Characterization of Na+-linked and Na+-independent Cl-/HCO3- exchange systems in Chinese hamster lung fibroblasts.

Cassel, Dan; Scharf, Orit; Rotman, Miriam; Cragoe, Edward J.; Katz, Michael

doi:10.1016/s0021-9258(18)68758-7

Cited by 55 publications

(1 citation statement)

References 36 publications

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“…Chinese hamster lung fibroblasts (Cassel et al, 1988) and monkey kidney Vero cells (Madshus & Olsnes, 1987). To simplify analysis of Cl"-dependent pH¡ regulation single-cell photometry experiments can be performed in the absence of Na+ to eliminate the contributions of the Na+-dependent system(s) (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

Novel Cl--Dependent Intracellular pH Regulation in Murine MDR 1 Transfectants and Potential Implications

Roepe

Weisburg²,

Luz³

et al. 1994

Biochemistry

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Previously [Luz et al. (1994) Biochemistry 33, 7239-7249], we determined that Cl(-)- and -HCO3-dependent pHi homeostasis was perturbed in multidrug resistant (MDR) cells created by transfecting LR73 Chinese hamster ovary fibroblasts with wild-type mu (murine) MDR 1 (Gros et al., 1991). Via single-cell photometry experiments performed under various conditions, we are now able to separate Na(+)-dependent and Na(+)-independent components of Cl-/-HCO3 exchange in the MDR transfectants and the parental LR73 cells. Cl(-)-dependent, Na(+)-independent reacidification of pHi, mediated by the anion exchanger 2 isoform in LR73 cells, is dramatically inhibited by mild overexpression of MDR protein. Analysis of H+ flux at different pHi shows that Cl(-)-dependent reacidification approaches 0.2 mM H+/s for LR73 cells at pHi = 8.0 but is at least 10-fold slower for MDR 1 transfectants that were never exposed to chemotherapeutics (EX4N7 cells). MDR 1 transfectants selected on the chemotherapeutic vinblastine (1-1 cells), which express approximately 10-fold more MDR protein relative to EX4N7 cells, exhibit similar behavior; however, alterations in Cl(-)-dependent pHi regulation are more severe. Hypotonic conditions, which have been shown to increase anomalous Cl- conductance in some cells overexpressing MDR protein (Valverde et al., 1992), are found to amplify the altered pHi homeostasis features in the primary transfectants that express lower levels of MDR protein such that they then mimic the behavior of the drug-selected cells that express substantially more MDR protein. Verapamil reverses the anomalous behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Novel Cl--Dependent Intracellular pH Regulation in Murine MDR 1 Transfectants and Potential Implications

Roepe

Weisburg²,

Luz³

et al. 1994

Biochemistry

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Thermotolerance and intracellular pH in two Chinese hamster cell lines adapted to growth at low pH

et al. 1996

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As an in vitro model for the low extracellular pH (pHe) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42 degrees C heating in pHe = 7.3 buffer, but not in pHe = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pHi) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pHi between cell lines. Despite this fundamental difference, relative changes in pHi were similar in that both lines showed a higher pHi in adapted than in unadapted cells, over the range of pHe values tested. The upregulation of pHi was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42 degrees heat at pHe = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pHi within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pHi in the two cell lines upon the removal of extracellular CO2/HCO3-. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells.

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Altered proton extrusion in cells adapted to growth at low extracellular pH

et al. 1997

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Intracellular pH (pHi) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pHi in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pHi relative to the extracellular pH (pHe). To test the ability of low pHe adapted cells to respond to a pHi lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pHi during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pHe = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pHi was consistently elevated in adapted cells as compared with cells grown at normal pHe = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pHi of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na+/ H+ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl-/HCO(-3)exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification.

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Characterization of Na+-linked and Na+-independent Cl-/HCO3- exchange systems in Chinese hamster lung fibroblasts.

Cited by 55 publications

References 36 publications

Novel Cl--Dependent Intracellular pH Regulation in Murine MDR 1 Transfectants and Potential Implications

Novel Cl--Dependent Intracellular pH Regulation in Murine MDR 1 Transfectants and Potential Implications

Thermotolerance and intracellular pH in two Chinese hamster cell lines adapted to growth at low pH

Altered proton extrusion in cells adapted to growth at low extracellular pH

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