Purinergic responses in HT29 colonic epithelial cells are mediated by G protein alpha-subunits

Using Fura-2 to measure changes in intracellular calcium ([Ca2+](i)), we sh ow that P-2U receptors in HT29 cells trigger an increase in [Ca2+](i) by pe rtussis toxin-insensitive G proteins. We then use replication-deficient ade noviruses 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 fr agment of beta-adrenergic receptor kinase (PARK-CT) to identify these G pro teins. We find the [Ca2+](i) response to UTP is not affected by increased e xpression of the wild-type G(alpha g), wild-type G(alpha i2) Or beta ARK-CT , while it is blocked by over-expression of dominant negative G(alpha q). T he 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 tha t the P-2U response is mediated, at least partially, by a G protein distinc t 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 bet a ARK-CT, a finding in agreement with our previous observation that the mus carinic 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 Ca2stores, suggesting that differential activation of G proteins can lead to C a2+ release from distinct stores. (C) 2000 Harcourt Publishers Ltd.