Effect of cytoplasmic pH on Ca(2+)-stimulated eicosanoid biosynthesis in human platelets

1. We have investigated the effect of cytoplasmic pH (pHi) 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 A2 (TXA2) as determined by radioimmunoassay of its stable hydrolysis product TXB2, and of 12-hydroxy eicosatetraenoic acid (12-HETE) measured by high performance liquid chromatography. 2. Varying pHi 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 pHi of 6.72 +/- 0.05, 7.31 +/- 0.02 and 7.71 +/- 0.04 (mean +/- s.e. mean, n = 5). Ionomycin (1.2 microM) increased [Ca2+]i by 97.1 +/- 17.6, 191.9 +/- 48.7 and 322.8 +/- 55.7 nM at the different values of pHi respectively; TXB2 production was 0.7 +/- 0.2, 2.1 +/- 0.4 and 10.7 +/- 3.3 ng micrograms-1 protein, and 12-HETE production was 150.9 +/- 68.2, 184.4 +/- 77.9 and 302.3 +/- 62.8 ng micrograms-1 protein. 3. Ammonium chloride (50 mM) caused a small reduction in pHo while increasing pHi from 7.32 +/- 0.04 to 7.89 +/- 0.05 and increasing ionomycin (1.2 microM)-induced [Ca2+]i responses from 94.1 +/- 67.3 to 721.6 +/- 288.3 nM. TXB2 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 microg-1 protein. Responses of [Ca2+]i and TXB2 production to epoxymethano prostaglandin H2 (U46619, an endoperoxide-thromboxane receptor agonist) increased significantly in the presence of NH4C1.4. Alterations of pHi (such as may occur under pathological conditions) influence [Ca2+]i responses and eicosanoid synthesis in human platelets.