FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1541252089> ?p ?o ?g. }

• W1541252089 endingPage "13663" @default.
• W1541252089 startingPage "13657" @default.
• W1541252089 abstract "Treatment of Madin-Darby canine kidney (MDCK) cells with the peptide hormone angiotensin II (Ang II) results in an increase in the concentrations of cytosolic free calcium ([Ca2+]i) and sodium ([Na+]i) with a concomitant decrease in cytosolic free Mg2+ concentration ([Mg2+]i). In the present study we demonstrate that this hormone-induced decrease in [Mg2+]i is independent of [Ca2+]i but dependent on extracellular Na+. [Mg2+]i, [Ca2+]i, and [Na+]i were measured in Ang II-stimulated MDCK cells by fluorescence digital imaging using the selective fluoroprobes mag-fura-2AM, fura-2AM, and sodium-binding benzofuran isophthalate (acetoxymethyl ester), respectively. Ang II decreased [Mg2+]i and increased [Na+]i in a dose-dependent manner. These effects were inhibited by irbesartan (selective AT1receptor blocker) but not by PD123319 (selective AT2receptor blocker). Imipramine and quinidine (putative inhibitors of the Na+/Mg2+ exchanger) and removal of extracellular Na+ abrogated Ang II-mediated [Mg2+]i effects. In cells pretreated with thapsigargin (reticular Ca2+-ATPase inhibitor), Ang II-stimulated [Ca2+]i transients were attenuated (p < 0.01), whereas agonist-induced [Mg2+]i responses were unchanged. Clamping the [Ca2+]i near 50 nmol/liter with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) inhibited Ang II-induced [Ca2+]i increases but failed to alter Ang II-induced [Mg2+]i responses. Benzamil, a selective blocker of the Na+/Ca2+ exchanger, inhibited [Na+]i but not [Mg2+]i responses. Our data demonstrate that in MDCK cells, AT1 receptors modulate [Mg2+]i via a Na+-dependent Mg2+ transporter that is not directly related to [Ca2+]i. These data support the notion that rapid modulation of [Mg2+]i is not simply a result of Mg2+ redistribution from intracellular buffering sites by Ca2+ and provide evidence for the existence of a Na+-dependent, hormonally regulated transporter for Mg2+ in renally derived cells. Treatment of Madin-Darby canine kidney (MDCK) cells with the peptide hormone angiotensin II (Ang II) results in an increase in the concentrations of cytosolic free calcium ([Ca2+]i) and sodium ([Na+]i) with a concomitant decrease in cytosolic free Mg2+ concentration ([Mg2+]i). In the present study we demonstrate that this hormone-induced decrease in [Mg2+]i is independent of [Ca2+]i but dependent on extracellular Na+. [Mg2+]i, [Ca2+]i, and [Na+]i were measured in Ang II-stimulated MDCK cells by fluorescence digital imaging using the selective fluoroprobes mag-fura-2AM, fura-2AM, and sodium-binding benzofuran isophthalate (acetoxymethyl ester), respectively. Ang II decreased [Mg2+]i and increased [Na+]i in a dose-dependent manner. These effects were inhibited by irbesartan (selective AT1receptor blocker) but not by PD123319 (selective AT2receptor blocker). Imipramine and quinidine (putative inhibitors of the Na+/Mg2+ exchanger) and removal of extracellular Na+ abrogated Ang II-mediated [Mg2+]i effects. In cells pretreated with thapsigargin (reticular Ca2+-ATPase inhibitor), Ang II-stimulated [Ca2+]i transients were attenuated (p < 0.01), whereas agonist-induced [Mg2+]i responses were unchanged. Clamping the [Ca2+]i near 50 nmol/liter with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) inhibited Ang II-induced [Ca2+]i increases but failed to alter Ang II-induced [Mg2+]i responses. Benzamil, a selective blocker of the Na+/Ca2+ exchanger, inhibited [Na+]i but not [Mg2+]i responses. Our data demonstrate that in MDCK cells, AT1 receptors modulate [Mg2+]i via a Na+-dependent Mg2+ transporter that is not directly related to [Ca2+]i. These