Agonist-dependent Regulation of Renal Na+, K+-ATPase Activity Is Modulated by Intracellular Sodium Concentration
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m108182200
ISSN1083-351X
AutoresRiad Efendiev, Alejandro M. Bertorello, Rubén O. Zandomeni, Angel R. Cinelli, Carlos H. Pedemonte,
Tópico(s)Ion channel regulation and function
ResumoWe tested the hypothesis that the level of intracellular sodium modulates the hormonal regulation of the Na+, K+-ATPase activity in proximal tubule cells. By using digital imaging fluorescence microscopy of a sodium-sensitive dye, we determined that the sodium ionophore monensin induced a dose-specific increase of intracellular sodium. A correspondence between the elevation of intracellular sodium and the level of dopamine-induced inhibition of Na+, K+-ATPase activity was determined. At basal intracellular sodium concentration, stimulation of cellular protein kinase C by phorbol 12-myristate 13-acetate (PMA) promoted a significant increase in Na+, K+-ATPase activity; however, this activation was gradually reduced as the concentration of intracellular sodium was increased to become a significant inhibition at concentrations of intracellular sodium higher than 16 mm. Under these conditions, PMA and dopamine share the same signaling pathway to inhibit the Na+, K+-ATPase. The effects of PMA and dopamine on the Na+, K+-ATPase activity and the modulation of these effects by different intracellular sodium concentrations were not modified when extracellular and intracellular calcium were almost eliminated. These results suggest that the level of intracellular sodium modulates whether hormones stimulate, inhibit, or have no effect on the Na+, K+-ATPase activity leading to a tight control of sodium reabsorption. We tested the hypothesis that the level of intracellular sodium modulates the hormonal regulation of the Na+, K+-ATPase activity in proximal tubule cells. By using digital imaging fluorescence microscopy of a sodium-sensitive dye, we determined that the sodium ionophore monensin induced a dose-specific increase of intracellular sodium. A correspondence between the elevation of intracellular sodium and the level of dopamine-induced inhibition of Na+, K+-ATPase activity was determined. At basal intracellular sodium concentration, stimulation of cellular protein kinase C by phorbol 12-myristate 13-acetate (PMA) promoted a significant increase in Na+, K+-ATPase activity; however, this activation was gradually reduced as the concentration of intracellular sodium was increased to become a significant inhibition at concentrations of intracellular sodium higher than 16 mm. Under these conditions, PMA and dopamine share the same signaling pathway to inhibit the Na+, K+-ATPase. The effects of PMA and dopamine on the Na+, K+-ATPase activity and the modulation of these effects by different intracellular sodium concentrations were not modified when extracellular and intracellular calcium were almost eliminated. These results suggest that the level of intracellular sodium modulates whether hormones stimulate, inhibit, or have no effect on the Na+, K+-ATPase activity leading to a tight control of sodium reabsorption. The molecular mechanism by which hormone receptors coupled to stimulation of protein kinase C (PKC) 1The abbreviations used are: PKCprotein kinase CPMAphorbol 12-myristate 13-acetateOKopossum kidneyBAPTA1,2-bis[o-aminophenoxy]ethane-N,N,N′, N′-tetraacetic acidSBFIsodium-binding benzofuran-isophthalateFura-21-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane -N,N,N′, N′tetraacetic acid20-HETE20-hydroxyeicosatetraenoic acidDMEMDulbecco's modified Eagle's medium 1The abbreviations used are: PKCprotein kinase CPMAphorbol 12-myristate 13-acetateOKopossum kidneyBAPTA1,2-bis[o-aminophenoxy]ethane-N,N,N′, N′-tetraacetic acidSBFIsodium-binding benzofuran-isophthalateFura-21-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane -N,N,N′, N′tetraacetic acid20-HETE20-hydroxyeicosatetraenoic acidDMEMDulbecco's modified Eagle's medium regulate sodium reabsorption in renal proximal convoluted tubules is not well understood. The Na+,K+-ATPase, located within the basolateral membrane of tubule epithelial cells, maintains a transmembrane concentration gradient for sodium, ensuring the net reabsorption of this cation. Hormonal short term regulation of Na+,K+-ATPase activity may contribute to the ability of the kidney to adjust sodium reabsorption. In recent years, an increasing number of publications (1.Bertorello A.M. Katz A.I. Am. J. Physiol. 1993; 265: F743-F755PubMed Google Scholar, 2.Hussain T. Lokhandwala M.F. Hypertension. 