Antisense co-suppression of Gα q and Gα 11 demonstrates that both isoforms mediate M3-receptor-activated Ca2+ signalling in intact epithelial cells
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We used replication-deficient adenoviruses overexpressing antisense against Gq class α-subunits to determine the roles of Gq and G11 in mediating M3-receptor-coupled Ca2+ mobilization in intact HT29 human colonic carcinoma epithelial cells. Western blot analysis and confocal microscopy showed that the viruses expressing antisense directed against the α-subunits of Gq or G11 produced isoform-specific reductions in the levels of these α-subunits. Fura-2 was used to measure changes in the Ca2+ response following activation of the M3 receptors by carbachol. The Gα q antisense virus suppressed the peak Ca2+ response by 70%, whereas the Gα 11 antisense virus reduced it by 34%. We then used co-infection with both viruses to determine the effect of concomitant suppression of both Gα q and Gα 11. Overexpression of antisense to both α-subunits reduced by approximately 50% the levels of both Gα q and Gα 11. It also almost completely inhibited the Ca2+ response to carbachol. These data show that both Gq and G11 are involved in mediating the action of the M3 receptor on cytosolic Ca2+ in HT29 cells. Furthermore, they suggest that the coupling of the M3 receptor to these G proteins is specific, in that Gα q cannot substitute for Gα 11, and vice versa.
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