GI PROTEINS AND THE RESPONSE TO 5-HYDROXYTRYPTAMINE IN PORCINE CULTURED ENDOTHELIAL-CELLS WITH IMPAIRED RELEASE OF EDRF

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 pro teins, is impaired in both regenerated endothelium of the coronary art ery following balloon catheterization and in porcine cultured endothel ial cells. This study used porcine cultered endothelial cells as a mod el 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 c ultured endothelial cells demonstrated specific binding sites for [H-3 ]-5-HT. Scatchard analyses revealed a single binding site for [3H]-5-H T 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 [H-3]-5-HT was displaced by methi othepin (5-HT1 and 5-HT2 antagonist; K-i = 6.2 +/- 1.2 nM), but not by ketanserin (preferential 5-HT2 antagonist). 3 Gi alpha 1 protein was expressed in cultured but not in native endothelial cells. Gi alpha 2 and Gi alpha 3 proteins were expressed to significant levels in porcin e native and cultured endothelial cells, as detected by Northern and W estern blot analysis. 4 In membranes from cultured endothelial cells, two bands of 40 and 41 kDa, which corresponded to the Gi alpha 2 and t he combination of Gi alpha 3-Gi alpha 1 proteins, respectively, were A DP-ribosylated by pertussis toxin. The labelling intensity was Gi alph a 2 > Gi alpha 3-Gi alpha 1 and the amount of ADP-ribosylation was not different between porcine native and cultured endothelial cells. Stim ulation of the cultured cells with 5-HT (3 x 10(-6) M; 4 min) decrease d significantly further ADP-ribosylation of Gi alpha 2 by pertussis to xin, but not that of Gi alpha 3 and/or Gi alpha 1. 5 The present resul ts suggest that porcine endothelial cell culture may lead to the abnor mal expression of Gi alpha 1 protein and that the dysfunctional releas e of EDRF from cultured porcine endothelial cells in response to 5-HT is not associated with the loss of Gi alpha proteins or the absence of 5-HT binding sites.