Antisense co-suppression of G(alpha q) and G(alpha 11) demonstrates that both isoforms mediate M-3-receptor-activated Ca2+ signalling in intact epithelial cells

Cummins, M. M., O'Mullane, L. M., Barden, J. A., Cook, D. I. and Poronnik, P. (2002) Antisense co-suppression of G(alpha q) and G(alpha 11) demonstrates that both isoforms mediate M-3-receptor-activated Ca2+ signalling in intact epithelial cells. Pflugers Archiv-european Journal of Physiology, 444 5: 644-653. doi:10.1007/s00424-002-0856-3


Author Cummins, M. M.
O'Mullane, L. M.
Barden, J. A.
Cook, D. I.
Poronnik, P.
Title Antisense co-suppression of G(alpha q) and G(alpha 11) demonstrates that both isoforms mediate M-3-receptor-activated Ca2+ signalling in intact epithelial cells
Journal name Pflugers Archiv-european Journal of Physiology   Check publisher's open access policy
ISSN 0031-6768
Publication date 2002-08
Sub-type Article (original research)
DOI 10.1007/s00424-002-0856-3
Volume 444
Issue 5
Start page 644
End page 653
Total pages 10
Place of publication New York
Publisher Springer-verlag
Language eng
Abstract We used replication-deficient adenoviruses overexpressing antisense against G(q) class alpha-subunits to determine the roles of G(q) and G(11) in mediating M-3-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 alpha-subunits of G(q) or G(11) produced isoform-specific reductions in the levels of these alpha-subunits. Fura-2 was used to measure changes in the Ca2+ response following activation of the M-3 receptors by carbachol. The G(alphaq) antisense virus suppressed the peak Ca2+ response by 70%, whereas the G(alpha11) antisense virus reduced it by 34%. We then used co-infection with both viruses to determine the effect of concomitant suppression of both G(alphaq) and G(alpha11). Overexpression of antisense to both alpha-subunits reduced by approximately 50% the levels of both G(alphaq) and G(alpha11). It also almost completely inhibited the Ca2+ response to carbachol. These data show that both G(q) and G(11) are involved in mediating the action of the M-3 receptor on cytosolic Ca2+ in HT29 cells. Furthermore, they suggest that the coupling of the M-3 receptor to these G proteins is specific, in that G(alphaq) cannot substitute for G(alpha11), and vice versa.
Keyword Physiology
antisense
Ca2+ signalling
colonic epithelia
G protein
muscarinic receptors
Replication-deficient Adenoviruses
Protein-alpha-subunits
Phosphatidylinositol 4,5-bisphosphate Hydrolysis
Portal-vein Myocytes
Phospholipase-c
Muscarinic Inhibition
Signaling Complexes
Down-regulation
Receptors
G(q)
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Biomedical Sciences Publications
 
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