data support the notion that rapid modulation of [Mg2+]i is not simply a result of Mg2+ redistribution from intracellular buffering sites by Ca2+ and provide evidence for the existence of a Na+-dependent, hormonally regulated transporter for Mg2+ in renally derived cells. Magnesium, the second most abundant intracellular cation, plays an important role in cellular function. Intracellular Mg2+ is an essential cofactor for many enzymes, it modulates various transporters and ion channels, it regulates lipid- and phosphoinositide-derived second messengers, and it influences DNA and protein synthesis (1O'Rourke B. Backx P.H. Marban E. Science. 1992; 257: 245-248Crossref PubMed Scopus (107) Google Scholar, 2Eskes R. Antonsson B. Osen Sand A. Montessuit S. Richter C. Sadoul R. Mazzei G. Nichols A. Martinou J.C. J. Cell Biol. 1998; 143: 217-224Crossref PubMed Scopus (583) Google Scholar, 3Mandel G. Goodman R.H. Nature. 1999; 398: 29-30Crossref PubMed Scopus (15) Google Scholar, 4Wolf F.I. Cittadini A. Front. Biosci. 1999; 4: 1-11Crossref Google Scholar, 5Morrill G.A. Gupta R.J. Kostellow A.B. Ma G.-Y. Zhang A. Altura B.T. Altura B.M. FEBS Lett. 1998; 440: 167-171Crossref PubMed Scopus (40) Google Scholar). Mg2+ is an important modulator of intracellular free Ca2+ concentration ([Ca2+]i) and pHi, which are major determinants of cell contraction, secretion, and motility (6Nakajima T. Iwasawa K. Hazama H. Asano M. Okuda Y. Omata M. Eur. J. Pharmacol. 1997; 320: 81-86Crossref PubMed Scopus (28) Google Scholar, 7Gilbert D'Angelo E.K. Singer H.A. Rembold C.M. J. Clin. Invest. 1992; 89: 1988-1994Crossref PubMed Google Scholar, 8Zhang A. Cheng T.P. Altura B.M. Biochim. Biophys. Acta. 1992; 1134: 25-29Crossref PubMed Scopus (126) Google Scholar). Furthermore, increasing evidence suggests that Mg2+ plays a major role in cell differentiation and proliferation by regulating transphosphorylations, DNA synthesis, and intracellular signaling (4Wolf F.I. Cittadini A. Front. Biosci. 1999; 4: 1-11Crossref Google Scholar). These physiological processes require that the intracellular free Mg2+ concentration ([Mg2+]i) be controlled within a narrow range. Mechanisms regulating [Mg2+]i have not been fully elucidated. Early reports suggested that intracellular sequestration of Mg2+does not contribute to [Mg2+]i homeostasis (9Grubbs R.D. Collins S.D. Maguire M.E. J. Biol. Chem. 1984; 259: 12184-12192Abstract Full Text PDF PubMed Google Scholar). However, these conclusions were based on studies of total cell Mg2+, not free [Mg2+]i, the physiologically important form of Mg2+. Mg2+ is not a static ion. It moves rapidly between intracellular compartments and across the plasma membrane (10Quamme G.A. Dai L.-J. Rabkin S.W. Am. J. Physiol. 1993; 265: H281-H288PubMed Google Scholar). Mg2+ enters cells along a concentration gradient and by facilitated diffusion (11Flatman P.W. Annu. Rev. Physiol. 1991; 53: 259-271Crossref PubMed Scopus (218) Google Scholar) and is extruded from cells via a Na+/Mg2+ exchanger (11Flatman P.W. Annu. Rev. Physiol. 1991; 53: 259-271Crossref PubMed Scopus (218) Google Scholar, 12Murphy E. Freudenrich C.C. Lieberman M. Annu. Rev. Physiol. 1991; 53: 273-287Crossref PubMed Scopus (111) Google Scholar, 13Gunther T. Mag. Bull. 1996; 18: 1-6Google Scholar). The Na+/Mg2+ exchanger was first demonstrated in the squid giant axon where Na+ entry is coupled to Mg2+ moving out of the cell (14DeWeer P. J. Gen. Physiol. 