Na+/H+ exchange in PAF-stimulated platelets

Abstract

In experiments on human platelets, inhibition of Na+/H+ exchange was caused either by equimolar substitution of external Na+ with choline or N-methyl-D-glucamine, by decreasing the pHo to 6.8, or by an inhibitor of the antiport 5-(N-ethyl-N-isopropyl)amiloride (EIPA). In all these cases a considerable inhibition of PAF-induced platelet aggregation and as a rule a more or less marked decrease in the cytoplasmic Ca2+ signal (quin-2-loaded platelets) occurred. Stimulation by 10(-7) M PAF caused biphasic pHi changes in human platelets loaded with the pH-sensitive fluorescent probe BCECF: a small transient decrease, followed by a sustained increase of 0.02 +/- 0.006 pH units, resulted from stimulation of the Na+/H+ exchange. Thrombin (0.1 U/ml) also caused biphasic pHi changes, but the alkalinization step was more pronounced (0.15 +/- 0.03 U). Every means of Na+/H+ exchange inhibition prevented a rise in pHi in stimulated platelets. Activation of the adenylate cyclase system by carbacyclin suppressed the agonist-induced pHi increase. The inhibition of neither cyclooxygenase by 10(-5) M indomethacin nor calmodulin-dependent enzymes by 10(-5) M calmidazolium affected the agonist-induced pHi signals. A decrease in temperature from 37 to 24 degrees C caused a considerable increase in the lag phase of the pHi signal induced by tetradecanoyl phorbol acetate (TPA), but did not affect the kinetics of the pHi signal induced by PAF. An inhibitor of protein kinase C (PKC), compound H-7 (60 microM), completely abolished the TPA-induced increase in pHi but caused only a partial inhibition of the pHi signal in about 50% of the experiments with PAF. On the basis of these results the conclusion is drawn that the activation of PKC is not the only pathway for the PAF-induced stimulation of Na+/H+ exchange. The PAF-induced pHi rise depended both on the presence of extracellular Ca2+ and on the [Ca2+]i increase. On the other hand, inhibition of Na+/H+ exchange decreased the magnitude of the Ca2+i signal in PAF-induced platelets loaded with quin-2, but did not influence the Ca2+ mobilization from intracellular stores as measured by quin-2 or chlortetracycline in experiments with thrombin-stimulated platelets. We conclude that in PAF-activated platelets some initial increase of [Ca2+]i is essential for Na+/H+ exchange activation while activated antiport potentiates a full-scale Ca2+ influx into the cells.