2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein and Breast-Neoplasms

Cells within solid tumors are known to exist in a microenvironment that may be acidic and depend on membrane-based mechanisms (Na+/H+ antiport and Na+-dependent Cl-/HCO3- exchanger) that regulate intracellular pH (pHi). We have used the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl) 5 (and 6)-carboxyfluorescein and flow cytometry to study the distribution of pHi and the activity of these pHi-regulating mechanisms among populations of murine mammary sarcoma (EMT6), human breast cancer (MCF-7), and Chinese hamster ovary cells exposed to different levels of extracellular pH (pHe). Cells were exposed to Na+ buffer in the presence or absence of HCO3- and of 5-(N-ethyl-N-isopropyl)-amiloride (a potent inhibitor of the Na+/H+ antiport) to determine the relative importance of each exchanger in the regulation of pHi. Our results indicate that: (a) the distribution of pHi at any value of pHe is broader than can be accounted for by machine noise; (b) cells maintain levels of pHi that are higher than pHe under acidic conditions; (c) the distribution of pHi is narrower when the Na+-dependent Cl-/HCO3- exchanger is active; and (d) populations that are derived from selected cells with values of pHi at lower and higher ends of the pHi distribution generate pHi distributions that are similar to those of controls, suggesting a stochastic variation in the activity of membrane-based mechanisms that regulate pHi. Our data suggest that the Na+-dependent Cl-/HCO3- exchanger is the dominant mechanism for regulation of pHi under moderately acidic conditions such as may occur in the microenvironment of solid tumors.

Topics: Amiloride; Animals; Antiporters; Bicarbonates; Breast Neoplasms; Chloride-Bicarbonate Antiporters; CHO Cells; Cricetinae; Cricetulus; Female; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Mammary Neoplasms, Animal; Mice; Potassium; Sarcoma; Sodium-Hydrogen Exchangers; Tumor Cells, Cultured

Na(+)-H+ exchange activity was studied in a human breast cancer cell line. At steady state, the intracellular pH (pHi) of the cells was 7.23 +/- 0.01, and intracellular buffering capacity (beta i) was 44 +/- 4 mM/pH unit. pHi was controlled by a Na(+)-H+ antiporter that was reversible, electroneutral, inhibited by 5-(N-methyl-N-isobutyl)amiloride, and dependent on extracellular Na+. The exchanger function depended on internal H+ concentration, according to an allosteric activation mechanism obeying the model of Hill. The exchanger was inactive at pHi > or = 7.22, and its maximal activity was reached at pHi < 6.60. The exchanger was stimulated by osmotic shrinking but was unaffected by growth factors (epidermal growth factor, insulin-like growth factor I) or by serum. When cells were grown in a medium supplemented with linoleic or alpha-linolenic acids, large quantities of the additional fatty acid accumulated in membranes, saturated fatty acids increased, and monounsaturated fatty acids decreased. These changes reduced cell proliferation but had no effect on the steady-state value of pHi, on beta i, or on the kinetic parameters of the Na(+)-H+ exchanger. Therefore, in this system, cell proliferation is not directly related to the activation of the Na(+)-H+ exchanger.

Topics: Acids; Blood; Breast Neoplasms; Cell Division; Cell Membrane; Extracellular Space; Fatty Acids; Fluoresceins; Fluorescent Dyes; Homeostasis; Humans; Hydrogen-Ion Concentration; Osmosis; Phospholipids; Sodium; Sodium-Hydrogen Exchangers; Tumor Cells, Cultured