1976; 68: 159-178Crossref PubMed Scopus (65) Google Scholar). Na+-dependent Mg2+ transport is inhibited by amiloride, quinidine, imipramine, and manganese, and it may be regulated by cAMP, protein kinase C, and the Na+/H+ antiporter (15Gunther T. Vormann J. FEBS Lett. 1992; 297: 132-134Crossref PubMed Scopus (71) Google Scholar, 16Feray J.C. Garay R. Biochim. Biophys. Acta. 1986; 856: 76-84Crossref PubMed Scopus (93) Google Scholar, 17Touyz R.M. Schiffrin E.L. J. Biol. Chem. 1996; 271: 24353-24358Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 18Romani A. Marfella C. Scarpa A. FEBS Lett. 1992; 296: 135-140Crossref PubMed Scopus (48) Google Scholar, 19Chen L. Mae Huang L.-Y. Nature. 1992; 356: 521-523Crossref PubMed Scopus (824) Google Scholar). Although the transporter has been functionally demonstrated, it has not yet been cloned or characterized. A P-type ATPase has been cloned from bacteria and has been implicated in Mg2+ transport; however, whether such an enzyme exists in mammalian cells is unclear (20Snavely M.D. Miller C.G. Maguire M.E. J. Biol. Chem. 1991; 256: 815-823Google Scholar). Another Mg2+ transporter, the Mg2+/H+exchanger, has recently been fully characterized in plant cells but not in mammalian cells (21Shaul O. Hilgemann D.W. de-Almeida-Engler J. Van Montagu M. Inze D. Galili G. EMBO. 1999; 18: 3973-3980Crossref PubMed Scopus (211) Google Scholar). Increasing evidence suggests that humoral factors influence transmembrane kinetics of Mg2+ to regulate cellular actions of these hormones by modulating signal transduction pathways. Small changes in [Mg2+]i can have significant effects on biological responses. Studies with perfused heart and liver, as well as with cardiac myocytes and hepatocytes, have demonstrated that Mg2+ fluxes are under the control of various hormones, such as insulin, endothelin-1, norepinephrine, epinephrine, aldosterone, and arginine vasopressin (22Murphy E. Freudenrich C.C. Levy L.A. London R.E. Lieberman M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2981-2984Crossref PubMed Scopus (92) Google Scholar, 23Romani A.M.P. Matthews V.D. Scarpa A. Circ. Res. 2000; 86: 326-333Crossref PubMed Scopus (60) Google Scholar, 24Romani A. Scarpa A. Nature. 1990; 346: 841-844Crossref PubMed Scopus (149) Google Scholar, 25Keenan D. Romani A. Scarpa A. Circ. Res. 1995; 77: 973-983Crossref PubMed Scopus (41) Google Scholar, 26Delva P. Pastori C. Degan M. Montesi G. Brazzarola P. Lechi A. Hypertension. 2000; 35: 113-117Crossref PubMed Scopus (54) Google Scholar, 27Okada K. Ishikawa S.E. Saito T. Am. J. Physiol. 1992; 263: C873-C878Crossref PubMed Google Scholar). We recently reported that angiotensin II (Ang II),1 a potent vasoactive agent, modulates [Mg2+]i in vascular smooth muscle cells and in platelets (17Touyz R.M. Schiffrin E.L. J. Biol. Chem. 1996; 271: 24353-24358Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 28Touyz R.M. Schiffrin E.L. Hypertension. 1999; 34: 442-449Crossref PubMed Scopus (45) Google Scholar, 29Touyz R.M. Schiffrin E.L. J. Hypertens. 1993; 11: 551-558Crossref PubMed Scopus (40) Google Scholar). However, very little is known about the mechanisms underlying Mg2+regulation by Ang II, and it is unknown whether Ang II influences Mg2+ metabolism in kidney cells, a major site for Ang II actions. The present study was therefore undertaken to examine Ang II effects on [Mg2+]i in Madin-Darby canine kidney (MDCK) cells, using the fluorescent indicator dye mag-fura-2 (30Raju B. Murphy E. Levy L.A. Hall R.D. London R.E. Am. J. Physiol. 