Thrombin Induces a Calcium Transient That Mediates an Activation of the Na⁺/H⁺ 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 (pH _i ) and intracellular calcium (Ca ²⁺ _i ) 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 Ca ²⁺ _i (325 +/- 39 nM, n = 12) that preceded the observed increase in pH _i . The increases in Ca ²⁺ _i and pH _i were dependent on the concentration of thrombin. The thrombin-induced increase in Ca ²⁺ _i occurred in the absence of external calcium indicating that thrombin released calcium from internal stores. Inhibition of the thrombin-induced increase in Ca ²⁺ _i 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 pH _i . The calcium ionophore ionomycin was used to increase Ca ²⁺ _i independent of growth factor stimulation. When Ca ²⁺ _i was elevated with ionomycin, a concomitant increase in pH _i was observed. The increase in pHi due to ionomycin was dependent on Na ⁺ and sensitive to amiloride. The removal of external Ca ²⁺ _i inhibited the ionomycin-induced elevation of both Ca ²⁺ _i and pH _i . The ionomycin-induced increases in Ca ²⁺ _i and pH _i 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 Ca ²⁺ _i .