Mechanisms and significance of calcium entry at positive membrane potentials in guinea-pig ventricular muscle cells.

Possible mechanisms for calcium entry at positive membrane potentials were investigated in single cells isolated from guinea-pig ventricular muscle. The cells were voltage clamped and contraction was measured by an optical technique. When prolonged (200 ms to 2 s) depolarizations at +60 mV were applied, contraction amplitude increased with pulse duration, in contrast to the contraction at 0 mV. When a 'pre-pulse' to 0 mV was applied for 200 ms to inactivate current through 'L-type' calcium channels, contraction nevertheless increased with membrane potential during a subsequent test pulse applied over the range -40 to +60 mV. Contraction during the test pulse at +60 mV was abolished when extracellular calcium was reduced to zero. This effect developed more rapidly than abolition of the contraction in response to the pre-pulse to 0 mV. Reduction of extracellular calcium from 2.5 to 1 mM reduced the contraction at +60 mV to a greater extent than that at 0 mV and caused an inward shift in the current at +60 mV. Nifedipine (5 microM) substantially reduced the contraction during the test pulse to 0 mV but had little effect on the contraction at +60 mV. Conversely, dodecylamine (20 microM) caused little or no decrease in the contraction at 0 mV but substantially reduced the contraction at +60 mV. Following a conditioning pre-pulse to 0 mV the contraction at +60 mV was not consistently reduced by exposure to 3 microM-ryanodine. The interpolation of a single 200 ms pulse to +60 mV in a train of pulses to 0 mV potentiated the following contraction to 0 mV. This potentiation decayed over the first four steps to 0 mV following an interpolated pulse and increased with the voltage of the interpolated pulse over the range -20 to +60 mV. Potentiation was abolished on exposure to 3 microM-ryanodine. These observations are consistent with entry of calcium at positive membrane potentials through voltage-dependent, non-inactivating pathways which are insensitive to nifedipine but inhibited by dodecylamine. The observations support the hypothesis that calcium entry via this mechanism may contribute, at least under some conditions, to the loading of intracellular stores of calcium during the late plateau of the action potential, and thus influence subsequent contraction. Calcium entry through Na+-Ca2+ exchange is a possibility which would allow calcium entry to increase over the range of membrane potentials at which contraction was increased. However, additional calcium entry through other nifedipine-insensitive pathways, such as calcium-activated non-selective channels, cannot be excluded.