from intracellular stores and the sustained component is due to calcium influx. They further suggest the possible existence of two intracellular calcium pools, one which is rapidly depleted in the absence of extracellular calcium and a second which is resistant to such depletion.