Na, K pump stimulation by intracellular Na in isolated, intact sheep cardiac purkinje fibers

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 (V_m) was measured by an open-tipped glass electrode and intracellular Na⁺ activity (aⁱ_Na) was calculated from the voltage difference between an Na⁺ microelectrode (ETH 227) and V_m. In some experiments, intracellular potassium (aⁱ_k) or chloride (aⁱ_cl) 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 aⁱ_Na ranging between 25 and 90 mM and V_m was -30.8 ± 2.2 mV. The Na, K pump was reactivated with 30 mM Rb⁺ or K⁺. V_m increased over 50-60 s to -77.4 ± 5.9 mV with Rb⁺ activation and to -66.0 ± 7.7 mV with K⁺ activation, aⁱ_Na decreased in both cases to 0.5 ± 0.2 mM in 5-15 rain. The maximum rate of aⁱ_Na decline (maximum δaⁱ_Na/δt) was the same with K⁺ and Rb⁺ at concentrations >20 mM. The response was abolished by 10^-5 M acetylstrophantidin. Maximum δaⁱ_Na/δt was independent of outside Na⁺, while ak was negatively correlated with aⁱ_Na, (aⁱ_K = 88.4 - 0.86·aⁱ_Na)· aⁱ_cl decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aⁱ_Na. This model provided a functional isolation of the Na, K pump, so that the relation between the pump rate (δaⁱ_Na/δt) and aⁱ_Na could be examined. A Hill plot allowed calculation of V_max 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 aⁱ_Na that gives δaⁱ_Na/δt = V_max/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 aⁱ_Na as described by the equation v/ V_max = a^i(1.94)_Na/(95.2 + a^i(1.94)_Na). The maximum sensitivity of the Na, K pump to aⁱ_Na occurred at ~6 mM.