The negative inotropic effect of acetylcholine on ferret ventricular myocardium.

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Abstract
The effects of acetylcholine (ACh) on developed tension and intracellular Ca2+ concentration (as measured with aequorin) were studied in ferret papillary muscles, and on twitch shortening, the action potential and membrane currents in ferret ventricular myocytes. Addition of ACh to ferret papillary muscles resulted in decreases in developed tension and the intracellular Ca2+ transient, both of which then partially recovered in the continued presence of ACh (''fade'' of the response). On wash-off of ACh both developed tension and the intracellular Ca2+ transient increased above control (''rebound'') before returning to control values. Addition of ACh to ferret ventricular myocytes resulted in a membrane hyperpolarization of 2 .+-. 0.5 mV (mean .+-. S.E.M.; n = 9), a decrease in action potential duration to 23 .+-. 6% of control and a decrease in twitch shortening to 31 .+-. 5% of control. In the continued presence of ACh these responses to ACh faded. Thirty seconds after the maximal effect of ACh, action potential duration had partially recovered to 34 .+-. 6% of control and twitch shortening to 46 .+-. 7% of control. The effects of ACh on twitch shortening could be mimicked under voltage clamp by varying clamp pulse duration to simulate the ACh-induced changes in action potential duration. When ACh was applied during a train of voltage clamp pulses of constant duration, 81% of the cells showed less than a 20% decrease in Ca2+ current and twitch shortening. However in 19% of the cells twitch shortening and the apparent Ca2+ current decreased by more than 30%. In the 81% of cells, the normal decrease in twitch shortening was wholly the result of the shortening of the action potential. This in turn was the result of an increase in an outward background current which increased the rate of repolarization during the action potential. The ACh-induced background current reversed at -89 .+-. 2 mV and showed inward-going rectification; these properties suggest that it was carried by K+. In the 19% of cells, the normal decrease in twitch shortening was only partly the result of the shortening of the action potential (due to both the increase in outward background current as well as the apparent decrease in Ca2+ current). In these cells the decrease in twitch shortening may also have been partly the direct result of the apparent decrease of Ca2+ current. After 7 h incubation in normal solution, the effect of ACh was still present, and could be blocked by the specific muscarinic receptor blocker atropine (20 .mu.mol/l). After 7 h incubation with pertussis toxin (200 ng/ml), ACh decreased shortening by less than 20%. It is concluded that ACh acts via the muscarinic receptor and its effect is mediated by a GTP-binding protein. A mechanism for the negative inotropic effect of ACh is proposed. The increase of outward background current (and in some cells a decrease of Ca2+ current) increases the rate of repolarization and decreases action potential duration. The shortening of the action potential in all cells and the reduction in Ca2+ current in a few cells decreases the size of the Ca2+ transient, and thus decreases twitch shortening or developed tension. Fade of all responses (apart from the decrease in Ca2+ current, which did not fade) is a result of the fade of the ACh-induced outward background current.