1. Individual cells were isolated from adult rats ventricular myocardium by a collagenase digestion procedure. 2. Steady membrane potentials recorded with conventional intracellular glass micro-electrodes from cells in a modified Krebs solution containing 3 . 8 mM-KCl and 0 . 5 mM-CaCl2 were less negative than -40 mV in most cells (-25 . 3 +/- 10 . 9 mV, mean +/- S.D., 211 cells). 3. After addition of the potassium selective ionophore valinomycin (60 nM) to the bathing solution all recorded membrane potentials were more negative than -60 mV (-74 . 8 +/- 7 . 0 mV, sixty-three cells). 4. The internal concentration of potassium in the cells was determined as 120 . 8 +/- 1 . 7 mM (+/- S.E., n = 24) by flame emission spectrometry after centrifugation through silicone oil, using tritiated water and D-[1-14C] mannitol to estimate total and extracellular water in the pellet. 5. In the majority of cells in the standard solution the membrane potential recorded within a few msec of penetration was more negative than -70 mV (-78 . 4 +/- 9 . 7 mV, seventy-three cells). In sixty-six cells penetration initiated an action potential which overshot zero by 31 . 3 +/- 7 . 1 mV. This overshoot was abolished by reducing the external sodium to 0 . 1 of the normal value, and reduced or abolished by addition of tetrodotoxin (30 microM). 6. Modifications of the standard bathing solution which increased the number of cells with steady recorded membrane potentials more negative than -60 mV were: isosmotic substitution of sucrose for NaCl; replacement of NaCl and KCl by sodium isethionate and potassium methyl sulphate; addition of 5 or 10 mM-CaCl2; addition of 10 mM-MnCl2. 7. For cells in solution containing 2 . 5 or 5 . 5 mM-CaCl2, input resistances estimated from the amplitude of hyperpolarizations evoked by 200 msec current pulses were approximately 40 M omega at a resting potential close to -80 mV and became much greater as cells were depolarized. Time constants measured at the resting potential were approximately 8 msec. 8. In certain conditions, repeated spontaneous action potentials were recorded from contracting cells, and in quiescent cells evoked action potentials could be initiated by applying brief depolarizing pulses through the micro-electrode. Action potentials were coincident with contractions. 9. It is concluded that the resting potential of these isolated cells is normally more negative than -70 mV, and that the cells retain the ionic mechanisms necessary for the generation of active currents.

No abstract

Action potentials were recorded from single cells isolated from rat and guinea‐pig ventricular muscle. In rat cells the repolarization showed two distinct phases, referred to as the early and late phases. In guinea‐pig cells there was a maintained plateau. Reducing external sodium by replacement with lithium or choline suppressed the late phase of the action potential in rat cells, and shortened the plateau of the action potential in guinea‐pig cells. Intracellular EGTA abolished contraction while suppressing the late phase of the action potential in rat cells, and shortening the plateau in guinea‐pig cells. Ryanodine (1 μm), which is thought to inhibit the release of calcium from internal stores, suppressed contraction and the late phase of the action potential in rat cells. In guinea‐pig cells, there was no substantial effect of ryanodine (1 μm) on either contraction or the time course of the action potential. The late phase of the action potential in rat cells was suppressed by increasing the external potassium concentration to 12 mm, and enhanced by reducing external potassium to 1.2 mm It is concluded that an inward current activated by internal calcium contributes to the late phase of the action potential in rat cells, and to the plateau in guinea‐pig cells. Two possibilities are a current arising from electrogenic sodium‐calcium exchange, and a current through ion channels activated by calcium. The effects of reducing external sodium would be consistent with either mechanism. The contribution of such an inward current would be expected to be modified by outward currents through a rectifying potassium conductance which varies with external potassium concentration. In the rat, but not the guinea‐pig, the rise in internal calcium which activates the inward current seems to be largely dependent on ryanodine‐sensitive release of calcium from internal stores.

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...

SUMMARY1. Isoprenaline (10 nM to 1 /M) and forskolin (06-100 /M) depolarized single guinea-pig myocytes studied in vitro. Under voltage clamp both agents caused an inward current to flow.2. These effects were abolished by propranolol (100 nM) and the /,1-antagonist metoprolol (100-200 nM), but not by the fl2-antagonist salbutamol (1 ,FM).3. The interaction of isoprenaline with forskolin, caffeine or isobutylmethylxanthine (IBMX) on current amplitude was as expected if all of these drugs were causing inward current by increasing intracellular levels of cyclic adenosine monophosphate (cyclic AMP). Low concentrations of forskolin (< 600 nM) or IBMX (< 20 /M) potentiated the effect of isoprenaline, whereas isoprenaline caused no further inward current in cells in which high concentrations of forskolin (600 nM-100 /SM) or IBMX (20 /SM-1 mM) were already evoking maximum inward current.4. Isoprenaline-induced inward current was reduced 30-50 % by acetylcholine (10-30 /tM). This action of acetylcholine was blocked by atropine (100 nM).5. The effect of isoprenaline on holding current was critically dependent on temperature. The onset of the current was delayed and its amplitude reduced as the myocyte was cooled from 37°C to ambient temperature (22-24°C).6. Isoprenaline-induced inward current was not affected by the potassium channel blockers barium (2 mM) or tetraethylammonium (TEA; 10-20 mM). T. M. EGAN AND OTHERS9. The inward current was absent when external sodium was replaced by the impermeant ion tetramethylammonium (TMA).10. Isoprenaline-and forskolin-induced inward currents were associated with an increase in both membrane chord conductance and noise. The increase in conductance was most readily measured at potentials where the inwardly rectifying potassium channel, iK1, was small, or when iKl was blocked by the addition of barium (2 mM).11. The I-V relationship for the total current caused by isoprenaline or forskolin also reflected the effects of these drugs on the calcium current (ica) and the delayed rectifier (iK). When the contributions of ica and iK to the I-V curve were minimized, the remaining inward current reversed at positive potentials. Extrapolation of the linear portion of the I-V curve suggested that the true reversal potential was close to that for sodium.12. These experiments suggest that agents which increase intracellular cyclic AMP open a channel in guinea-pig ventricular sarcolemma which is permeable to sodium ions.