1998; 32: 187-197Crossref PubMed Scopus (170) Google Scholar, 3.Aperia A.C. Annu. Rev. Physiol. 2000; 62: 621-647Crossref PubMed Scopus (223) Google Scholar, 4.Therien A.G. Blostein R. Am. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar) have reported the short term regulation of kidney Na+,K+-ATPase by hormones and intracellular second messengers that modulate proximal tubule sodium reabsorption. Renal Na+,K+-ATPase activity is regulated by phosphorylation/dephosphorylation processes, and both cAMP-dependent protein kinase and protein kinase C (PKC) phosphorylate the Na+,K+-ATPase (1.Bertorello A.M. Katz A.I. Am. J. Physiol. 1993; 265: F743-F755PubMed Google Scholar, 2.Hussain T. Lokhandwala M.F. Hypertension. 1998; 32: 187-197Crossref PubMed Scopus (170) Google Scholar, 3.Aperia A.C. Annu. Rev. Physiol. 2000; 62: 621-647Crossref PubMed Scopus (223) Google Scholar, 4.Therien A.G. Blostein R. Am. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar, 5.Bertorello A.M. Aperia A. Walaas S.I. Nairn A.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11359-11362Crossref PubMed Scopus (284) Google Scholar, 6.Middleton J.P. Khan W.A. Collinsworth G. Hannun Y.A. Medford R.M. J. Biol. Chem. 1993; 268: 15958-15964Abstract Full Text PDF PubMed Google Scholar, 7.Fisone G. Cheng S.X.-H. Nairn A.C. Czernik A.H. Hemmings Jr., H.C. Hoog J.-O. Bertorello A.M. Kaiser R. 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Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 11.Efendiev R. Bertorello A.M. Pedemonte C.H. FEBS Lett. 1999; 456: 45-48Crossref PubMed Scopus (91) Google Scholar, 12.Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (70) Google Scholar, 13.Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (37) Google Scholar, 14.Chibalin A.V. Ogimoto G. Pedemonte C.H. Pressley T.A. Katz A.I. Féraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1999; 274: 1920-1927Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15.Efendiev R. Bertorello A.M. Pressley T.A. Rousselot M. Ferraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (81) Google Scholar). These amino acids are phosphorylated by different PKC isoforms during these processes (10.Chibalin A.V. Pedemonte C.H. Katz A.I. Féraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 11.Efendiev R. Bertorello A.M. Pedemonte C.H. FEBS Lett. 1999; 456: 45-48Crossref PubMed Scopus (91) Google Scholar, 12.Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (70) Google Scholar, 13.Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (37) Google Scholar, 14.Chibalin A.V. Ogimoto G. Pedemonte C.H. Pressley T.A. Katz A.I. Féraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1999; 274: 1920-1927Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15.Efendiev R. Bertorello A.M. Pressley T.A. Rousselot M. Ferraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (81) Google Scholar). We also described that although dopamine inhibition of Na+,K+-ATPase is mediated by endocytosis of plasma membrane Na+,K+-ATPase molecules, PMA stimulation is due to recruitment of Na+,K+-ATPase molecules from intracellular compartments to the plasma membrane (10.Chibalin A.V. Pedemonte C.H. Katz A.I. Féraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1998; 273: 8814-8819Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15.Efendiev R. Bertorello A.M. Pressley T.A. Rousselot M. Ferraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (81) Google Scholar). protein kinase C phorbol 12-myristate 13-acetate opossum kidney 1,2-bis[o-aminophenoxy]ethane-N,N,N′, N′-tetraacetic acid sodium-binding benzofuran-isophthalate 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane -N,N,N′, N′tetraacetic acid 20-hydroxyeicosatetraenoic acid Dulbecco's modified Eagle's medium protein kinase C phorbol 12-myristate 13-acetate opossum kidney 1,2-bis[o-aminophenoxy]ethane-N,N,N′, N′-tetraacetic acid sodium-binding benzofuran-isophthalate 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane -N,N,N′, N′tetraacetic acid 20-hydroxyeicosatetraenoic acid Dulbecco's modified Eagle's medium Decreased Na+,K+-ATPase activity induced by dopamine is partly responsible for reduced sodium reabsorption during a high salt diet, and impaired regulation of the Na+,K+-ATPase activity in renal tubules has been linked to the development of high blood pressure (1.Bertorello A.M. Katz A.I. Am. J. Physiol. 1993; 265: F743-F755PubMed Google Scholar, 2.Hussain T. Lokhandwala M.F. Hypertension. 1998; 32: 187-197Crossref PubMed Scopus (170) Google Scholar, 3.Aperia A.C. Annu. Rev. Physiol. 2000; 62: 621-647Crossref PubMed Scopus (223) Google Scholar, 4.Therien A.G. Blostein R. Am. