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SUMMARY1. A double sucrose-gap voltage-clamp technique has been used to study the effects of acetylcholine on the membrane currents in atrial trabeculae of the bullfrog, Rana catesbeiana.2. The second, or slow inward (Ca2+/Na+) current, was found to be markedly reduced by concentrations of acetylcholine greater than approximately 2-0 x 10-8 M. The resulting decrease in net calcium entry provides a straightforward explanation for the negative inotropic action of acetylcholine in atrial muscle.3. Measurements of membrane resistance near the resting potential showed that relatively high doses of acetylcholine (approximately 10-7 M) decrease membrane resistance by about twofold. This effect is shown to be the result of an increase in a time-independent background current which appears to be carried mainly by potassium ions.4. Using appropriate pharmacological techniques, it has been possible to demonstrate: (i) that the peak slow inward current is reduced to about half its initial value before any significant increase in background current occurs; (ii) that even when a sufficient dose of acetylcholine to produce an increase in background current is used, the background current shows inward-going rectification and cannot account for the observed reduction in the slow inward current.5. No consistent change was observed in the degree of activation of the time-dependent outward membrane currents after application of concentrations of acetylcholine which produced large decreases in the peak slow inward current.6. These results are discussed in relation to previous electro-physiological and radioisotope studies of the mechanism of the negative inotropic effect of acetylcholine in cardiac muscle.

SUMMARY1. In atrial wall trabeculae of Rana catesbeiana and R. ridibunda very slowly decaying membrane currents have been consistently observed in decay tails following voltage clamp depolarizing and hyperpolarizing pulses. It is not thought that these currents are carried by time-dependent conductance channels but rather result from potassium ion accumulation or depletion.2. Since voltage clamp techniques generally impose a non-physiological situation on the membranes of excitable cells, evidence that potassium ion accumulation occurs in unclamped atrial tissue is presented.3. When potassium ions accumulate, the reversal potentials for both atrial delayed conductance mechanisms, iXfat and ix,, should be shifted in a positive direction, the magnitude of the shifts being a function of the charge transferred during depolarization. Experiments have been performed to test this prediction quantitatively, and as a result, a simple accumulation model is developed. 4. A second important effect of accumulation should be upon the timeindependent potassium conductance, iK.. It was found that this effect produces current tails whose decay becomes exponential when the amount of accumulation is small. The time constant of this exponential is shown to be equal to the time constant of decay of accumulation, Tacc. Experimental verification of this assumption is presented. This 'crossover' effect allows current changes due to accumulation to show an apparent 'reversal potential' and so to appear like a conductance mechanism. 6. Potassium depletion is shown to occur during hyperpolarizing pulses.This depletion process must be allowed for in a direct kinetic analysis of the pace-maker current, ih51, at potentials negative to the resting potential (ER).

SUMMARY1. Slow inward tail currents attributable to electrogenic sodium-calcium exchange can be recorded by imposing hyperpolarizing voltage clamp pulses during the normal action potential of isolated guinea-pig ventricular cells. The hyperpolarizations return the membrane to the resting potential (between -65 and -88 m V) allowing an inward current to be recorded. This current usually has peak amplitude when repolarization is imposed during the first 50 ms after the action potential upstroke, but becomes negligible once the final phase of repolarization is reached. The envelope of peak current tail amplitudes strongly resembles that of the intracellular calcium transient recorded in other studies.2. Repetitive stimulation producing normal action potentials at a frequency of 2 Hz progressively augments the tail current recorded immediately after the stimulus train. Conversely, if each action potential is prematurely terminated at 01 Hz, repetitive stimulation produces a tail current much smaller than the control value. The control amplitude of inward current is only maintained if interrupted action potentials are separated by at least one full 'repriming' action potential. These effects mimic those on cell contraction (Arlock & Wohlfart, 1986) and suggest that progressive changes in tail current are controlled by variations in the amplitude and time course of the intracellular calcium transient.3. When intracellular calcium is buffered sufficiently to abolish contraction, the tail current is abolished. Substitution of calcium with strontium greatly reduces the tail current.4. The inward tail current can also be recorded at more positive membrane potentials using standard voltage clamp pulse protocols. In this way it was found that temperature has a large effect on the tail current, which can change from net inward at 22°C to net outward at 37 'C. The largest inward currents are usually recorded at about 30 'C. It is shown that this effect is attributable predominantly to the temperature sensitivity of activation of the delayed potassium current, iK, whose decay can then mask the slow tail current at high temperatures.5. Studies of the relationship between the tail current and the membrane calcium current, iCa, have been performed using a method of drug application which is capable of perturbing ica in a very rapid and highly reversible manner. Partial block of iCa with cadmium does not initially alter the size of the associated inward current T. M. EGAN AND OTHERS tail. When iCa is increased by applying isoprenaline, the percentage augmentation of the associated tail current is much greater but occurs more slowly. Similarly, the tail current recovers to its initial value more slowly than does ica.6. These results are interpreted to indicate that the sodium-calcium exchange current flows during the time course of the cardiac action potential and that its amplitude is more closely related to intracellular calcium release than to the membrane calcium current per se. Calculation of the exchange current flowing durin...

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