Role of G‐proteins in muscarinic receptor inward and outward currents in rabbit jejunal smooth muscle.

Abstract

1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of rabbit jejunum were held under voltage clamp using patch pipettes and membrane currents measured. The effects of carbachol or caffeine applied externally were examined in cells dialysed with normal pipette solutions or with a solution containing heparin (which blocks receptors for D-myo-inositol 1,4,5-trisphosphate, InsP3), guanosine 5-O-(.gamma.-thio)triphosphate (GTP.gamma.s) or guanosine 5-O-(.beta.-thio)diphosphate (GDP.beta.S). 2. Outward current in response to application of carbachol or caffeine was considered to represent the opening of calcium-activated potassium channels in response to a localized rise in the free ionized calcium concentration occasioned by the rapid discharge of stored calcium (Ca) by these agents. 3. Heparin included in the pipette solution blocked outward current to muscarinic receptor activation by carbachol but not that to caffeine, suggesting that receptor-evoked discharge of stored cellular Ca is caused by InsP3 action. However, heparin did not affect muscarinic-receptor inward current. 4. After dialysis with 0.1-0.5 mM-GTP.gamma.S, carbachol inward current was evoked in two out of three of the cells; after dialysis with 0.1-0.2 mM GTP.gamma.S for an average of 7.7 min it was 80% of the normal response; after dialysis for an average of 8.6 min with 0.5 mM-GTP.gamma.S it was 31% of the normal response. In contrast, 0.1 mM-GTP.gamma.S reduced caffeine outward current by 93% after an average 4.5 min dialysis and spontaneous transient outward currents (STOCs) were abolished in 2.9 min on average. 5. Carbachol inward current (at -40 or -50 mV) and carbachol outward current (at 0 mV) in responding cells were reduced only by half after 8-10 min dialysis with 1 mM-GDP.beta.S which has been shown in portal vein cells to antagonize the depletion of Ca stores by intracellular GTP.gamma.S (Komori and Bolton, 1989). After 8-10 min dialysis with 5 mM-GDP.beta.S outward current was 27% of normal. However, if GDP.beta.S was present, outward current generally could not be evoked by a second application of carbachol. 6. The discharge of Ca stores by dialysis with 0.1 mM-GTP.gamma.S was prevented completely by heparin included in the pipette solution, suggesting that activation of a G-protein associated with phospholipase C(PLC) enzyme accelerates PLC activity, InsP3 production and depletion of Ca stores. However, dialysis with GDP.beta.S or heparin did not affect Ca storage or discharge of STOCs suggesting that any tonic production of InsP3 is without effect on Ca storage and that InsP3 does not contribute to the cyclical process which gives rise to STOCs. 7. The results suggest that a G-protein is associated with PLC and InsP3 production. Upon muscarinic receptor activation, InsP3 production is essential for Ca store release. The opening of cationic (inward current) channels in response to muscarinic receptor activation did not have properties typical of a G-protein-mediated system, and if a G-protein is involved it does not seem to be the G-protein-mediated system, and if a G-tproein is involved it does not seem to be the G-protein by which PLC is activated when GTP.gamma.S is introduced into the cell.