Characterization of Na(+)-K+ homeostasis of cultured human skin fibroblasts in the presence and absence of fetal bovine serum.

Previously, we demonstrated that removal of fetal bovine serum (FBS) from the medium of human skin fibroblasts resulted in an accelerated 86Rb+ washout, decreased cellular K+, and increased Na+ contents. In the present study we examined the mechanism underlying these changes. The efflux rate constant for 86Rb+, and the cellular contents of Na+ and K+ were measured. Verapamil (K1/2 = 15 microM) and chlorpromazine (K1/2 = 1 microM) reduced by approximately 70% the increased 86Rb+ washout evoked by FBS removal. The effect of the two drugs was additive at low, but not high, concentrations. Verapamil and chlorpromazine also attenuated the decrease in cellular K+ content and prevented the increase in cellular Na+ content associated with FBS depletion. Bumetanide (50 microM) was only partially effective in offsetting the enhanced 86Rb+ efflux and was completely without any effect on the cellular Na+ and K+ changes induced by FBS removal. In the presence of FBS, A-23187 produced a slight and transient increase of the 86Rb+ washout. The protein kinase C activator phorbol 12-myristate 13-acetate enhanced the 86Rb+ efflux in FBS-containing medium for a prolonged period but this increase was only a fraction of that caused by serum removal. Cellular Na+ and K+ contents were not changed by the phorbol ester. We conclude that FBS removal raises the cellular Na+ content, and enhances 86Rb+ efflux, through a calmodulin-dependent pathway activated by calcium influx.

Kinetics of Ca2(+)-ATPase activation in platelet membranes of essential hypertensives and normotensives.

To explore the etiology of altered Ca metabolism in essential hypertension, we studied parameters, i.e., maximal initial reaction velocity (Vmax) and Michaelis constant (Km), of Ca activation kinetics of Ca2(+)-ATPase in membrane fractions (isolated by a sucrose gradient) from platelets of blacks and whites, 27 of whom were essential hypertensives, 17 of whom were normotensives with a family history of essential hypertension, and 10 of whom were normotensives without a family history of the disease. The Vmax of hypertensives was significantly lower than in normotensives without a family history of essential hypertension (hypertensives, 14.99 +/- 1.71 nmol Pi.mg protein-1.min-1; normotensives, positive family history, 22.67 +/- 3.17 nmol Pi.mg protein-1.min-1; normotensives, negative family history, 27.54 +/- 4.37 nmol Pi.mg protein-1.min-1; overall, P = 0.0078). The Km was lower in both hypertensives and normotensives with a positive family history of essential hypertension as compared with normotensives with a negative family history of the disease (hypertensives, 1.70 +/- 0.23 microM; normotensives, positive family history, 1.38 +/- 0.2 microM; normotensives, negative family history, 2.79 +/- 0.58 microM; overall, P = 0.0251). Furthermore, the Km in whites was inversely related to plasma renin activity (r = 0.50; P less than 0.005). We propose that a lower Vmax for Ca2(+)-ATPase may play a role in the higher level of free Ca in platelets of essential hypertensives and that a higher affinity of the enzyme to Ca may reflect a process compensating for the lower Vmax. We also suggest that a higher Km for Ca2(+)-ATPase in juxtaglomerular cells of whites would result in blunting the release of renin.

[3H]ouabain binding of red blood cells in whites and blacks.

In a previous study, we demonstrated that the red blood cell Na+ concentration and Na+,K+-ATPase activity are sex-dependent and race-dependent: a higher intracellular Na+ concentration in blacks and men was associated with a lower Na+,K+-ATPase activity. To examine whether the low Na+,K+-ATPase activity is due to a decreased number of enzyme units, altered structure of the enzyme, or the presence of an endogenous digoxinlike substance, ouabain binding studies were performed on the same subject group. The measurements included displacement of [3H]ouabain from its specific binding sites by unlabeled ouabain or potassium. The results demonstrate that groups with lower enzyme activity manifest lower numbers of total specific ouabain binding sites on the surface of the red blood cell (mean +/- SD: blacks, 654 +/- 24.4; whites, 806 +/- 18.3; women, 806 +/- 26.9; men, 728 +/- 21.2). Other kinetic parameters of [3H]ouabain displacement appear to be the same among the groups. The respective red blood cell Na+ and K+ concentrations were negatively and positively correlated with the number of ouabain binding sites. Our findings suggest that the lower activity of red blood cell Na+,K+-ATPase in blacks and men is a function of a lower number of Na+-K+ pump units. The results also indicate that sex and race should be considered when red blood cell ouabain binding is examined.

Race and sex differences in erythrocyte Na+, K+, and Na+-K+-adenosine triphosphatase.

Several reports indicate that erythrocytes (RBCs) from blacks and men have higher sodium concentrations than those from whites and women. One possible mechanism to explain this finding is a difference in the activity of Na+-K+-ATPase. To explore this possibility, we have studied the Na+ and K+ kinetics of RBC Na+-K+-ATPase and RBC Na+ and K+ concentrations in 37 normotensive blacks and whites, both males and females. The maximal initial reaction velocity (Vmax) values for RBC Na+-K+-ATPase were lower in blacks and men as compared with whites and women. Higher RBC Na+ levels were observed in blacks and males vs. whites and females. Significant inverse correlations were noted between the Na+-K+-ATPase activity and RBC Na+ concentrations. These findings indicate that cellular Na+ homeostasis is different in blacks and men as compared with whites and women. Since higher RBC Na+ concentrations have also been observed in patients with essential hypertension as compared with normotensive subjects, the higher intracellular Na+ concentrations in blacks and men may contribute to the greater predisposition of these groups to essential hypertension.