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 Endothelium-dependent responses of porcine coronary arteries were examined in vitro by use of conventional organ chambers. Morphological analysis was performed by isolating and culturing the endothelial cells from these arteries. The expression of Gi-proteins in regenerated endothelial cells was measured by Western blots and immunolabelling. The function of G-proteins was assessed by measuring the GTPase activity of cultured endothelial cells. 3 Eight days following denudation, endothelial regrowth was confirmed by histological examination and by demonstrating the presence of endothelium-dependent relaxations to bradykinin and 5-hydroxytryptamine (5-HT). In primary culture, the regenerated endothelial cells displayed a 'cobblestone' pattern as seen with native endothelial cells. 4 Twenty eight days after denudation, the endothelium-dependent relaxations induced by 5-HT were impaired, but those to bradykinin were maintained. However, the latter were reduced when endothelium-dependent hyperpolarization was prevented. 5 Twenty eight days after denudation, muitinucleated giant cells were present in the regenerated but not in the native cultured endothelial cell populations. These regenerated endothelial cells incorporated less tritiated thymidine than native endothelial cells. 6 The intensities of the bands on the immunoblot 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 same antibodies was similar between the giant cells and the regenerated endothelial cells of normal size. The hydrolysis of GTP was lower in regenerated 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 regeneration cannot be explained by a reduced expression of Gi proteins but rather reflects an abnormal function of the G-proteins in the regenerated endothelium.