Refined Evaluation of the Exponential Curve Parameters and Initial Exchange Rate Constant for 22Na+ Washout in Cultured Human Skin Fibroblasts

Abstract

A technique is proposed to evaluate the exponential curve parameters and the initial exchange rate constant (kk) for 22Na+ washout from cultured human skin fibroblasts. After loading with the isotope, the cells were subjected to cold washing and warming steps. A desaturation curve for 22Na+ washout was developed including the activity in the warming medium that corresponded to t = 0 min. Using nonlinear regression analysis, a general three exponential function adequately described the 22Na+ washout in the time interval of 0-70 min. A back extrapolation was performed to estimate the initial time (ti; a negative number) when the total activity was present in the cells. The ti was substituted into the first derivative function of the three exponents to yield the k;. Calculated from the equilibrium distribution of 22Na+ and the specific activity of the medium, the concentration of Na+ (in mM; mean k SD) for fibroblasts of two individuals were 13.3 k 2.3, n = 3, and 19.0 f 5.2, n = 4. This indicates that the washout

Differences of Ca2+ regulation in skin fibroblasts from blacks and whites

“…Assuming that under our experimental conditions all exponents of the washout represent 45Ca2+ that is transported across the cell membrane, the first derivative of the exponential function at the initiation of the washout represents the apparent, overall 45Ca2 + exchange rate constant (I&). We have used this approach previously in studying 22Na+ washout (Kino et al, 1986;Hopp et al, 1987Hopp et al, , 1988Kuriyama et al, 1988Kuriyama et al, , 1988a. Results presented in Tables 3 and 5 indicate that this parameter is significantly higher in are presented as fi-dCtioIIS of total cellular activity ( x 10-'); 0.3 mM EGTA serum-stimulated fibroblasts from blacks than in serum-stimulated fibroblasts from whites.…”

“…Approximations of the various parameters were obtained by sequential peeling (Van Liew, 1967). The values obtained were used as initial estimations for nonlinear regression analyses in which the exponential coefficients (al-s) represent apparent cellular pools of 45Ca2 + and the exponential factors (klJ represent apparent washout-rate constants associated with these pools (see Kin0 et al, 1986, andHopp et al, 1988 for specific equations).…”

“…The 45Ca2+ washout is based primarily on the 46Ca2 + gaining access to the washout medium. Our approach €or determination of the parameters of the washout curve compensates, to a large extent, for the 45Ca2 ' lost during the removal of the loading medium and the subsequent washing of the cells before the initiation of the washout (for details, see Hopp et al, 1988) In contrast, the uptake data rely on the amount of isotope associated with the cells. A portion of the 45Ca2 + associated with the cells during the uptake is likely to be isotope bound to the plasma membrane, To examine this possibility, cells were washed after the uptake experiments either with 0.1 M MgClz or with 0.09 M MgClz plus 10 mM LaCl3.…”