MORPHOLOGICAL HETEROGENEITY WITH NORMAL EXPRESSION BUT ALTERED FUNCTION OF G-PROTEINS IN PORCINE CULTURED REGENERATED CORONARY ENDOTHELIAL-CELLS

1 Experiments were designed to investigate whether che pertussis toxin -dependent endothelial dysfunction following balloon injury is due to a reduced expression or an insufficient function of G-proteins. 2 Endo thelium-dependent responses of porcine coronary arteries were examined in vitro by use of conventional organ chambers. Morphological analysi s was performed by isolating and culturing the endothelial cells from these arteries. The expression of Gi-proteins in regenerated endotheli al cells was measured by Western blots and immunolabelling. The functi on of G-proteins was assessed by measuring the GTPase activity of cult ured endothelial cells. 3 Eight days following denudation, endothelial regrowth was confirmed by histological examination and by demonstrati ng the presence of endothelium-dependent relaxations to bradykinin and 5-hydroxytryptamine (5-HT). In primary culture, the regenerated endot helial cells displayed a 'cobblestone' pattern as seen with native end othelial cells. 4 Twenty eight days after denudation, the endothelium- dependent relaxations induced by 5-HT were impaired, but those to brad ykinin were maintained. However, the latter were reduced when endothel ium-dependent hyperpolarization was prevented. 5 Twenty eight days aft er denudation, muitinucleated giant cells were present in the regenera ted but not in the native cultured endothelial cell populations. These regenerated endothelial cells incorporated less tritiated thymidine t han native endothelial cells. 6 The intensities of the bands on the im munoblot of the regenerated endothelial cells, when several antibodies against Gi alpha 1/alpha 2/alpha 3 were used, were the same as those obtained in native endothelial cells. The immunolabelling with the sam e antibodies was similar between the giant cells and the regenerated e ndothelial cells of normal size. The hydrolysis of GTP was lower in re generated than in native endothelial cell membranes. 7 In conclusion, endothelium-dependent relaxations mediated by Gi-proteins are impaired in balloon denuded coronary arteries. This dysfunction following rege neration cannot be explained by a reduced expression of Gi proteins bu t rather reflects an abnormal function of the G-proteins in the regene rated endothelium.