Effect of acute serum depletion on Na⁺ ‐K⁺ homeostasis in cultured human skin fibroblasts

Abstract

In order to elucidate changes in cell transport behavior of cultured human skin fibroblasts in response to acute serum depletion, we performed uptake and washout of 22Na+ and 86Rb+ as well as measurements of the intracellular Na+ and K+ levels in the presence and absence of ouabain. Pronounced and lasting increase in cellular Na+ and decrease in K+ were observed after removal of fetal bovine serum (FBS) from the medium. The sum of the Na+ and K+ contents (nEq/10(5) cells) was lower in FBS-free medium (mean +/- SD; 17.3 +/- 2.2) than in FBS-containing medium (26.2 +/- 3.8; P less than .02). Simultaneously, a decrease in cellular water volume was detected in the FBS-free medium. The cation uptake and washout data suggest that FBS removal primarily renders the cells more permeable to Na+ and K+ with a secondary stimulation of the ouabain-sensitive Na+ extrusion mechanism. FBS at a concentration of 0.2% prevented approximately 50% of the maximal increase in the 86Rb+ washout rate constant associated with FBS depletion. Ouabain (2 microM) produced an increase in the 86Rb+ washout rate constant. This effect was substantially larger in cells subjected to medium without FBS (from 0.0303 to 0.2500 min-1) than in fibroblasts incubated in medium with FBS (from 0.0107 to 0.0487 min-1). The cellular K+ content was drastically reduced by ouabain to a level not different in medium with or without FBS (33.9 +/- 4.5 to 1.75 +/- 0.38 and 16.7 +/- 1.4 to 1.4 +/- 0.13 nEq/10(5) cells, respectively). The 22Na+ washout data exhibited a three-exponential pattern. Analytical solutions of the washout data by means of two models (serial and parallel) with three compartments showed that FBS depletion resulted in increase of the size of all three compartments. It is concluded that in cultured human skin fibroblasts, FBS is essential to the maintenance of a normal Na+ and K+ homeostasis. The removal of FBS results in dramatic permutation of this homeostasis that develops within minutes and lasts for hours.

A higher cellular sodium turnover rate in cultured skin fibroblasts from blacks.

“…Whereas the k% values are derived from a model that is dependent on values of intracellular Na + concentration, the ki is independent of this parameter. In previous investigations we found that the first and fastest exponent of "Na + washout from cultured rat fibroblasts and vascular smooth muscle cells 3 ' " as well as human skin fibroblasts 16 primarily represented the Na + -K + pump and its respective cellular compartment, inasmuch as subjecting these cells to ouabain or K + -deficient medium resulted in essentially complete disappearance of the fastest component. Moreover, based on the equilibrium distribution ratio of ^Na + between the medium and the cellular milieu at the initiation of the washout experiments, the calculated cellular Na + concentrations agreed quite well with direct measurements of the ion using atomic absorption spectrometry.…”

“…16 Thus, the ouabain-sensitive Na + and K + (Rb + ) transport may not be sensitive and reliable indictors for the Na + -K + pump activity. One possible explanation for the observed differences in fibroblasts between blacks and whites could relate to differing effects of the cardiac glycoside on Na + -K + pump independent transports.…”

Characterization of Na+‐K+ homeostasis of cultured human skin fibroblasts in the presence and absence of fetal bovine serum

“…Cellular Na ' and K t contents were not changed by the phorbol ester. We conclude that FBS removal raises the cellular Na+ content, and enhances "Rb+ efflux, through a calmodulin-dependent pathway activated by calcium influx.Removal of fetal bovine serum (FBS) causes marked changes in the Nat and K+ homeostasis of cultured human fibroblasts (Dall'Asta et al, 1986;Hopp et al, 1987). In a previous study (Hopp et al, 19871, with cultured human skin fibroblasts, we showed that FBS removal resulted in a 3 4 times increase in the intracellular Na+ concentration caused by an accelerated uptake rate and decreased efflux rate constant for the ion.…”

Effects of serum on calcium mobilization in the submandibular cell line A253

“…Serum is also an important factor in maintaining cell function in many cell lines, and its deprivation can induce striking alterations in cell function. For example, removal of FBS from culture medium can cause a significant increase in the intracellular Na ϩ and a decrease in intracellular K ϩ in fibroblasts [Hopp et al, 1987]. Moreover, the mitogenic effect of serum may be responsible for some pathological alterations as well.…”