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Department of Pharmacology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.

1. The effects of cyclic AMP on contraction and Ca(2+)-activated K+ currents induced by carbachol (CCh), caffeine and inositol 1,4,5-trisphosphate (InsP3) were examined in intact and skinned smooth muscle fibres and in whole-cell voltage-clamped smooth muscle cells of the rat stomach. Intracellular Ca2+ level, [Ca2+]i, was monitored in intact muscle fibres loaded with Fura-2. 2. In intact muscle fibres, dibutyryl cyclic AMP, 8-bromo-cyclic AMP and forskolin inhibited a phasic contraction induced by CCh (100 microM) much more extensively than that induced by caffeine (30 mM) in Ca(2+)-free solution containing 2 mM-EGTA. A rise in [Ca2+]i evoked by CCh was also reduced by dibutyryl cyclic AMP. 3. In skinned muscle fibres, InsP3 (40 microM) produced a contraction of amplitude similar to that evoked by caffeine (30 mM) in Ca(2+)-free solution containing 0.05 mM-EGTA. Cyclic AMP suppressed the InsP3-induced contraction to a much greater extent than that induced by caffeine. 4. In cells voltage-clamped at 0 mV, CCh (100 microM) evoked a transient Ca(2+)-activated outward K+ current in 61% of cells tested. After wash-out of CCh, caffeine (10 mM) evoked a similar K+ current in all cells. In cells loaded with cyclic AMP (100 microM), the percentage of cells responding to CCh was reduced to 26% and the magnitude of current response tended to decrease. Cyclic AMP caused a small increase in the caffeine-induced K+ current. 5. An outward current was elicited immediately after the patch membrane was ruptured at a holding potential of 0 mV, using a patch pipette containing InsP3 (40 microM), in 76% of cells tested. In cells treated with dibutyryl cyclic AMP, the percentage of cells responding to InsP3 was reduced to 50% and the magnitude of current response tended to decrease. 6. In intact muscle fibres loaded with Fura-2, the relationship between [Ca2+]i and tension development shifted to the right in the presence of dibutyryl cyclic AMP. In skinned muscle fibres, cyclic AMP shifted the pCa-tension relation to the right, suggesting that cyclic AMP inhibits the contractile machinery directly. 7. These results suggest that the inhibitory effect of cyclic AMP on muscarinic receptors mediating both the contraction and the Ca(2+)-activated K+ current, is partly due to the inhibition of InsP3-induced Ca2+ release from intracellular stores in rat gastric smooth muscle cells.

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