Endothelin influences pHi of human platelets through protein kinase C mediated pathways

Stimulation of human platelets with endothelin raises cytosolic pH (pHi). In order to determine whether this effect is mediated via protein kinase C and Na(+)-H+ linked pathways, the effects of staurosporine and calphostin C (protein kinase C inhibitors) and 5-(N,N-hexamethylene) amiloride (Na(+)-H+ exchange blocker) on endothelin-induced pHi responses in human platelets were assessed. In addition, platelet endothelin receptor subtypes were determined pharmacologically using the selective ETA receptor antagonist BQ-123 and the ETB receptor agonist sarafotoxin S6c. pHi was measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets obtained from 15 healthy subjects. Endothelin-1 at a fixed concentration of 10(-9) M significantly increased pHi from 7.11 +/- 0.01 ([H+]i = 77 +/- 0.9 nM) to 7.19 +/- 0.04 ([H+]i = 64 +/- 0.9 nM) (p < 0.01). The pHi-stimulating effect of endothelin-1 was inhibited by 10(-7) M staurosporine, calphostin C and 5-(N,N-hexamethylene) amiloride. BQ-123 (10(-7) M) abolished the pHi responses to endothelin-1, whereas sarafotoxin S6c had no effect on platelet pHi. These data suggest that in vitro effects of endothelin-1 on platelet pHi are receptor-mediated via a pathway involving protein kinase C. Platelet endothelin receptors appear to be of the ETA subtype.