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

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 G_iα-2 and G_iα-3 protein. Immunoprecipitation of solubilized, ₃₂P-ADP ribosylated protein was achieved using antisera specific for the G_i-2 α-subunit, but not with antisera specific for the G_i-3 α-subunit. Serotonin, bradykinin, and mastoparan (a direct G-protein activator) increased the release of a dilator mediator from porcine endothelial cells. The dilator activity was abolished when the endothelial cells were incubated with L-NAME (3 × 10₋₅M), an inhibitor of EDRF-NO synthase. Treatment of the endothelial cells with pertussis toxin (100 ng/ml) did not affect the basal release of EDRF-NO nor the endothelial response to bradykinin but it markedly reduced the responses evoked by serotonin or mastoparan. Therefore, these results suggest that serotonergic and α₂-adrenergic receptors are coupled to a G_i-2 protein in porcine endothelial cells and that activation of this G-protein either via membrane-bound receptors or directly by mastoparan causes the release of EDRF-NO.