1989; 256: C540-C548Crossref PubMed Google Scholar). Regulatory mechanisms whereby Ang II modulates [Mg2+]i were also investigated. Our findings demonstrate that in MDCK cells Ang II decreases [Mg2+]i via AT1 receptors. These effects are not related to changes in [Ca2+]i but are dependent on extracellular Na+ and a quinidine- and imipramine-sensitive transporter. These data suggest that rapid modulation of [Mg2+]i is not simply a result of Mg2+ redistribution from intracellular buffering sites by Ca2+ and provide evidence for the existence of a Na+-dependent, Ang II-regulated transporter for Mg2+ in renally derived cells. Mag-fura-2AM, fura-2AM, mag-fura-2, sodium-binding benzofuran isophthalate (acetoxymethyl ester), and gramicidin were purchased from Molecular Probes (Eugene, OR). PD123319, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester), Bay K 8644, and thapsigargin were purchased from Calbiochem (San Diego, CA). Irbesartan was a kind gift from Bristol-Myers Squibb Co. All other reagents were from Sigma and ICN Biomedicals Inc. (Irvine, CA). MDCK cells were purchased from the American Type Culture Collection (ATTC, Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium containing fetal calf serum, l-glutamine, HEPES, penicillin, and streptomycin and grown on round glass coverslips and maintained at 37 °C in a humidified incubator (95% air, 5% CO2) as previously described (31Touyz R.M. Tolloczko B. Schiffrin E.L. J. Hypertens. 1994; 12: 663-673Crossref PubMed Scopus (93) Google Scholar). At subconfluence, cells were rendered quiescent by serum deprivation for 30 h prior to experimentation. The selective fluorescent probe mag-fura-2AM was used to measure [Mg2+]i. Cells were washed with modified Hanks' buffered saline solution containing (in mmol/liter): 137 NaCl, 5.4 KCl, 4.2 NaHCO3, 3 Na2HPO4, 0.4 KH2PO4, 1.3 CaCl2, 0.5 MgCl2, 0.8 MgSO4, 10 glucose, Hepes, pH 7.4 and loaded with mag-fura-2AM (4 μmol/liter), which was dissolved in dimethyl sulfoxide with 0.02% pluronic acid. Cells were incubated for 30 min at room temperature and then washed with warmed buffer and incubated for a further 15 min to ensure complete deesterification. Under these loading conditions, the ratiometric (343/380 nm) fluorescence cell images were homogeneous, indicating that there was no significant intracellular compartmentation of mag-fura-2. The coverslip containing cells was placed in a stainless steel chamber and mounted on the stage of an inverted microscope (Axiovert 135, Zeiss) as previously described (31Touyz R.M. Tolloczko B. Schiffrin E.L. J. Hypertens. 1994; 12: 663-673Crossref PubMed Scopus (93) Google Scholar). Cells were exposed to increasing concentrations of Ang II (10−12–10−6mol/liter). In some experiments, cells were pretreated (15 min) with 10−5 mol/liter irbesartan (AT1receptor antagonist) or PD123319 (AT2 receptor blocker). [Mg2+]i was measured in multiple cells simultaneously by fluorescence digital imaging (Attofluor Ratiovision, Zeiss) using an emission wavelength of 520 nm and alternating excitatory wavelengths of 343 and 380 nm (30Raju B. Murphy E. Levy L.A. Hall R.D. London R.E. Am. J. Physiol. 1989; 256: C540-C548Crossref PubMed Google Scholar). The Attofluor system was calibrated by viewing mag-fura-2, tetrapotassium salt solutions containing zero and saturating Mg2+ concentrations and including these data in the ratio calculations for construction of a standard curve relating Mg2+ concentration to the 343/380 ratio. The curve was derived from the equation [Mg2+]i (in mmol/liter) =Kd [(R − Rmin)/(Rmax− R)] × β (30Raju B. Murphy E. Levy L.A. Hall R.D. London R.E. Am. J. Physiol. 1989; 256: C540-C548Crossref PubMed Google Scholar), where R is the ratio of fluorescence at 343 and 380 nm, Rmax and Rmin are the ratios for mag-fura free acid at 343 and 380 nm in the presence of saturating Mg2+ and zero Mg2+, respectively, and β is the ratio of fluorescence of mag-fura-2 at 380 nm in zero and saturating magnesium. Kd is the dissociation constant of mag-fura-2 for Mg2+ and is assumed to be 1.5 mmol/liter (30Raju B. Murphy E. Levy L.A. Hall R.D. London R.E. Am. J. Physiol. 