Role of Na+/H+ exchange in thrombin-induced platelet-activating factor production by human endothelial cells.
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.
Ghigo
D
D
Department of Genetics, Biology, and Medical Chemistry, University of Torino, Italy.
Bussolino
F
F
Garbarino
G
G
Heller
R
R
Turrini
F
F
Pescarmona
G
G
Cragoe
E J
EJ
Pegoraro
L
L
Bosia
A
A
D000123:Acetyltransferases / Q000378:metabolism; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008985:Monensin / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D017923:Sodium-Hydrogen Exchangers; D013917:Thrombin / Q000502:physiology*; D014471:Umbilical Veins / Q000378:metabolism
D002352:Carrier Proteins; D010972:Platelet Activating Factor; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D008985:Monensin; D000123:Acetyltransferases; D013917:Thrombin; D002118:Calcium; C039614:ethylisopropylamiloride