Pucovsky_1998_Eur.J.Pharmacol_346_323

Cationic current (Icat) and inhibition of the voltage-dependent Ca2+ current (ICa) evoked by muscarinic receptor activation with carbachol were studied using whole-cell patch clamp technique in smooth muscle cells isolated from longitudinal muscle of guinea pig small intestine. With low buffering of [Ca2+]i (0.1 mM BAPTA [1,2-bis-(2-aminophenoxy)-ethane-N,N, N', N'-tetraacetic acid] in pipette solution) Icat and ICa inhibitory responses had a rapid onset to an initial peak followed by a sustained phase. The sustained phase of ICa suppression was bigger than in the case when [Ca2+]i was clamped to 100 nM, but decreased with repeated stimulation. Upon repeated stimulation with 50 microM carbachol in cells where [Ca2+]i was clamped to 100 nM and when GTP was absent, Icat amplitude decreased strongly and more substantially compared to ICa inhibition, but both responses declined only slightly when 1 mM GTP was present in the pipette solution. GDP-betaS (1 or 5 mM) in pipette solution or pre-treatment of cells with pertussis toxin (6 microg/ml, for 4 h or longer) blocked Icat more than ICa suppression by carbachol, whereas L-NAME (N-omega-nitro-L-arginine methyl ester hydrochloride) (100 microM in pipette solution) affected neither of them significantly. We conclude that the cationic current and the suppression of the voltage-dependent Ca2+ current evoked by muscarinic receptor activation are mediated by pertussis toxin-sensitive G-protein(s) but the latter response was less sensitive to blockade by GDP-betaS and to GTP deficiency in the cell.