Apparent affinity of the Na/K pump for ouabain in cultured chick cardiac myocytes. Effects of Nai and Ko.

Abstract

The measured apparent affinity (K0.5) of the Na/K pump for ouabain has been reported to vary over a wide range. In a previous report we found that changing Na~ could alter apparent affinity by at least an order of magnitude and that the model presented predicted this variability. To increase our understanding of this variability, isolated cells or two-to three-cell clusters of cardiac myocytes from 11-d embryonic chick were used to measure the effects of Na~ and K o on the K05 of the Na/K pump for ouabain. Myocytes were whole-cell patch clamped and Na/K pump current (Ip) was measured in preparations exposed to a Ca-free modified Hank's solution (HBSS) that contained 1 mM Ba, 10 mM Cs, and 0.1 mM Cd. Under these conditions there are no Ko-sensitive currents other than Ip because removal of Ko in the presence of ouabain had no effect on the current-voltage (I-V) relation. The I-V relation for Ip showed that in the presence of 5.4 mM K o and 51 mM Na~, Ip has a slight voltage dependence, decreasing ~ 30% from 0 to -130 mV. Increasing Na~ in the patch pipette from 6 to 51 mM (K o --5.4 mM) caused Ip to increase from 0.46 _ 0.07 (n = 5) to 1.34 -+ 0.08 ~/cm 2 (n = 13) with a K05 for Na i of 17.4 mM and decreased the K05 for ouabain from 18.5 _+ 1.8 (n = 4) to 3.1 _+ 0.4 ~M (n = 3). Similarly, varying Ko between 0.3 and 10.8 mM (Nai = 24 mM) increased Ip from 0.13 _ 0.01 (n = 5) to 0.90 -+ 0.05 txA/cm 2 (n = 5) with a K05 for Ko of 1.94 mM and increased K05 for ouabain from 0.56 -0.14 (n = 3-6) to 10.0 -1.1 I~M (n = 6). All of these changes are predicted by the model presented. A qualitative explanation of these results is that Na~ and Ko interact with the Na/K pump to shift the steady-state distribution of the Na/K pump molecules among the kinetic states. This shift in state distribution alters the probability that the Na/K pump will be in the conformation that binds ouabain with high affinity, thus altering the apparent affinity. In intact cells, the measured apparent affinity represents a combination of all the rate constants in the model and does not equate to simple first-order binding kinetics. The results show that the apparent affinity for ouabain is variable (0.5-20 ~M) and depends on Na i and Ko. Although this range is large, the data can be completely accounted for by the model presented.

Na+‐K+ pump cycle during β‐adrenergic stimulation of adult rat cardiac myocytes

“…To analyse our data further a six-state cyclic model of the Na¤-K¤ pump was used (Chapman, Johnson & Kootsey, 1983;Stimers, Liu & Lieberman, 1991). In the present work the only modification of the model was to include voltage dependence on the rate constants f× and b× for the Na¤ translocation step.…”

“…Additionally, each of the other rate constants were varied to determine if changes in any other rate constants might yield simulations that are consistent with the results of our present and previous work. Table 1 lists initial parameters obtained from previous reports in the literature (Chapman et al 1983;Stimers et al 1991). To determine if this model could predict an increase in Na¤-K¤ pump current observed at 0 mÒ K¸without changing voltage dependence (see previous section) we varied sequentially forward and backward rate constants with indices: 2, 3, 5 and 6.…”

Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors.

“…Previous studies have demonstrated that activation of adenosine receptors in these cultured heart cells produces physiologic effects similar to those elicited by adenosine in the adult mammalian heart (25)(26)(27)(28). The cultured ventricular myocytes contain predominantly ( Ͼ 90%) myocytes (21; our unpublished data) and are largely devoid of neuronal, blood, or vascular cells, thus the confounding influence of changes in blood flow is avoided (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). The present study aims to develop the chick ventricular cell culture as a model to investigate the role of adenosine receptor subtypes in the preconditioning process and to study the mechanism(s) by which preconditioning of the ventricular myocytes can be modulated.…”

“…Ventricular myocytes cultured from chick embryos retain many of the properties of the intact heart and have served as a useful model for a variety of experimental paradigms (18)(19)(20)(21)(22)(23)(24). Previous studies have demonstrated that activation of adenosine receptors in these cultured heart cells produces physiologic effects similar to those elicited by adenosine in the adult mammalian heart (25)(26)(27)(28).…”

The antagonistic effect of K+o and dihydro‐ouabain on the Na+ pump current of single rat and guinea‐pig cardiac cells.

“…N. Hermanrs, H. G. Glitsch and F Verdonck voltage clamp measurements (Stimers, Lobaugh, Liu, Shigeto & Lieberman, 1990) and whole-cell recording (Hainill, Alarty, Neher, Sakmann & Sigworth, 1981) [Na+]i (Stimers, Liu & Lieberman, 1991;Bielen, Glitsch & Verdonck, 1992 [K+]o and tlhus a decrease in the apparent affinitv of the Na+-K+ pump for the drugs (Stimers et al 1991;Bielen et al 1992). According to the latter authors the antagonistic effect of K+ on the inhibition of Ip by dilhydro-ouabain (DHO) in rabbit Purkinje cells is almost entirely due to a reduction of the apparent DHO association rate constant, w7hich is in agreement with an earlier study on a multicellular guinea-pig ventricular preparation (Daut, 1983).…”