Analysis of Pertussis Toxin-Sensitive Receptor: G-Protein Interactions in Native Porcine Endothelial Cells

Abstract

Endothelium-dependent relaxations evoked by activation of α2-adrenergic and serotonergic receptors are reduced by pertussis toxin, which irreversibly inhibits the activity of certain G-proteins. The present experiments were conducted in order to further characterize the inhibitory effect of the toxin and to directly analyze receptor: G-protein interactions in native porcine endothelial cells. Cell membranes, prepared from freshly harvested endothelial cells, were incubated with pertussis toxin in the presence of 32P-NAD and labelled proteins were separated on SDS-PAGE. Pertussis toxin catalyzed the transfer of 32P-ADP-ribose from NAD to a 40 kD protein. The toxin-catalyzed ADP-ribosylation was not significantly affected by bradykinin but was reduced by serotonin, mastoparan (a direct G-protein activator) or UK 14, 304, an α2-adrenergic agonist. Western blot analysis demonstrated that porcine endothelial cells express Giα-2 and Giα-3 protein. Immunoprecipitation of solubilized, 32P-ADP ribosylated protein ...