Thrombin Induces a Calcium Transient That Mediates an Activation of the Na\u3csup\u3e+\u3c/sup\u3e/H\u3csup\u3e+\u3c/sup\u3e Exchanger in Human Fibroblasts

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 (Ca2+i) were measured using the fluorescent dyes 2\u27,7\u27-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 Ca2+i (325 +/- 39 nM, n = 12) that preceded the observed increase in pHi. The increases in Ca2+i and pHi were dependent on the concentration of thrombin. The thrombin-induced increase in Ca2+i occurred in the absence of external calcium indicating that thrombin released calcium from internal stores. Inhibition of the thrombin-induced increase in Ca2+i with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride or bis-(o-aminophenoxy)ethane-N,N,N\u27,N\u27- tetraacetic acid also inhibited the thrombin-stimulated increase in pHi. The calcium ionophore ionomycin was used to increase Ca2+i independent of growth factor stimulation. When Ca2+i was elevated with ionomycin, 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 Ca2+i inhibited the ionomycin-induced elevation of both Ca2+i and pHi. The ionomycin-induced increases in Ca2+i and pHi were not inhibited by 8-diethylaminooctyl 3,4,5-trimethoxy-benzoate hydrochloride. The results suggest that thrombin treatment can activate the Na+/H+ exchanger, and this activation is mediated by an increase in Ca2+i

Thrombin Induces a Calcium Transient That Mediates an Activation of the Na\u3csup\u3e+\u3c/sup\u3e/H\u3csup\u3e+\u3c/sup\u3e Exchanger in Human Fibroblasts

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 (Ca2+i) were measured using the fluorescent dyes 2\u27,7\u27-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 Ca2+i (325 +/- 39 nM, n = 12) that preceded the observed increase in pHi. The increases in Ca2+i and pHi were dependent on the concentration of thrombin. The thrombin-induced increase in Ca2+i occurred in the absence of external calcium indicating that thrombin released calcium from internal stores. Inhibition of the thrombin-induced increase in Ca2+i with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride or bis-(o-aminophenoxy)ethane-N,N,N\u27,N\u27- tetraacetic acid also inhibited the thrombin-stimulated increase in pHi. The calcium ionophore ionomycin was used to increase Ca2+i independent of growth factor stimulation. When Ca2+i was elevated with ionomycin, 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 Ca2+i inhibited the ionomycin-induced elevation of both Ca2+i and pHi. The ionomycin-induced increases in Ca2+i and pHi were not inhibited by 8-diethylaminooctyl 3,4,5-trimethoxy-benzoate hydrochloride. The results suggest that thrombin treatment can activate the Na+/H+ exchanger, and this activation is mediated by an increase in Ca2+i