Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells

Abstract

In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks is 0.58 +/- 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4(-20)] curve (0.106 +/- 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.

Phosphate transport processes in eukaryotic cells

“…Studies have now been initiated on P~ transport processes in heart and skeletal muscle (Nuutinen & Hassinen, 1981;Medina & Illingsworth, 1980), nerve fibers (Jirounek et al, 1982(Jirounek et al, , 1984, across the placenta (Stulc & Stulcova, 1984;Brunette & Allard, 1985), and in a variety of cultured cell lines. In addition, uptake of P~ by cancer cells has been studied (Wehrle & Pedersen, 1982;Bowen & Levinson, 1983), partly to determine whether Pi uptake plays a role in rapid growth. A derepressible, sodium-dependent, high-affinity Pi transport system is also found in yeast (Roomans, Blasco & Borst-Pauwels, 1977).…”

Phosphate transport in Ehrlich ascites tumor cells: Inhibition by H+

“…The time-dependent decrease in pHi shown in the upper curve of Figure 1 indicates that inhibition of Pi uptake was manifest when pHi reached a value below about 6.5. The rate of Pi transport during the 15-to 25-minute period following the change in pHo corresponds closely to the P i flux in cells equilibrated at pHo 5.5 (Bowen and Levinson, 1983). Clearly, the rapid decrease of pHo does not result in the inhibition of Pi transport.…”

“…Previous studies of the effect of pH on Pi transport (Bowen and Levinson, 1983) showed that the maximal rate of transport, which occurs at pHo 6.9, is inhibited by both a n increase and a decrease in H f concentration. These results were obtained only when cells were equilibrated at pHo between 5.5 and 7.9.…”

“…Since evidence has been presented that HzP04-is the transported species (Bowen and Levinson, 1983), the extracellular Pi concentration was maintained at 5 mM, so that the [HZPOJ was maintained at a saturating level over the entire pHo range. were maintained at pH, 7.3.…”

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

“…The extracellular space containing trapped [14C]-DMO was estimated to be 21% of the total volume of the centrifuged cell pellet. This value was obtained from measurements using [32p] p~ or [3H]-methoxyinulin as extracellular space markers (Bowen & Levinson, 1983). The appropriate correction was applied to the intracellular [14C]-DMO radioactivity and the ratio (cpm/ml cell water)/(cpm/ml medium) was calculated.…”