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

J. E. Freeman, W. Y. Kuo, G. Milligan, C. J. Lowenstein, M. A. Levine, N. A. Flavahan

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


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 was achieved using antisera specific for the Gi-2 α-subunit, but not with antisera specific for the Gi-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 Gi-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.

Original languageEnglish (US)
Pages (from-to)321-330
Number of pages10
Issue number4
StatePublished - Jan 1 1995


  • NO
  • bacterial toxins
  • coronary artery
  • endothelial dysfunction
  • immunoprecipitation
  • nitric oxide synthase

ASJC Scopus subject areas

  • Physiology
  • Cell Biology


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