1989; 256: C540-C548Crossref PubMed Google Scholar). Video images of fluorescence at 520 nm emission were obtained using an intensified charge-coupled device camera system with the output digitized to a resolution of 512 × 480 pixels. Images of fluorescence ratios were obtained by dividing, pixel by pixel, the 343 nm image after background subtraction by the 380 nm image after background subtraction. For determination of the mag-fura-2 fluorescence spectrum, mag-fura-2-loaded cells were exposed to graded Mg2+ buffers or graded Ca2+ buffers. The buffers were identical in composition with the Hanks' buffer as indicated above, apart from a variable concentration of MgCl2/MgSO4 or CaCl2. Cells were lysed by addition of 0.1% Triton X-100 (final concentration). Excitation spectra were recorded, and the 343:380 nm ratio was determined. In some experiments [Ca2+]i was measured. The method used has been previously described in detail (31Touyz R.M. Tolloczko B. Schiffrin E.L. J. Hypertens. 1994; 12: 663-673Crossref PubMed Scopus (93) Google Scholar) and was similar to that for [Mg2+]i described above. Briefly, cells were loaded with fura-2AM (4 μmol/liter) and incubated at room temperature in the dark for 30 min. [Ca2+]i was measured in washed cells by fluorescence digital imaging using excitation wavelengths of 343/380 nm and an emission wavelength of 520 nm. Fura 2-pentapotassium salt was used for [Ca2+]i calibration, and the absolute Ca2+ concentration was determined from the Grynkiewicz formula as stated above for [Mg2+]i (32Grynkiewicz G. Poene M. Tsien T.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). The Kd for the fura-2-Ca2+ complex was assumed to be 224 nmol/liter (32Grynkiewicz G. Poene M. Tsien T.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). [Na+]i was measured using the Na+-selective fluorescent probe SBFI-AM (33Harootunian A.T. Kao J.P.Y. Eckert B.K. Tsien R.Y. J. Biol. Chem. 1989; 264: 19458-19467Abstract Full Text PDF PubMed Google Scholar). Cells were washed with modified Hanks' buffered solution and loaded with SBFI-AM (4 μmol/liter) for 90 min at room temperature. The loaded cells were washed and incubated for a further 15 min. [Na+]iwas measured using an emission wavelength of 20 nm and alternating excitatory wavelengths of 343 and 380 nm. [Na+]iwas calibrated by equilibrating [Na+]i with the extracellular Na+ concentration using the monovalent cation ionophore gramicidin D (1 μmol/liter) (33Harootunian A.T. Kao J.P.Y. Eckert B.K. Tsien R.Y. J. Biol. Chem. 1989; 264: 19458-19467Abstract Full Text PDF PubMed Google Scholar). Na+calibration solutions were made from appropriate mixtures of high Na+ and high K+ solutions. The former contained 90 mmol/liter sodium gluconate, 60 mmol/liter NaCl, 1.2 mmol/liter CaCl2, 10 mmol/liter Hepes; the high K+solution was identical except for complete replacement of Na+ by K+. The reference standards were adjusted to give final concentrations of 0–100 mmol/liter Na+ or K+. Cells were exposed to buffers in which the extracellular Na+ concentration ([Na+]e) was varied from 10 to 80 mmol/liter. Thereafter, gramicidin D was added to clamp [Na+]i to [Na+]e. The final values of the 343/380 excitation ratio were plotted versus[Na+]e. The calibration curve, which was linear between 10 and 80 mmol/liter (r = 0.97;n = 5), was used to obtain [Na+]i. To determine the dependence of [Mg2+]i on Na+, [Mg2+]i responses to Ang II were measured in Na+-free solution. Cells were preincubated in Na+-free Hanks' buffer (Na+ isosmotically replaced with N-methylglucamine) for 15 min before [Mg2+]i measurement. To further assess the relationship between Mg2+ and Na+, [Mg2+]i responses were determined in cells that had been pre-exposed to imipramine or quinidine (0.5 mmol/liter), putative inhibitors of the Na+/Mg2+ exchanger (5Morrill G.A. Gupta R.J. Kostellow A.B. Ma G.-Y. Zhang A. Altura B.T. Altura B.M. FEBS Lett. 1998; 440: 167-171Crossref PubMed Scopus (40) Google Scholar, 10Quamme G.A. Dai L.