Perturbation of Na+ and K+ gradients in human fibroblasts incubated in unsupplemented saline solutions (original) (raw)
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Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase
Journal of Biological Chemistry, 1994
The objective of this study has been to delineate the side-specific effects of Na' and K+ on the transport kinetics of tissue-specific NaM pumps. Two experimental systems have been used. In one, NaM pumps of exogenous microsomal membrane sources (rat axolemma, kidney) were delivered by membrane fusion into dog erythrocytes, and in the other, the three isoforms of the catalytic subunit of the rat enzyme were individually transfected into HeLa cells as in previous studies (Jewell, E. A, and Lingrel, J. B (1991) J. Biol. Chern. 266, 16925-16930), with the a, and a, isoforms rendered relatively resistant to ouabain by site-directed mutagenesis. Whereas the kidney microsomes comprise the a1 catalytic isoform, the axolemma microsomes were predominantly as (4 0 %) with lesser amounts of a2 (4 5 %) and a1 (-15%) as measured by the ouabain-sensitive profile of phosphoenzyme as well as by immunoblotting with isoform-specific antibodies using membranes of known specific activity as standards (a1 of kidney, a, and a, of muscle). Both systems were analyzed with respect to the effects of varying concentrations of cytoplasmic Na' and extracellular K' on pump-mediated ssRb+(K+) influx. With the individual isoform-transfected HeLa cells and monensin added to vary and control the intracellular Na+ concentration, differences in apparent affinities of the a, isoform compared with the a1 and a2 isoforms were observed, i.e. a &-fold higher affinity for extracellular K' and 4-fold lower affinity for cytoplasmic Na+. Thus, in the presence of 10 m~ extracellular Na' , apparent values for extracellular K' activation of K'(Rb+) influxes were 0.22 * 0.02 m~ for aI, 0.20 t 0.02 m~ for a2, and 0.09 * 0.01 m~ for a,. At high intracellular K+ (2100 m~) and saturating extracellular K+ concentrations, apparent KO, values for cytoplasmic Na+ activation were 17.6 2 1.1 m~ for al, 19.7 = 1.0 m~ for a,, and 63.5 9.1 m~ for a ,. The functional differences observed with the individual isoform-transfected cells were completely consistent with the kinetic differences observed with the axolemma and kidney pumps fused into erythrocytes. kvolemma pumps had a 4-f o l d lower for extracellular K+ and a &-fold higher for cytoplasmic Na'. In the HeLa transfectants, differences in affinities for cytoplasmic Na' were associated with differences in the steady-state intracellular Na' concentration, Le. 27.5 m~ in a,-transfected cells compared with 15.7 and 19.7 m~ in al-and a,-transfected cells, respectively. Research Council of Canada (to R. B.) and ROHIL28573 from the Na-*This work was supported by Grants MT-3876 from the Medical tional Institutes of Health (to J. B. L.). The costs of publication of this article must therefore be hereby marked "aduertisement" in accordance article were defrayed in part by the payment of page charges. This The abbreviations used are: Na,K-ATPase or Na/K pump, sodiumpotassium adenosine triphosphatase; EP, the phosphorylated form of the Na,K-ATPase enzyme;
Journal of Cellular Physiology, 1992
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 μM) and chlorpromazine (K1/2 = 1 μM) 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 μM) 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 i...
Proceedings of the National Academy of Sciences, 1985
The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on Na+ influx were investigated in cultured human foreskin fibroblasts (HSWP cells). We report here that in serum-deprived HSWP cells the addition of PMA alone has no significant effect on Na+ influx. However, the addition of PMA to cells whose Na+/H+ exchanger is partially activated with a submaximal dose of the Ca2+ ionophore A23187 leads to a larger stimulation than seen with A23187 alone. These data suggest that although stimulation of protein kinase C is not a sufficient signal to activate the Na+/H' exchanger in HSWP cells or in another human foreskin line (Jackson fibroblasts) studied, there are some cooperative effects of protein kinase C activation with a rise in Ca2+ to stimulate Na+/H' exchange. In addition, we found that PMA actually inhibits the mitogen-induced stimulation of Na+ influx in HSWP and Jackson fibroblasts. This observation strengthens the argument that in these cells activation of protein kinase C is not sufficient to activate Na+/H+ exchange and suggests that there is a negative feedback control via protein kinase C that inhibits some signal that is necessary for
The regulation of sodium-dependent transport of anionic amino acids in cultured human fibroblasts
FEBS Letters, 1994
In cultured human fibroblasts the transport of anionic amino acids through the sodium-dependent system Xi, is stimulated rapidly and transiently by phorbol 12,13-dibutyrate. Transport stimulation is consistent with an effect due to the activation of protein kinase C. Bradykinin (1 PM) and PDGF-AA (100 &ml) also stimulate the activity of system Xi,. The bradykinin effect appears to be fully dependent upon PKC activation whereas the stimulation of aspartate transport by PDGF-AA is also due to PKC-independent mechanisms.
Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells
American Journal of Physiology-cell Physiology, 1987
Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells. Am. J. Physiol. 251 (Cell Physiol. 20): Cl86-Cl90, 1986.-Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. We examined the effect of aldosterone and insulin on Na'-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TBGC) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (ISc) in TBGC cells. Aldosterone increases Na+-K'-ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in ISo has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I,, in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na+-K'-ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na+ entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na+-K+-ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on &. aldosterone; insulin; short-circuit current; synergism ALDOSTERONE AND INSULIN BOTH stimulate active Na+ transport in a variety of target tissues (3,5). Aldosterone is thought to have an action dependent upon synthesis of new proteins, and recently a number of studies have pointed to an effect on the enzyme Na+-K+-ATPase (4,