Purinergic responses in HT29 colonic epithelial cells are mediated by G protein alpha -subunits.
Using Fura-2 to measure changes in intracellular calcium ([Ca(2+)](i)), we show that P(2U)receptors in HT29 cells trigger an increase in [Ca(2+)](i)by pertussis toxin-insensitive G proteins. We then use replication-deficient adenoviruses expressing wild-type and dominant negative mutants of G(alpha q)and G(alpha i2), antisense directed against G(alpha q)or the C-terminal fragment of beta-adrenergic receptor kinase (beta ARK-CT) to identify these G proteins. We find the [Ca(2+)](i)response to UTP is not affected by increased expression of the wild-type G(alpha q), wild-type G(alpha i2)or beta ARK-CT, while it is blocked by over-expression of dominant negative G(alpha q). The timecourse of the UTP response is, however, altered by wild-type G(alpha q)and is only weakly inhibited by antisense G(alpha q). This suggests that the P(2U)response is mediated, at least partially, by a G protein distinct from G(alpha q). In contrast, the M(3)muscarinic response is inhibited by over-expression of antisense against G(alpha q), or over-expression of beta ARK-CT, a finding in agreement with our previous observation that the muscarinic response in HT29 cells is mediated by the beta gamma-subunits of G(q). We also find that P(2U)and M(3)receptors do not control identical Ca(2+)stores, suggesting that differential activation of G proteins can lead to Ca(2+)release from distinct stores.
Cummins
M M
MM
Department of Physiology, University of Sydney, NSW, 2006, Australia.
O'Mullane
L M
LM
Barden
J A
JA
Cook
D I
DI
Poronnik
P
P
D000256:Adenoviridae; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D016373:DNA, Antisense / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D019073:HT29 Cells / Q000187:drug effects; D006801:Humans; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014544:Uridine Triphosphate / Q000494:pharmacology; D051552:beta-Adrenergic Receptor Kinases
D018679:Cholinergic Agonists; D016373:DNA, Antisense; C546385:P2RY2 protein, human; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D002217:Carbachol; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins; D002118:Calcium; D014544:Uridine Triphosphate