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar). In vivo, increases in dietary sodium intake or acute sodium loading lead to natriuresis accompanied by elevated urinary dopamine excretion, which suggested that dopamine produced endogenously by the epithelial proximal tubule cells might contribute to the natriuretic response (2.Hussain T. Lokhandwala M.F. Hypertension. 1998; 32: 187-197Crossref PubMed Scopus (170) Google Scholar, 3.Aperia A.C. Annu. Rev. Physiol. 2000; 62: 621-647Crossref PubMed Scopus (223) Google Scholar, 4.Therien A.G. Blostein R. Am. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar). In this model, endogenously produced dopamine would be transported outside the proximal tubule cells where it binds to specific cell membrane receptors. The question arising is how an external effect, the acute sodium load, is translated into activation of the intracellular dopaminergic system. Our hypothesis is that an increased filtered load of sodium may lead to a transient elevation in intracellular sodium concentration that triggers the dopaminergic response. The present study was performed to test the hypothesis that the level of intracellular sodium modulates the tight control of Na+,K+-ATPase activity by different agonists. We present evidence that the intracellular sodium concentration of kidney cells determines the level of inhibition of the Na+,K+-ATPase activity by dopamine. We also demonstrate that direct stimulation of cellular protein kinase C by the phorbol ester PMA leads to either activation or inhibition of Na+,K+-ATPase activity depending on the intracellular sodium concentration. Cell culture supplies were purchased from Invitrogen and HyClone Laboratories (Logan, UT). Molecular biology reagents were from New England Biolabs (Beverly, MA), Promega (Madison, WI), Stratagene (La Jolla, CA), and Sigma. Ouabain was purchased fromCalbiochem. PMA, ethoxyresorufin, and dopamine were obtained from Sigma. [86Rb+]RbCl was obtained from PerkinElmer Life Sciences. Other reagents were of the highest quality available. Opossum kidney (OK) cells were maintained at 37 °C (10% CO2) in Dulbecco's modified Eagle's medium with 10% calf serum and antibiotics (DMEM-10). The expression vector pCMV containing the rodent Na+-pump α1-subunit cDNA was obtained from PharMingen. Mutants of α1 were prepared, as previously described (12.Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (70) Google Scholar, 13.Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (37) Google Scholar, 14.Chibalin A.V. Ogimoto G. Pedemonte C.H. Pressley T.A. Katz A.I. Féraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1999; 274: 1920-1927Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15.Efendiev R. Bertorello A.M. Pressley T.A. Rousselot M. Ferraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (81) Google Scholar), from a plasmid containing the wild type α-subunit sequence and complementary oligonucleotides containing the desired change. Briefly, annealed plasmid and oligonucleotides were subjected to PCR amplification with Pfu polymerase, followed by restriction of the original wild type template with DpnI. After transformation of bacteria, the recovered mutant plasmids were evaluated by restriction analysis and direct sequencing of the altered region. Plasmids containing the wild type and mutated α-subunit cDNAs were transfected into OK cells using liposomes, as described previously (12.Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (70) Google Scholar, 13.Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (37) Google Scholar, 14.Chibalin A.V. Ogimoto G. Pedemonte C.H. Pressley T.A. Katz A.I. Féraille E. Berggren P.-O. Bertorello A.M. J. Biol. Chem. 1999; 274: 1920-1927Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15.Efendiev R. Bertorello A.M. Pressley T.A. Rousselot M. Ferraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (81) Google Scholar). Selection for cells expressing the highest level of rodent α-subunit was achieved by exposing them to a medium containing 3 μm ouabain. Because the endogenous Na+-pump of OK cells is completely inhibited by this concentration of ouabain, only successful recipients of transfected rodent α-subunit would be able to survive. Resistant colonies were expanded and maintained in DMEM-10 containing 3 μmouabain. Experiments were performed with a mix of at least 20 independent clones for each cell line. The Na+,K+-ATPase of mock-transfected cells (vector alone, vector plus liposomes, or liposomes alone) had the same activity and sensitivity to ouabain as non-transfected host cells. Cells were solubilized with SDS, and aliquots were used for protein determination. Protein concentration was determined by the bicinchoninic acid method (Pierce) using bovine serum albumin as a standard. Measurements of Na+,K+-ATPase-mediated transport by Rb+ uptake were performed with attached cells as described previously (12.Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (70) Google Scholar, 13.Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (37) Google Scholar, 15.Efendiev R. Bertorello A.M. Pressley T.A. Rousselot M. Ferraille E. Pedemonte C.H. Biochemistry. 2000; 39: 9884-9892Crossref PubMed Scopus (81) Google Scholar). Transfected cells grown in DMEM-10 were exposed for 2 h to 2 mm EGTA to facilitate access of ligands to Na+,K+-ATPase. To measure Rb+ transport, cells were transferred to serum-free DMEM containing 50 mm HEPES, pH 7.4 (DMEM/HEPES), and either 3 μm or 5 mm ouabain (incubation medium). Cells were incubated with these amounts of ouabain first for 20 min at 37 °C in an air atmosphere and then for 10 min at 25 °C. All treatments and determinations were performed at 25 °C. After treatment, a trace amount of [86Rb+]RbCl was added to the cell medium. After 20 min, cells were washed four times with ice-cold saline and dissolved with SDS, and accumulated radioactivity was determined. Na+,K+-ATPase-mediated Rb+transport was calculated from the difference in tracer uptake between samples incubated in 3 μm and 5 mm ouabain. The ouabain-insensitive Rb+ transport was 25–30% of the total Rb+ transport measured. As PMA was dissolved in Me2SO, the same volume of solvent was added to control samples. For the experiments with ethoxyresorufin, the drug was dissolved in ethanol. The volume of solvent used did not alter the Rb+ transport of control samples. Each experiment was made in triplicate, and results are the mean ± S.D. of at least three independent experiments. Data are expressed as nanomoles of Rb+ transported per mg of protein per min, or as the percentage of Rb+ transported with respect to a control sample. When monensin was used, control sample was the ouabain-sensitive Rb+ transport of cells treated with monensin. Several experiments were performed with cells in a low calcium medium. DMEM has a concentration of calcium of 1.8 mm. To reduce calcium concentration, 4 mm EGTA was added to the medium. Assuming that the total calcium in DMEM is free calcium, 4 mm EGTA would reduce the free calcium concentration to 0.12 μm. Several hours of incubation in this low calcium medium does not affect the attachment of the cells or their morphology as observed by microscopy. To chelate intracellular calcium, 1,2-bis[o-aminophenoxy]ethane-N,N,N′, N′-tetraacetic acid (BAPTA) was used. The membrane-permeant acetoxymethyl form of BAPTA (BAPTA-AM) was added to the cell incubation solution at the concentration of 100 μm and incubated for 1 h at 25 °C, as indicated by the manufacturer (Molecular Probes, Eugene, OR). Optical determinations of intracellular sodium with a sodium-sensitive dye were performed as described previously (12.Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (70) Google Scholar, 13.Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (37) Google Scholar). Fluorescence measurements of [Na+]i were performed using the membrane-permeant tetra(acetoxymethyl) ester of the sodium-binding benzofuran-isophthalate (SBFI-AM, Molecular Probes, Eugene, OR) following standard protocols (16.Harootunian A.T. Kao J.P. Eckert B.K. Tsien R.Y. J. Biol. Chem. 1989; 264: 19458-19467Abstract Full Text PDF PubMed Google Scholar, 17.Moore E.D. Fay F.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8058-8062Crossref PubMed Scopus (26) Google Scholar). Cells were loaded for 3 h with the dye at room temperature in DMEM/HEPES medium containing 2–5 μm SBFI-AM and 0.1% w/v Pluronic F-127 (Molecular Probes, Eugene, OR). After loading, the cells were washed several times with DMEM/HEPES medium and incubated for 30 min in the same medium to allow de-esterification of SBFI-AM. The complete hydrolysis of SBFI-AM to SBFI was judged by changes in the excitation and emission spectra (16.Harootunian A.T. Kao J.P. Eckert B.K. Tsien R.Y. J. Biol. Chem. 1989; 264: 19458-19467Abstract Full Text PDF PubMed Google Scholar). Optical measurements were performed in DMEM/HEPES medium at room temperature. Fluorescence measurements of [Ca2+]iwere performed using 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane -N,N,N′, N′-tetraacetic acid (Fura-2). OK cells were loaded with the membrane-permeant tetra(acetoxymethyl) ester of this dye (Fura-2-AM, Molecular Probes, Eugene, OR) following similar protocols as with the SBFI-AM dye. Cells were loaded for 1–2 h with the dye at room temperature in