-J. Rabkin S.W. Am. J. Physiol. 1993; 265: H281-H288PubMed Google Scholar, 16Feray J.C. Garay R. Biochim. Biophys. Acta. 1986; 856: 76-84Crossref PubMed Scopus (93) Google Scholar, 17Touyz R.M. Schiffrin E.L. J. Biol. Chem. 1996; 271: 24353-24358Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 34Zhang G.H. Melvin J.E. J. Biol. Chem. 1996; 271: 29067-29072Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 35Gunther T. Vorkman J. Hollriegl V. Biochim. Biophys. Acta. 1990; 1023: 455-461Crossref PubMed Scopus (54) Google Scholar, 36Wolf F.I. Di Francesco A. Covacci V. Cittadini A. Biochem. Biophys. Res. Commun. 1994; 202: 1209-1214Crossref PubMed Scopus (24) Google Scholar). In some experiments, cells were pre-exposed to benzamil (10−5 mol/liter), which selectively inhibits the Na+/Ca2+ exchanger (37Bkaily G. Jaalouk D. Sader S. Shbaklo H. Pothier P. Jacques D. D'Orleans-Juste P. Cragoe E.J. Bose R. Mol. Cell. Biochem. 1998; 188: 187-197Crossref PubMed Scopus (35) Google Scholar). Intracellular free Ca2+concentration was manipulated with 10−5mol/liter thapsigargin (endoplasmic reticular Ca2+-ATPase inhibitor) (38Jan C.R. Ho C.M. Wu S.N. Tseng C.J. Life Sci. 1999; 64: 259-267Crossref PubMed Scopus (63) Google Scholar) or BAPTA-AM (50 μmol/liter), a high capacity chelator that selectively binds Ca2+ about 105 over Mg2+ (39Adams S.R. Kao J.P.Y. Tsien R.Y. J. Am. Chem. Soc. 1989; 111: 7959-7968Google Scholar). In these experiments cells were exposed to either agent for 10–15 min in Ca2+-free buffer (Hanks' buffer without CaCl2) before Ang II stimulation. Each experiment was repeated at least three times using different cell preparations. Data were calculated as the mean response per experiment and then as the mean of multiple experiments. Results are presented as the mean ± S.E. and compared by Student's t test or by analysis of variance where appropriate. Tukey-Kramers' correction was used to compensate for multiple testing procedures. The Ang II concentration (mol/liter) eliciting 50% of the maximal response (EC50) or minimal response (IC50) was determined from concentration-response curves, which were fitted by nonlinear regression. Maximal (or minimal) responses to Ang II were expressed as Emax (or Emin) (in mmol/liter). Sensitivity to Ang II was expressed as pD2 = −log [EC50] (or pI2 = −log IC50) (in mol/liter). p < 0.05 was significant. The specificity of mag-fura for Mg2+ within the physiological Ca2+ range was studied by recording the fluorescence spectrum of mag-fura-2 in graded Ca2+ and Mg2+buffers. With increasing Mg2+ concentrations, the mag-fura-2 excitation ratio increased linearly, whereas the mag-fura-2 fluorescence spectrum was not influenced by Ca2+ (Fig.1A). To further demonstrate the specificity of mag-fura-2 for Mg2+, cells were exposed to the Ca2+ channel agonist Bay K 8644, which increases [Ca2+]i but not [Mg2+]i(37Bkaily G. Jaalouk D. Sader S. Shbaklo H. Pothier P. Jacques D. D'Orleans-Juste P. Cragoe E.J. Bose R. Mol. Cell. Biochem. 1998; 188: 187-197Crossref PubMed Scopus (35) Google Scholar). Bay K 8644 did not alter mag-fura-2 fluorescence but significantly increased fura-2 fluorescence (Fig. 1, B andC), indicating that elevations in [Ca2+]i do not influence mag-fura-2 fluorescence, at least within the range of concentrations tested. Resting levels of Mg2+, Na+, and Ca2+ in MDCK cells were 0.51 ± 0.02 mmol/liter (n = 135 cells), 17.6 ± 0.12 mmol/liter (n = 17 cells), and 84 ± 8.8 nmol/liter (n = 70 cells), respectively, consistent with previously reported values (40Quamme G.A. Dai L.-J. Am. J. Physiol. 