EFFECT OF CYTOPLASMIC PH ON CA2+-STIMULATED EICOSANOID BIOSYNTHESIS IN HUMAN PLATELETS

1 We have investigated the effect of cytoplasmic pH (pH(i)) on the relationship between platelet cytoplasmic Ca2+ concentration ([Ca2+](i)) and eicosanoid biosynthesis. Stirred gel-filtered human platelets loaded with fluorescent indicators of Ca2+ and H+ were suspended in balanced salt solutions at 37 degrees C. [Ca2+](i) was controlled by calcium ionophore (ionomycin). Increased [Ca2+](i) was associated with increased production of thromboxane A(2) (TXA(2)) as determined by radioimmunoassay of its stable hydrolysis product TXB(2), and of 12-hydroxy eicosatetraenoic acid (12-HETE) measured by high performance liquid chromatography. 2 Varying pH(i) with a K+/H+ ionophore (nigericin) in platelets suspended in K+ rich solutions of pH 6.8, 7.4 or 7.8 with subsequent resuspension in solution of pH 7.4 containing albumin (1 g l-(1)) and Ca2+ (1 mM) resulted in pH(i) of 6.72 +/- 0.05, 7.31 +/- 0.02 and 7.71 +/- 0.04 (mean +/- s.e. mean, n = 5). Ionomycin (1.2 mu M) increased [Ca2+](i) by 97.1 +/- 17.6, 191.9 +/- 48.7 and 322.8 +/- 55.7 nM at the different values of pH(i) respectively; TXB(2) production was 0.7 +/- 0.2, 2.1 +/- 0.4 and 10.7 +/- 3.3 ng mu g(-1) protein, and 12-HETE production was 150.9 +/- 68.2, 184.4 +/- 77.9 and 302.3 +/- 62.8 ng mu g(-1) protein. 3 Ammonium chloride (50 mM) caused a small reduction in pH(o) while increasing pH(i) from 7.32 +/- 0.04 to 7.89 +/- 0.05 and increasing ionomycin (1.2 mu M)-induced [Ca2+](i) responses from 94.1 +/- 67.3 to 721.6 +/- 288.3 nM. TXB(2) production increased from 3.1 +/- 2.1 to 17.3 +/- 8.2 and 12-HETE production increased from 100.5 +/- 26.7 to 203.2 +/- 36.4 ng mu g(-1) protein. Responses of [Ca2+](i) and TXB(2) production to epoxymethano prostaglandin H-2 (U46619, an endoperoxide-thromboxane receptor agonist) increased significantly in the presence of NH4Cl. 4 Alterations of pH(i) (such as may occur under pathological conditions) influence [Ca2+](i) responses and eicosanoid synthesis in human platelets.