DMEM/HEPES medium containing 2 μm Fura-2-AM and 0.1% w/v Pluronic F-127. Cells were then washed several times with DMEM/HEPES and incubated for 30 min in the same medium to allow de-esterification of the dye. Optical signals were acquired in DMEM/HEPES medium at room temperature. The general plan of the optical setup used to monitor Na+ and Ca2+ cytosolic levels in OK cells was based on standard methods using a dual-excitation fluorescence imaging system. Essentially the optical system consists of an inverted fluorescence microscope (Olympus, Melville, NY) with a video camera (Pentamax, Princeton Instruments, Trenton, NJ) attached to its video port. Light from a 75-watt xenon lamp (model 1600, Optic Quip, Highland Mills, NY) is collimated and rendered quasi-monochromatic by one of several interference filters, focused by means of a quartz UV-grade condenser and reflected to the preparation by a dichroic mirror. Excitation wavelengths were selected through a computer-controlled filter changer using excitation filters having wavelengths of 340 and 380 nm (5 nm bandwidth, Omega Optical, Brattleboro, VT). These excitation filters were selected because they are adequate for ratio measurements using either SBFI or Fura-2 indicators. By using these indicators, the fluorescence emission was detected above 420 nm after passing through a dichroic mirror (400 nm, Omega Optical, Brattleboro, VT) and a 420 nm highpass filter (Omega Optical, Brattleboro, VT). To ensure optical stability in the recordings and avoid possible photobleaching effects, the excitation light levels were reduced by neutral density filters until the emitted fluorescence intensity remained constant for 200 s of illumination. No significant levels of auto-fluorescence were observed, and drugs at the concentrations used did not affect or quench fluorescence levels. No detectable change in the SBFI or Fura-2 ratios was observed in the pH range (7.4–7.1) tested in the present study. To improve efficiency, fluorescent light from the cells was collected by high numerical aperture (×20 or 40, Fluo; Nikon), which formed a real image on the CCD sensor of the video camera located in the image plane of the microscope. The camera sensitivity was optimized by controlling exposure times according to background fluorescence levels of the cells and the size of the fluorescence changes to be detected. By using the protocol previously described, fluorescence measurements of Na+ levels in cells loaded with SBFI usually required image exposures of 250–300 ms. For free Ca2+ determinations with Fura-2, exposures in the order of 100–120 ms were needed. The chambers containing loaded cells were alternately excited at 340 and 380 nm by rapidly switching optical filters, and ratiometric determinations usually corresponded to image pairs taken within 800 ms. Sequential image pairs were usually collected every 6 s, although in some experiments faster time resolution was used. Fluorescence measurements of [Na+]i and [Ca2+]i were performed using traditional ratiometric determination protocols (12.Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (70) Google Scholar,13.Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (37) Google Scholar, 18.Grynkiewics G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Google Scholar, 19.Cinelli A.R. Neff S.R. Kauer J.S. J. Neurophysiol. 1995; 73: 2017-2032Crossref PubMed Scopus (22) Google Scholar). Terms of the equation were assessed by in situcalibration at the end of each experiment with solutions of known ionic concentrations. Cytosolic Na+ levels were calculated according to the original equation described previously (18.Grynkiewics G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Google Scholar) with a Kd value for SBFI-Na+ of 18 mm. Calibrations of the excitation ratio were performed with cells permeabilized with gramicidin D (10 μm) and superfused with different standard Na+ concentrations. Similarly, intracellular free Ca2+ concentration was measured by determining the ratio of Fura-2 fluorescence at 340 nm (F340) and 380 nm (F380) excitations. There was no detectable photobleaching during measurements as determined by the isosbestic wavelength for both dyes. The fluorescence ratio F340/F380 of Fura-2 was calibrated in situ according to standard protocols using the same equation (18.Grynkiewics G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Google Scholar). In this case, calibrations were performed in OK cells permeabilized with the Ca2+ ionophore ionomycin. These calibrations were confirmed with cells permeabilized with either digitonin or saponin. Fmax and Fmin were determined in Ringer's solution (1 mm Ca2+) to saturate the Ca2+ indicator and then bathing the cell in low Ca2+ Ringer's solution supplemented with 5 mm EGTA. Standard computer-based image analysis software was used for the analysis of Na+ and Ca2+ images. Video images were acquired at 8 bits resolution and stored in real time in a Pentium IBM-compatible computer system. Final ionic determinations were obtained applying standard ratiometric processing algorithms. In the figures, ionic changes in single cells are illustrated by pseudocolors reflecting ionic concentration ranges as determined according to ratiometric determinations (see above). Temporal plots of Na+ and Ca2+ transients were obtained from averaged values over 6 × 6 pixel kernels. To improve signal-noise ratio of SBFI measurements, [Na+]i determinations at each time results from the averaging of multiple samples acquired at faster time resolutions. Usually, individual points represent the average of 6 ratio determinations taken every 10 s (6 paired-frame/min), although in some experiments faster time resolution was used. Comparisons between groups were performed by Student's t test for unpaired data. The sodium ionophore monensin was used to produce stable incremental elevations of intracellular sodium in OK cells. To determine the intracellular concentration of free sodium, OK cells were loaded with a sodium-sensitive dye, and the level of emitted fluorescence was monitored using a video imaging system, as described previously (12.Pedemonte C.H. Pressley T.A. Lokhandwala M.F. Cinelli A.R. J. Membr. Biol. 1997; 155: 219-227Crossref PubMed Scopus (70) Google Scholar, 13.Pedemonte C.H. Pressley T.A. Cinelli A.R. Lokhandwala M.F. Mol. Pharmacol. 1997; 52: 88-97Crossref PubMed Scopus (37) Google Scholar). Fluorescent images of OK cells loaded with SBFI and excited at 380 nm are shown at the left of Fig. 1, A—C. Upon excitation at 340 and 380 nm, the level of intracellular sodium was calculated from the ratio of emitted fluorescence that was calibrated by loading the cells with standard sodium concentrations. Ratiometric images of SBFI-loaded cells, displayed in pseudocolor, were obtained at different times of treatment and concentrations of monensin. Images in the center of Fig. 1, A–C (X1), illustrate the basal sodium concentration (no monensin treatment). Basal levels of intracellular sodium ranged from 5.3 to 13.1 mm with an average of 7.8 ± 3.3 mm (n = 24). Images on the right of Fig. 1, A–C (X2) illustrate the intracellular sodium concentration 5 min after the addition of 6, 9, or 12 μm monensin, respectively, to the cell medium. Fig. 1, D and E, illustrate intracellular sodium levels at various times after addition of different monensin concentrations to the cell medium. In situ calibration of the excitation ratio of SBFI at various intracellular sodium concentrations indicated that monensin produced a steady increase in the intracellular sodium concentration of OK cells up to about 30 min, and then the concentration of sodium was stable for at least another 40 min (Fig. 1E). As expected, higher intracellular concentrations of sodium were determined at higher levels of monensin in the cell medium. Once the steady state of intracellular sodium concentration was reached, a linear relation between the concentration of monensin in the cell medium and the concentration of intracellular sodium was calculated (Fig. 1F). The linear equation from this plot ([Na+]i = (2.25 ± 0.11)[monensin] 103 + (8.92 ± 0.71) mm) was used to calculate the intracellular sodium concentrations that correspond to the concentrations of monensin in the cell medium. Because the new steady state of intracellular sodium produced by monensin is reached at about 30 min, determinations of Rb+transport were always started at this time. Because the activity of the Na+,K+-ATPase is limited by the level of intracellular sodium (3.Aperia A.C. Annu. Rev. Physiol. 2000; 62: 621-647Crossref PubMed Scopus (223) Google Scholar), the elevated intracellular sodium concentration elicited by monensin produced stimulation of the Na+,K+-ATPase activity (Fig. 2A). The basal Na+,K+-ATPase activity was 9.8 ± 0.7 nmol/mg/min, and it was stimulated to 18.5 ± 1.0 by 5 μm monensin (Fig. 2A). To determine the effect of intracellular sodium concentration on the inhibition of Na+,K+-ATPase activity by dopamine, cells were treated with monensin for 30 min before the addition of 1 μm dopamine. Five minutes later, radioactive Rb+ was added to the cell medium to determine the ouabain-sensitive ion transport for 20 min. At basal concentrations of intracellular sodium
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