THROMBIN INDUCES A CALCIUM TRANSIENT THAT MEDIATES AN ACTIVATION OF THE NA+/H+ EXCHANGER IN HUMAN-FIBROBLASTS

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  • 25 November 1989
    • journal article
    • research article
    • Vol. 264  (33) , 19540-19547
Abstract
The calcium dependence of growth factor-induced cytoplasmic alkalinization was determined in serum-deprived human fibroblasts (WS-1 cells). Intracellular pH (pHi) and intracellular calcium (Cai2+) were measured using the fluorescent dyes 2'',7''-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein and fura2, respectively. Thrombin (10 nM) induced an alkalinization (0.18 .+-. 0.01 pH units, n = 23) that was Na+-dependent and amiloride-sensitive, suggesting that the alkalinization was mediated by the Na+/H+ exchanger. Thrombin treatment caused a transient increase in Cai2+ (325 .+-. 39 nM, n = 12) that preceded the observed increase in pHi. The increases in Cai2+ and pH were dependent on the concentration of thrombin. The thrombin-induced increase in Cai2+ occurred in the absence of external calcium indicating that thrombin released calcium from internal stores. Inhibition induced increase in Cai2+ with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride or bis-(o-aminophenoxy)ethane-N,N,N'',N''-tetraacetic acid also inhibited the thrombin-stimulated increase in pHi. The calcium ionophore ionomycin was used to increase Cai2+ independent of growth factor stimulation. When Cai2+ was elevated with ionomycin, in a concomitant increase in pHi was observed. The increase in pHi due to ionomycin was dependent on Na+ and sensitive to amiloride. The removal of external Cai2+ inhibited the ionomycin-induced elevation of both Cai2+ and pHi. The ionomycin-induced increase in Cai2+ and pHi were not inhibited by 8-diethylaminooctyl 3, 4, 5-trimethoxybenzoate hydrochloride. The results suggest that thrombin treatment can activate the Na+/H+ exchanger, and this activation is mediated by an increase in Cai2+.