Characterization of basolateral Na/H exchange (Na/H-1) in MDCK cells
Pflügers Archiv - 1992
Tóm tắt
MDCK cells were grown to confluent monolayers on permeant filter supports; pH was analysed by using the pH-sensitive fluorescent probe 2′7′-biscarboxyethyl-5,6-carboxyfluorescein and a routine spectrofluorometer equipped with a perfusion cuvette [Krayer-Pawlowska et al. (1990) J Membr Biol 120:173–183]. Superfusion of the basolateral (but not apical) cell surface with Na+-containing solutions led to immediate recovery of pHi from an acid load (NH4 prepulse). This pHi recovery was reversibly inhibited by ethylisopropylamiloride indicating Na/H exchange activity. Na/H exchange activity showed an apparent K
m for Na+ of about 25 mM Na+ and an apparent K
i for inhibition by dimethylamiloride of around 0.2 μM; inhibition by dimethylamiloride was competitive with Na+ interaction. Lowering pHi prior to analysis of Na/H exchange leads to sharp activation of Na/H exchange; the apparent V
max for Na/H exchange is increased more than tenfold by lowering the pHi from 7.0 to 6.7 without an effect on apparent K
m values for Na+ interaction. It is concluded that MDCK cells (strain I) grown on a permeant support contain only basolateral Na/H exchange activity, most likely Na/H-1 [for nomenclature see Igarashi et al. (1991) Kidney Int 40:S84–S89].
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Tài liệu tham khảo
Alpern RJ (1985) Mechanism of basolateral membrane H+/OH−/HCO −3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process. J Gen Physiol 86:613–636
Alpern RJ (1990) Cell mechanisms of proximal tubule acidification. Physiol Rev 70:79–114
Boron WF, DeWeer P (1976) Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J Gen Physiol 67:91–112
Boyarsky G, Ganz MB, Sterzel RB, Boron WF (1988) pH regulation in single glomerular mesangial cells: I. Acid extrusion in absence and presence of HCO −3 . Am J Physiol 255:C844-C856
Casavola V, Helmle-Kolb C, Murer H (1989) Separate regulatory control of apical and basolateral Na+/H+-exchange in renal epithelial cells. Biochem Biophys Res Commun 165:833–837
Casavola V, Helmle-Kolb C, Montrose MH, Murer H (1991) Polarized expression of Na+/H+ exchange activities in clonal LLC-PK1 cells (Clone4 and PKE20) Pflügers Arch 418:276–283
Casavola V, Helmle-Kolb C, Murer H (1991) Comparison between apical and basolateral transporters in tubular epithelia: a dual location of Na+/H+-exchange with different properties. In: Hatano M (ed) Nephrology, vol II. Springer, Tokyo, pp 950–955
Cereijido M, Contreras RG, Gonzalez-Mariscal L (1989) Development and alteration of polarity. Annu Rev Physiol 51:785–795
Frelin C, Vigne P, Lazdunski M (1985) The role of the Na+/H+ exchange system in the regulation of the internal pH in cultured cardiac cells. Eur J Biochem 149:1–4
Frelin C, Vigne P, Ladoux A, Lazdunski M (1988) The regulation of the intracellular pH in cells from vertebrates. Eur J Biochem 174:3–14
Gstraunthaler JA (1988) Epithelial cells in tissue culture. Renal Physiol Biochem 11:1–42
Haggerty JG, Agarwal N, Reilly RF, Adelberg EA, Slayman CW (1988) Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1) Proc Natl Acad Sci USA 85:6797–6801
Hart D, Nord EP (1991) Polarized distribution of Na+/H+ antiport and Na+/HCO −3 cotransport in primary cultures of renal inner medullary collecting duct cells. J Biol Chem 266:2374–2382
Helmle-Kolb C, Montrose MH, Murer H (1990) PTH-regulation of Na+/H+-exchange in opossum kidney cells: polarity and mechansims. Pflügers Arch 416:615–623
Igarashi P, Reilly RF, Hildebrandt F, Biemesderfer D, Rebouças NA, Slaymann CW, Aronson PS (1991) Molecular biology of renal Na+-H+ exchangers. Kidney Int 40:S84-S89
Krayer-Pawlowska D, Helmle-Kolb C, Montrose MH, Krapf R, Murer H (1991) Studies on the kinetics of Na+/H+-exchange in OK-cells: introduction of a new device for the analysis of polarized transport in cultured epithelia. J Membr Biol 120:173–183
Kulanthaivel P, Leibach FH, Mahesh VB, Cragoe EJ Jr, Ganapathy V 1990) The Na+H+ exchanger of the placental brush-border membrane is pharmacologically distinct from that of the renal brush border membrane. J Biol Chem 265:1249–1252
Kurtz J (1987) Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule. J Clin Invest 80:928–935
Montrose MH, Murer H (1990) Regulation of intracellular pH by cultured opossum kidney (OK) cells. Am J Physiol 259:C110-C120
Montrose MH, Murer H (1990) Polarity and kinetics of Na+/H+ exchange in cultured opossum kidney (OK) cells. Am J Physiol 259:C121-C133
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
Nord EP, Brown SES, Crandall ED (1988) Cl−/HCO −3 exchange modulates intracellular pH in rat type II alveolar epithelial cells. J Biol Chem 263:5599–5606
Oberleithner H, Vogel U, Kersting U, Steigner W (1990) Madine-Darby canine kidney cells: II. Aldesterone stimulates Na+/H+ and Cl−/HCO −3 exchange. Pflügers Arch 416:533–539
Preisig PA, Alpern RJ (1991) Basolateral membrane H/HCO3 transport in renal tubules. Kidney Int 39:1077–1086
Renner EL, Lake JR, Perscio M, Scharschmidt BF (1989) Na+ -H+ exchange activity in rat hepatocytes: role in regulation of intracellular pH. Am J Physiol 256:G44-G52
Rindler MJ, Saier MH Jr (1981) Evidence for Na+/H+ antiport in cultured dog kidney cells (MDCK). J Biol Chem 256:10820–10825
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
Sardet C, Franchi A, Pouysségur J (1989) Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter. Cell 56:271–280
Selvaggio AM, Schwartz JH, Bengele HH, Alexander EA (1986) Kinetics of the Na-H antiporter as assessed by the change in intracellular pH in MDCK cells. Am J Physiol 251:C558-C562
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
Vigne P, Frelin C, Cragoe EJ, Lazdunski M (1983) Ethylisopropylamiloride: a new and highly potent derivative of amiloride for the inhibition of the Na+/H+ exchange system in various cell types. Biochem Biophys Res Commun 116:86–90
Vigne P, Frelin C, Cragoe EJ, Lazdunski M (1984) Structure-activity relationships of amiloride and certain of its analogues in relation to the blockade of the Na+/H+ exchange system. Mol Pharmacol 25:131–136
Weintraub WH, Machen TE (1989) pH regulation in hepatoma cells: roles for Na-H exchange Cl-HCO3 exchange, and Na-HCO3 cotransport. Am J Physiol 257:G317-G327