pHi regulation in Ehrlich mouse ascites tumor cells: Role of sodium-dependent and sodium-independent chloride-bicarbonate exchange

Abstract

pHi recovery in acid-loaded Ehrlich ascites tumor cells and pHi maintenance at steady-state were studied using the fluorescent probe BCECF. Both in nominally HCO3(-)-free media and at 25 mM HCO3-, the measured pHi (7.26 and 7.82, respectively) was significantly more alkaline than the pHi value calculated assuming the transmembrane HCO3- gradient to be equal to the Cl- gradient. Thus, pHi in these cells is not determined by the Cl- gradient and by Cl-/HCO3- exchange. pHi recovery following acid loading by propionate exposure, NH4+ withdrawal, or CO2 exposure is mediated by amiloride-sensitive Na+/H+ exchange in HCO3(-)-free media, and in the presence of HCO3- (25 mM) by DIDS-sensitive, Na+)-dependent Cl-/HCO3- exchange. A significant residual pHi recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H+ pump in pHi regulation. pHi maintenance at steady-state involves both Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange. Acute removal of external Cl- induces a DIDS-sensitive, Na(+)-dependent alkalinization, taken to represent HCO3- influx in exchange for cellular Cl-. Measurements of 36Cl- efflux into Cl(-)-free gluconate media with and without Na+ and/or HCO3- (10 mM) directly demonstrate a DIDS-sensitive, Na(+)-dependent Cl-/HCO3- exchange operating at slightly acidic pHi (pHo 6.8), and a DIDS-sensitive, Na+)-independent Cl-/HCO3- exchange operating at alkaline pHi (pHo 8.2).

pH Regulation in Sensitive and Multidrug Resistant Ehrlich Ascites Tumor Cells

“…Thus, we conclude that there is no correlation between drug resistance or expression of P-gp, and steady-state pH i in Ehrlich ascites tumor cells. Furthermore, in agreement with previous observations on wild-type Ehrlich cells [22], amiloride, an inhibitor of the Na + /H + exchanger, had no significant effect on steadystate pH i . This makes it unlikely that putative differences in proton efflux via P-gp had been compensated by an altered activity of the exchanger under steady-state conditions.…”

“…In order to see whether this could be a result of compensation via different levels of activity of the Na + /H + exchanger in the different cell lines, we investigated the effect on steady-state pH i of amiloride, an inhibitor of the Na + /H + exchanger. As seen, addition of amiloride (200 ÌM, 5 min) did not significantly affect pH i (table 1), in agreement with the previous observation that at steady-state pH i , the Na + / H + exchanger is virtually quiescent in Ehrlich cells [22].…”

“…(3) An increased level of activity of the Na + /H + exchanger after intracellular acidification in resistant compared to wild-type cells has previously been observed in CHO cells and in a myeloma cell line [14,34]. This could also explain the observation that resistant cells acidify less after an acid load, since Na + /H + exchange is a major mechanism of recovery of pH i in most cell types, including Ehrlich cells [22,30,31]. However, in the present study, acidificationinduced Na + /H + exchanger activity was, if anything, reduced in the resistant as compared to the wild-type cells, hence, this does not explain our observations.…”

“…In most cell types, including Ehrlich cells, a Na + /H + exchanger is activated by acidic pH i , and is an important mechanism of recovery of pH i after an acid load [22,30,31]. Alterations in Na + /H + exchanger activity in drug-resistant tumor cell lines has previously been reported [14,19].…”

“…The cell suspensions were preincubated in standard medium for 15-30 min before initiation of the experiments. Intracellular pH pH i of cell suspensions was assessed spectrophotometrically using the pH-sensitive fluorescent probe 2),7)-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), as previously described [22,23]. Cell suspensions (4 ml, cytocrit 4%) were incubated at 37°C for 30-35 min with BCECF-tetraacetoxymethyl ester (BCECF-AM) (3.6 ÌM), followed by washing (twice, 700 g, 30 s) in standard medium containing bovine serum albumin (BSA) (1 mg/ml), and resuspension at a cytocrit of 8% in standard medium containing BSA.…”