Abstract
Confocal laserscan microscopy with a dual-excitation device was used to record intracellular pH (pHi) regulation in rat proximal convoluted tubules microperfused in vivo. Cells were loaded with the pH-sensitive, fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Single cells could be distinguished within the tubules and separate measurements were possible. Application of an NH4Cl pulse by peritubular perfusion caused an immediate increase in intracellular pH. Intraluminal injection of NH4Cl led to a slower increase in intracellular pH. In both cases, cessation of perfusion led to an immediate acidification. Peritubular perfusion with 300 μM 4,4′-diisothiocanatodihydrostilbene-2, 2′-disulphonic acid (H2DIDS) caused an intracellular alkalinisation. Microperfusion, pH-sensitive dyes and confocal laserscan microscopy provide a new non-invasive method to measure intracellular pH effectively in individual cells of near-surface structures of the intact kidney.
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Allen CN, Harpur ES, Gray TJB, Simmons NL, Hirst BH (1990) Efflux of bis-carboxyethyl-carboxyfluorescein (BCECF) by a novel ATP-dependent transport mechanism in epithelial cells. Biochem Biophys Res Commun 172:262–267
Alpern RJ (1985) Mechanism of basolateral membrane H+/OH−/ HCO −3 transport in the rat proximal convoluted tubule. J Gen Physiol 86:613–636
Alpern RJ (1990) Cell mechanisms of proximal tubule acidification. Physiol Rev 70:79–114
Alpern RJ, Chambers M (1986) Cell pH in the rat proximal convoluted tubule. Regulation by luminal and peritubular pH and sodium concentration. J Clin Invest 78:502–510
Aronson PS (1983) Mechanisms of active H+ secretion in the proximal tubule. Am J Physiol 245:F647-F659
Busa WB, Nuccitelli R (1984) Metabolic regulation via intracellular pH. Am J Physiol 246:R409-R438
Brakenhoff GJ, Blom P, Barends P (1979) Confocal scanning light microscopy with high aperture immersion lenses. J Microsc 117:219–232
Bright GR, Fisher GW, Rogowska J, Taylor DL (1987) Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH. J Cell Biol 104:1019–1033
Capasso G, Kinne-Saffran E, De Santo NG, Kinne R (1985) Regulation of volume reabsorption by thyroid hormones in the proximal tubule of rat: minor role of luminal sodium permeability. Pflügers Arch 403:97–104
Carrington WA, Fogarty KE, Lifschitz L, Fay FS (1990) Three dimensional imaging on confocal and wide-field microscopes. In: Pawley J (ed) Handbook of biological confocal microscopy. Plenum, New York, pp 151–161
Deutsch C, Taylor JS, Price M (1984) pH homeostasis in human lymphocytes: modulation by ions and mitogen. J Cell Biol 98:885–893
Frömter E (1984) Viewing the kidney through microelectrodes. Am J Physiol 247:F695-F705
Garvin JL, Burg MB, Knepper MA (1987) NH3 and NH +4 transport by rabbit renal proximal straight tubules. Am J Physiol 252:F232–F239
Golchini K, Kurtz I (1988) NH3 permeation through the apical membrane of MDCK cells is via a lipid pathway. Am J Physiol 255:F135–141
Kikeri D, Sun A, Zeidel ML, Hebert SC (1989) Cell membranes impermeable to NH3. Nature 339:478–480
Kinne R, Kinne-Saffran E (1992) Renal plasma membranes: isolation, general properties, and biochemical components. In: Windhager EE (ed) Handbook of physiology:renal physiology. Oxford University Press, New York, pp 2083–2117
Kinne R, Kinne-Saffran E, Schütz H, Schölermann B (1986) Ammonium transport in medullary thick ascending limb of rabbit kidney. Involvement of the Na+,K+,Cl− cotransporter. J Membr Biol 94:279–284
Montrose MH, 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 Membr Biol 97:63–78
Musgrove E, Rugg C, Hedley D (1986) Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes. Cytometry 7:347–355
Opitz N, Weinlich M, Mooren F, Keinemann F-K, Kinne R (1991) Dual wavelength excitation in confocal laser scanning microscopy: intracellular pH measurements with a fluorescent indicator. Biomed Technik 36:132–133
Paradiso AM, Tsien RY, Demarest JR, Machen TE (1987) Na-H and Cl-HCO3 exchange in rabbit oxyntic cells using fluorescence microscopy. Am J Physiol 253:C30-C36
Pawley J (1990) Handbook of biological confocal microscopy. Plenum, New York
Pfaller W, Gstraunthaler G, Kotanko P (1985) Compartments and surfaces in renal cells. In: Kinne RKH (ed) Renal biochemistry. Cells, membranes, molecules. Elsevier, Amsterdam, pp 1–62
Roos A, Boron WF (1981) Intracellular pH. Physiol Rev 61:296–434
Rosenberg SO, Berkowitz PA, Li L, Schuster VL (1991) Imaging of filter-grown epithelial cells: MDCK Na+-H+ exchanger is basolateral. Am J Physiol 260:C868-C876
Shotton DM (1989) Confocal scanning optical microscopy and its applications for biological specimens. J Cell Sci 94:175–206
Thomas JA, Buchsbaum RN, Zimniak A, Racker E (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210–2218
Tsien RY (1989) Fluorescent indicators of ion concentrations. Methods Cell Biol 30:127–156
Wang X, Kurtz I (1990) H+/base transport in principal cells characterized by confocal fluorescence imaging. Am J Physiol 259:C365-C373
Weiner ID, Hamm LL (1989) Use of fluorescent dye BCECF to measure intracellular pH in cortical collecting tubule. Am J Physiol 256:F957-F964
Weinlich M, Capasso G, Kinne RKH (1992) Confocal microscopy. In: Giebisch G, Ussing HH, Kristensen P (eds) Membrane transport in biology, vol 6 (in press)
White JG, Amos WB, Fordham M (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 105:41–48
Wilson T (1989) Trends in confocal microscopy. Trends Neurosci 12:486–493
Yoshitomi K, Frömter E (1984) Cell pH of rat renal proximal tubule in vivo and the conductive nature of peritubular HCO −3 (OH−) exit. Pflügers Arch 402:300–305
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Weinlich, M., Capasso, G. & Kinne, R.K.H. Intracellular pH in renal tubules in situ: single-cell measurements by confocal laserscan microscopy. Pflugers Arch. 422, 523–529 (1993). https://doi.org/10.1007/BF00375081
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DOI: https://doi.org/10.1007/BF00375081