Effect of internal sodium and cellular calcium load on voltage-dependence of the Indo-1 transient in guinea-pig ventricular myocytes

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Abstract
To investigate the effect of altering internal Na and cell Ca load on the voltage-dependence of the intracellular Ca transient.Methods: Ventricular myocytes were isolated enzymatically from the guinea-pig heart. They were patch clamped and dialysed internally with pipette solutions which contained either 0 or 10 or 20 mM Na. Intracellular Ca was monitored with Indo-1 and experiments were carried out at 36 °C. A standard level of Ca loading was established before each test pulse by applying a train of conditioning pulses. The voltage-dependence of the Ca i (Indo-1) transient provided information about the mechanisms which trigger Ca release from the sarcoplasmic reticulum (SR).Results: The voltage-dependence of L-type Ca current (ICa,L) was assessed in separate experiments by dialysing myocytes with a Cs-based solution. ICa,L had a maximum amplitude at 0 mV, declined at more positive potentials and there was little net inward ICa,L at + 100 mV. The rapid initial phasic component of the Indo-1 transient was abolished by ryanodine/thapsigargin; therefore, this component reflected the magnitude of SR Ca release. In cells dialysed with 10 mM Na, the voltage-dependence of the Indo-1 transient was different from ICa,L. The Indo-1 transient became maximal at +20 mV, and the decline of the Indo-1 transient at positive potentials was less steep than the decline of ICa,L. A large proportion of the phasic Indo-1 transient could remain at positive potentials where there was no detectable ICa,L. Increasing dialysing Na from 10 to 20 mM led to a marked change in voltage-dependence of the Indo-1 transient. With 20 mM Na, the amplitude of the phasic Indo-1 transient remained large between +20 and +100 mV. Removing Na from the pipette dialysis solution had the opposite effect on voltage-dependence of the transient. For each dialysing [Na], the level of cellular and SR Ca content was altered by varying the potential of conditioning pulses applied before each test pulse. There was no significant effect on voltage-dependence of the Indo-1 transient of either increasing or reducing the cellular Ca content.Conclusion: These data are consistent with the hypothesis that the voltage-dependence of the Ca i transient results from the sum of the voltage-dependencies of the two main trigger mechanisms— ICa,L and reverse Na/Ca exchange. When a myocyte was dialysed with Na-free solution, the voltage-dependence of the Ca i transient became more similar (but not identical) to that for ICa,L. With 20 mM Na dialysis, the altered voltage-dependence of the Ca i transient may reflect an increased trigger influence of reverse Na/Ca exchange.

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