Gi proteins and the response to 5-hydroxytryptamine in porcine cultured endothelial cells with impaired release of EDRF

Abstract

1. The receptor-mediated release of endothelium-derived relaxing factor(s) (EDRF) requires the presence of different functional G proteins in endothelial cells. Release of EDRF in response to 5-hydroxytryptamine (5-HT), which involves activation of pertussis toxin-sensitive Gi proteins, is impaired in both regenerated endothelium of the coronary artery following balloon catheterization and in porcine cultured endothelial cells. This study used porcine cultered endothelial cells as a model of regenerated endothelium to determine if the abnormal release of EDRF in response to 5-HT may be associated with the loss of functional pertussis toxin-sensitive Gi proteins. 2. Binding studies on porcine cultured endothelial cells demonstrated specific binding sites for [3H]-5-HT. Scatchard analyses revealed a single binding site for [3H]-5-HT with K(d) of 7.2 ± 3.5 nM and maximal binding (B(max)) of 121.4 ± 51.3 fmol mg-1 protein. Binding of [3H]-5-HT was displaced by methiothepin (5-HT1 and 5-HT2 antagonist; K(i) = 6.2 ± 1.2 nM), but not by ketanserin (preferential 5-HT2 antagonist). 3. Giα1 protein was expressed in cultured but not in native endothelial cells. Giα2 and Giα3 proteins were expressed to significant levels in porcine native and cultured endothelial cells, as defected by Northern and Western blot analysis. 4. In membranes from cultured endothelial cells, two bands of 40 and 41 kDa. which corresponded to the Giα2 and the combination of Giα3-Giα1 proteins, respectively, were ADP-ribosylated by pertussis toxin. The labelling intensity was Giα2 > Giα3-Giα1 and the amount of ADP-ribosylation was not different between porcine native and cultured endothelial cells. Stimulation of the cultured cells with 5-HT (3 x 10-6 M; 4 min) decreased significantly further ADP-ribosylation of Giα2 by pertussis toxin, but not that of Giα3 and/or Giα1. 5. The present results suggest that porcine endothelial cell culture may lead to the abnormal expression of Giα1 protein and that the dysfunctional release of EDRF from cultured porcine endothelial cells in response to 5-HT is not associated with the loss of Giα proteins or the absence of 5-HT binding sites.