TY - JOUR
T1 - Analysis of Pertussis Toxin-Sensitive Receptor
T2 - G-Protein Interactions in Native Porcine Endothelial Cells
AU - Freeman, J. E.
AU - Kuo, W. Y.
AU - Milligan, G.
AU - Lowenstein, C. J.
AU - Levine, M. A.
AU - Flavahan, N. A.
N1 - Funding Information:
We would like to thank Allen Spiegel (NIH) for the gift of specific antisera to the G-protein a-subunits. We are also grateful to Ken Stoltzfus and everyone at Stoltzfus Meats, Intercourse, PA for their help in harvesting porcine aortas. This work was supported in part by National Heart, Lung and Blood Institute Grants HL 43365 (NAF) and P50 HL 47212 (NAF) and DK 34281 (MAL).
PY - 1995/1/1
Y1 - 1995/1/1
N2 - 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.
AB - 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.
KW - NO
KW - bacterial toxins
KW - coronary artery
KW - endothelial dysfunction
KW - immunoprecipitation
KW - nitric oxide synthase
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U2 - 10.3109/10623329509024674
DO - 10.3109/10623329509024674
M3 - Article
AN - SCOPUS:0029591260
VL - 3
SP - 321
EP - 330
JO - Endothelium: Journal of Endothelial Cell Research
JF - Endothelium: Journal of Endothelial Cell Research
SN - 1062-3329
IS - 4
ER -