Summary
Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2′,7′-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH4 prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na+ removal or EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na+/H+ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na+/H+ exchange. Despite this heterogeneity, the Na+/H+ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4–7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na+/H+ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations.
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Aronson, P.S., Nee, J., Suhm, M.A. 1982. Modifier role of internal H+ in activating the Na+−H+ exchanger in renal membrane vesicles.Nature (London) 299:161–163
Cantiello, H.F., Scott, J.A., Rabito, C.A. 1986. Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubule cells.J. Biol. Chem. 261:3252–3258
Crissman, H.A., Steinkamp, J.A. 1973. Rapid simultaneous measurement of DNA, protein and cell volume in single cells from large mammalian cell populations.J. Cell Biol. 59:766–771
Frankfurt, O.S. 1980. Flow cytometric analysis of double-stranded RNA content distributions.J. Histochem. Cytochem. 28:663–669
Gerson, D.F. 1982. Determination of intracellular pH changes in lymphocytes with 4-methylumbelliferone by flow microfluorometry.In: Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions. R. Nuccitelli and D.W. Deamer, editors. pp. 125–133. Alan R. Liss, New York
Gmaj, P., Murer, H. 1986. Cellular mechanisms of inorganic phosphate transport in kidney.Physiol. Rev. 66:36–70
Grinstein, S., Rothstein, A. 1986. Mechanisms of regulation of the Na+/H+ exchanger.J. Membrane Biol. 90:1–12
Iino, Y., Burg, M.B. 1979. Effect of parathyroid hormone on bicarbonate absorption by proximal tubules in vitro.Am. J. Physiol. 236:F378-F391
Kahn, A.M., Dolson, G.M., Hise, M.K., Bennett, S.C., Weinman, E.J. 1985. Parathyroid hormone and dibutryl cAMP inhibit Na−H exchange in renal brush border vesicles.Am. J. Physiol. 248:F212-F218
Koyama, H., Goodpasture, C., Miller, M.M., Teplitz, R.L., Riggs, A.D. 1978. Establishment and characterization of a cell line from the American opossum (Didelphys virginiana).In Vitro 14:239–246
Malmström, K., Murer, H. 1986. Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC-12.P3 cells.Am. J. Physiol. 251:C23-C31
Malmström, K., Murer, H. 1987. Parathyroid hormone regulates phosphate transport in OK cells via an irreversible inactivation of a membrane protein.FEBS Lett. 216:257–260
Malmström, K., Stange, G., Murer, H. 1987. Identification of proximal tubular transport functions in the established kidney cell line, OK.Biochim. Biophys. Acta 902:269–277
Malmström, K., Stange, G., Murer, H. 1988. Intracellular cascades in the parathyroid-hormone-dependent regulation of Na+/phosphate cotransport in OK cells.Biochem. J. 251:207–213
Miller, R.T., Pollock, A.S. 1987. Modification of the internal pH sensitivity of the Na+/H+ antiporter by parathyroid hormone in a cultured renal cell line.J. Biol. Chem. 262:9115–9120
Montrose, M.H., Friedrich, T., Murer, H. 1987. Measurements of intracellular pH in single LLC-PK1 cells: Recovery from an acid load via basolateral Na+/H+ exchange.J. Membrane Biol. 97:63–78
Montrose, M.H., Knoblauch, C., Murer, H. 1988. Separate control of regulatory volume increase and Na+/H+ exchange by cultured renal cells.Am. J. Physiol. 255:C76-C85
Moran, A., Montrose, M.H., Murer, H. 1988. Regulation of Na+−H+ exchange in cultured opossum kidney cells by parathyroid hormone, atrial natriuretic peptide and cyclic nucleotides.Biochim. Biophys. Acta 969:48–56
Musgrove, E., Rugg, C., Hedley, D. 1986. Flow cytometric measurement of cytoplasmic pH: A critical evaluation of available fluorochromes.Cytometry 7:347–355
Pollock, A.S., Warnock, D.G., Strewler, G.J. 1986. Parathyroid hormone inhibition of Na+−H+ antiporter activity in a cultured renal cell line.Am. J. Physiol. 250:F217-F225
Shapiro, H.M. 1986. Practical Flow Cytometry. A.R. Liss, New York
Valet, G., Raffael, A., Moroder, L. 1981. Fast intracellular pH determination in single cells by flow cytometry.Naturwissenschaften 68:265–267
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Montrose, M.H., Condrau, M.A. & Murer, H. Flow cytometric analysis of intracellular pH in cultured opossum kidney (OK) cells. J. Membrain Biol. 108, 31–43 (1989). https://doi.org/10.1007/BF01870423
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DOI: https://doi.org/10.1007/BF01870423