calcimycin and ethylisopropylamiloride

Effects of extracellular Na+ on platelet responses were examined. Cytoplasmic pH decreased by stimulation with thrombin, TPA and AA in Na(+)-containing buffer as well as in Na(+)-free buffer. Thrombin-induced aggregation and serotonin release were higher in Na(+)-containing buffer, but TPA- and A23187-induced responses exhibited lower values. Thrombin-stimulated platelet in Na(+)-free buffer started to aggregate by the addition of NaCl. Inhibition of thrombin-induced aggregation by indomethacin was apparently weaker than that by removal of Na+. These results suggest that the changes of platelet responses by removal of Na+ is due to the effect of Na+ ion itself, not by the inhibition of Na+/H+ exchanger, and that Na+ affects on mechanisms other than arachidonic acid mobilization.

Topics: Amiloride; Arachidonic Acids; Blood Platelets; Calcimycin; Cytoplasm; Humans; Hydrogen-Ion Concentration; Indomethacin; Platelet Aggregation; Serotonin; Sodium; Thrombin; Tissue Plasminogen Activator

1. The release of prostacyclin (PGI2) from bovine aortic endothelial cells stimulated by adenosine 5'-triphosphate (ATP) was decreased by amiloride analogues bearing alkyl groups on the 5-amino nitrogen atom, like 5-(N-ethyl-N-isopropyl)amiloride (EIPA), which are inhibitors of the Na+/H+ exchanger. Analogues substituted on a terminal guanidino nitrogen atom were not inhibitory. 2. The release of PGI2 induced by ATP was not significantly depressed in a Na+-poor medium or in a medium acidified to pH 6.9, two conditions known to inhibit the Na+/H+ exchanger. 3. Cytoplasmic alkalinization by ammonium chloride did not suppress the inhibitory action of EIPA. By itself, ammonium chloride decreased the response of endothelial cells to ionophore A23187 and ATP, whereas sodium acetate had no effect. 4. EIPA did not decrease the mobilization of free arachidonic acid induced by ATP. It inhibited the conversion of exogenous arachidonate into PGI2 and prostaglandin E2 (PGE2). 5. Although the intracellular pH was not measured in this study, it seems unlikely that cytoplasmic alkalinization via the activation of the Na+/H+ exchanger plays a significant role in the stimulatory action of ATP on the release of PGI2 from endothelial cells. The inhibition of that release by EIPA and other amiloride analogues might involve a direct effect on cyclo-oxygenase, although an action on the reacylation of free arachidonic acid cannot be excluded.

Topics: Adenosine Triphosphate; Amiloride; Animals; Aorta, Thoracic; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cattle; Cells, Cultured; Dinoprost; Endothelium, Vascular; Epoprostenol; Hydrogen-Ion Concentration; Radioimmunoassay

Rabbit coronary microvascular endothelial (RCME) cells synthesize prostaglandin (PG) I2 and PGE2 in response to stimulation with human thrombin, ATP, and the Ca2+ ionophore, A23187. Replacement of extracellular Na+ with choline or N-methylglucamine reduced thrombin-stimulated PG secretion but did not significantly affect either ATP- or A23187-stimulated PG secretion. Pretreatment of RCME cells with Na+ channel or Na+ -Ca2+ exchange blockers did not alter PG release in response to any of these three agonists. Pretreatment of RCME cells with the specific Na+ -H+ antiport blockers 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 microM) and 5-(N,N-hexamethylene)-amiloride (HMA, 0.1 microM) significantly reduced thrombin but not A23187- or ATP-stimulated PG secretion. Studies with the intracellular pH indicator dye 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein demonstrated thrombin activation of Na+ -H+ antiport, an effect blocked by either HMA or EIPA. Since manipulations known to inhibit Na+ -H+ exchange (EIPA, HMA, replacement of Na+ with choline or N-methylglucamine) reduced thrombin-stimulated RCME cell PG release, we conclude that activation of Na+ -H+ exchange is involved in the coupling of thrombin interaction with RCME cells to subsequent phospholipase activation and PG release.

Topics: Adenosine Triphosphate; Amiloride; Animals; Biological Transport; Calcimycin; Carrier Proteins; Cells, Cultured; Coronary Vessels; Dinoprostone; Endothelium, Vascular; Epoprostenol; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Microcirculation; Prostaglandins; Protons; Rabbits; Sodium; Sodium-Hydrogen Exchangers; Thrombin