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Pflügers Archiv

, Volume 444, Issue 5, pp 644–653 | Cite as

Antisense co-suppression of Gα q and Gα 11 demonstrates that both isoforms mediate M3-receptor-activated Ca2+ signalling in intact epithelial cells

  •  M. Cummins
  •  L. O'Mullane
  •  J. Barden
  •  D. Cook
  •  P. Poronnik
Original Article

Abstract.

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.

Antisense Ca2+ signalling Colonic epithelia G protein Muscarinic receptors 

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Copyright information

© Springer-Verlag 2002

Authors and Affiliations

  •  M. Cummins
    • 1
  •  L. O'Mullane
    • 1
  •  J. Barden
    • 2
  •  D. Cook
    • 1
  •  P. Poronnik
    • 1
  1. 1.Department of Physiology, University of Sydney, Sydney, 2006 Australia
  2. 2.Department of Anatomy, University of Sydney, Sydney, 2006 Australia

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