Self‐inhibition of chloride transport in ehrlich ascites tumor cells

Abstract

Previous studies have shown that mediated Cl- transport which occurs by at least two processes (Cl- -dependent cation cotransport and Cl- self-exchange) becomes progressively inhibited when extracellular Cl- exceeds about 60 mM (Hoffmann et al., 1979). To account for this type of kinetic behavior, that is, self-inhibition, an anion transport system possessing two sites, a high affinity transport site and a lower affinity modifier site is suggested (Dalmark, 1976). In the present experiments we have attempted to determine which of the mediated transport pathways is susceptible to self-inhibition by studying the dependence of the steady state Cl- flux on the extracellular Cl- concentration and how DIDS, an inhibitor of Cl- self-exchange, and H + affect this relationship. Addition of DIDS to Ehrlich cells results in inhibition of Cl- transport at every Cl- concentration tested (40-150 mM). Moreover, the Cl- flux/Cl- concentration relationship no longer exhibits self-inhibition, suggesting that this phenomenon is a characteristic of the Cl- self-exchanger rather than of the Cl- -dependent cation cotransport system. Lowering the extracellular pH (pHo) from 7.35 to 5.30 stimulates Cl- transport by a process that saturates with respect to [H +]. Half-maximal stimulation occurs at pHo 6.34. A comparison of the kinetic parameters, Ks and Jmax, calculated from the ascending limb of the Cl- flux/Cl- concentration curve at pHo 7.30 to those at pHo 5.50 show that the values for Ks are almost identical (23.6 mM and 21.3 mM, respectively), while the values for Jmax [22.2 mEq/Kg dry wt) X min] differ by only 15%. This finding along with the observation that DIDS completely blocks H + stimulation of Cl- transport is compatible with the suggestion that H + interact at the modifer site of the Cl- self-exchanger and thereby prevents self-inhibition.

Identification of the anion exchange protein of ehrlich cells: A kinetic analysis of the inhibitory effects of 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS) and labeling of membrane proteins with3H-DIDS

“…Since the saturation curve which we obtain by replacing NaCI with sucrose (see Hoffmann et al, 1979), is identical to the one obtained by Levinson (1984), using gluconate to replace chloride, we suggest that the results at high or low external chloride in the present report are only weakly influenced by the changes in Na + or ionic strength.…”

“…In Hoffmann et al (1979) we found a KI/2 (the chloride concentration for half-maximal flux) of 15 mM from a saturation curve, where sucrose substituted for NaCI. The fluxes in these experiments were a sum of the exchange fluxes and the fluxes through the cotransport system (Levinson, 1984) so K~/2 might overestimate K, of the exchange system. It thus seems likely that the competition between DIDS and chloride is on the substrate site.…”

Sidium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells

“…The unidirectional C1-influx was calculated from the time-dependent uptake of 36C1 as previously described (Levinson, 1984). In the present experiments the sampling procedure, as well as the measurement of 36C1 trapped within the extracellular space, was identical to that described for 86Rb uptake.…”

“…Ln (1 -relative specific activity of C1 ) is plotted as a function of time (Levinson, 1984). The efflux rate coefficients (ke; min -I) which…”

“…In the absence of net ion and water movements approximately 50% of K + transport is ouabain insensitive (Tupper, 1975;Geck et al, 1978) while about 45% of C1-transport persists in the presence of DIDS, an inhibitor of the anion exchanger (Aull et al, 1977;Levinson, 1978Levinson, , 1984. The observations that ouabain-insensitive K + transport is abolished when C1-is replaced by NO3 and that both K + and C1 transport are inhibited by furosemide suggests that K + and Cl are cotransported (Aull, 1982;Bakker-Grunwald, 1978;Bakker-Grunwald et al, 1980).…”