Regulation of the Na, K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37°C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aiNa) was calculated from the voltage difference between an Na+ microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aik) or chloride (aicl) was measured by a third separate microelectrode. Strands were loaded by Na, K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10-8 M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aiNa ranging between 25 and 90 mM and Vm was -30.8 ± 2.2 mV. The Na, K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 ± 5.9 mV with Rb+ activation and to -66.0 ± 7.7 mV with K+ activation, aiNa decreased in both cases to 0.5 ± 0.2 mM in 5-15 rain. The maximum rate of aiNa decline (maximum δaiNa/δt) was the same with K+ and Rb+ at concentrations >20 mM. The response was abolished by 10-5 M acetylstrophantidin. Maximum δaiNa/δt was independent of outside Na+, while ak was negatively correlated with aiNa, (aiK = 88.4 - 0.86·aiNa)· aicl decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aiNa. This model provided a functional isolation of the Na, K pump, so that the relation between the pump rate (δaiNa/δt) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1. K0.5 was 10.5 ± 0.6 mM (the aiNa that gives δaiNa/δt = Vmax/2) and n equaled 1.94 ± 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g−, giving a maximum Na+ turnover of 300 s-1. The Na, K pump in intact Purkinje strands exhibited typical sig-moidal saturation kinetics with regard to aiNa as described by the equation v/ Vmax = ai(1.94)Na/(95.2 + ai(1.94)Na). The maximum sensitivity of the Na, K pump to aiNa occurred at ~6 mM.
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