Thrombin-stimulated endothelial cells produce platelet-activating factor (PAF) in a dose-dependent manner: the activation of a Ca2+-dependent lyso-PAF acetyltransferase is the rate-limiting step in this process. The present study shows that acetyltransferase activation and consequent PAF production induced by thrombin in human endothelial cells are markedly inhibited in Na+-free media or after addition of the amiloride analog 5-(N-ethyl-N-isopropyl)amiloride, suggesting that a Na+/H+ antiport system is present in endothelial cells and plays a prominent role in thrombin-induced PAF synthesis. Accordingly, thrombin elicits a sustained alkalinization in 6-carboxyfluorescein-loaded endothelial cells, that is abolished in either Na+-free or 5-(N-ethyl-N-isopropyl)amiloride-containing medium. Extracellular Ca2+ influx induced by thrombin (as measured by quin2 and 45Ca methods) is completely blocked in the same experimental conditions, and monensin, a Na+/H+ ionophore mimicking the effects of the antiporter activation, evokes a dose-dependent PAF synthesis and a marked Ca2+ influx, which are abolished in Ca2+-free medium. An amiloride-inhibitable Na+/H+ exchanger is present in the membrane of human endothelial cells, its apparent Km for extracellular Na+ is 25 mM, and its activity is greatly enhanced when the cytoplasm is acidified. These results suggest that Na+/H+ exchange activation by thrombin and the resulting intracellular alkalinization play a direct role in the induction of Ca2+ influx and PAF synthesis in human endothelial cells.

Role of Na+/H+ exchange in thrombin-induced platelet-activating factor production by human endothelial cells.

GHIGO, Dario Antonio;BUSSOLINO, Federico;GARBARINO, Giovanni;TURRINI, Francesco Michelangelo;PESCARMONA, Gianpiero;BOSIA, Amalia
1988

Abstract

Thrombin-stimulated endothelial cells produce platelet-activating factor (PAF) in a dose-dependent manner: the activation of a Ca2+-dependent lyso-PAF acetyltransferase is the rate-limiting step in this process. The present study shows that acetyltransferase activation and consequent PAF production induced by thrombin in human endothelial cells are markedly inhibited in Na+-free media or after addition of the amiloride analog 5-(N-ethyl-N-isopropyl)amiloride, suggesting that a Na+/H+ antiport system is present in endothelial cells and plays a prominent role in thrombin-induced PAF synthesis. Accordingly, thrombin elicits a sustained alkalinization in 6-carboxyfluorescein-loaded endothelial cells, that is abolished in either Na+-free or 5-(N-ethyl-N-isopropyl)amiloride-containing medium. Extracellular Ca2+ influx induced by thrombin (as measured by quin2 and 45Ca methods) is completely blocked in the same experimental conditions, and monensin, a Na+/H+ ionophore mimicking the effects of the antiporter activation, evokes a dose-dependent PAF synthesis and a marked Ca2+ influx, which are abolished in Ca2+-free medium. An amiloride-inhibitable Na+/H+ exchanger is present in the membrane of human endothelial cells, its apparent Km for extracellular Na+ is 25 mM, and its activity is greatly enhanced when the cytoplasm is acidified. These results suggest that Na+/H+ exchange activation by thrombin and the resulting intracellular alkalinization play a direct role in the induction of Ca2+ influx and PAF synthesis in human endothelial cells.
263
19437
19446
GHIGO D; F. BUSSOLINO; GARBARINO G; HELLER R; TURRINI F; PESCARMONA G; CRAGOE EJ JR; PEGORARO L; BOSIA A
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/39569
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