1990; 259: C521-C525Crossref PubMed Google Scholar). Fig.2A demonstrates representative fluorescence tracings of Ang II effects on [Mg2+]i, [Na+]i, and [Ca2+]i in cells loaded with mag-fura-2AM, SBFI-AM, and fura-2AM, respectively. Ang II induced a small transient followed by a rapid decrease in mag-fura-2 fluorescence. This effect was sustained for up to 300 s following Ang II addition. SBFI-AM and fura-2AM fluorescence were increased by Ang II, indicating increased [Na+]i and [Ca2+]i. Maximal [Na+]i and [Ca2+]i responses, which occurred within 90 and 40 s, respectively, were sustained for up to 400 s following Ang II addition. Minimum fluorescence values for [Mg2+]i and maximum fluorescence values for [Na+]i and [Ca2+]i were used to generate full dose-response curves. As shown in Fig.2B, Ang II decreased [Mg2+]i(Emin = 0.38 ± 0.01 mmol/liter; pI2 = 8.8 ± 0.2) and increased [Na+]i (Emax = 53 ± 4 mmol/liter; pD2 = 8.5 ± 0.15) and [Ca2+]i (Emax = 618 ± 43 nmol/liter; pD2 = 8.0 ± 0.2) in a dose-dependent manner. To ascertain the receptor subtype through which Ang II mediates [Mg2+]i effects, cells were exposed to the AT1 receptor antagonist irbesartan or the selective AT2 receptor blocker PD123319. In cells pre-exposed to irbesartan, Ang II failed to elicit changes in [Mg2+]i, whereas in cells pretreated with PD123319, Ang II effects were unaltered (Fig.3). These data suggest that Ang II actions are mediated exclusively via the AT1 receptor subtype. Two approaches were used to determine whether Ang II modulates [Mg2+]i via Na+-dependent mechanisms. First, cells were incubated in Na+-free buffer, and second, cells were exposed to quinidine and imipramine, two different inhibitors of the Na+/Mg2+exchanger. As shown in Figs. 4 and 5, exposing cells to Na+-free buffer inhibited the Ang II-induced [Mg2+]i response. [Na+]ieffects were also blunted in Na+-free medium but were not completely abolished. In the absence of extracellular Na+, the magnitude of the [Ca2+]i response was unchanged, even though the time course was altered (Fig. 4). Taken together, these results indicate that Ang II-induced mobilization of intracellular Mg2+ does not directly involve an increase in [Ca2+]i, whereas changes in [Na+]i are required. The Na+dependence of Ang II-elicited [Mg2+]i responses suggests that a Na+/Mg2+ exchange mechanism may be operational.Figure 5Bar graphs demonstrate effects of Ang II (10−7 mol/liter) on MDCK cell [Mg2+]i, [Na+]i, and [Ca2+]i in the presence or absence of extracellular Na+. Na+-free Hanks' buffer was prepared by replacing Na+ withN-methylglucamine. Results are means ± S.E. of four to six experiments, with each experimental field comprising many cells. +,p < 0.05; **, p < 0.01versus basal counterpart.View Large Image Figure ViewerDownload (PPT) As demonstrated in Figs. 6 and 7, quinidine and imipramine abrogated Ang II-elicited [Mg2+]ieffects. [Na+]i responses were reduced by both agents but were not completely inhibited (Figs. 6 and 7), indicating that in addition to the Na+/Mg2+ exchanger, other mechanisms control [Na+]i, possibly the Na+/Ca2+ exchanger. To investigate whether this antiporter contributes to Na+ and Mg2+regulation by Ang II, the effects of benzamil, a selective inhibitor of the Na+/Ca2+ exchanger, were assessed. Benzamil significantly inhibited Ang II-induced Na+ responses but did not alter [Mg2+]i effects (Fig. 7). When cells were pretreated with imipramine and benzamil together, Ang II-induced [Na+]i responses were almost completely abolished (Fig. 7). These data suggest that whereas Mg2+ is regulated primarily via a Na+-dependent Mg2+ transporter, Na+ regulation by Ang II is mediated in large part via the Na+/Ca2+ exchanger.Figure 7Line graphs demonstrate effects of Ang II on [Mg2+]i and [Na+]i in MDCK cells in the absence and presence of imipramine (5 × 10−4mol/liter), the most effective inhibitor of the Na+/Mg2+ exchanger (36Wolf F.I. Di Francesco A. Covacci V. Cittadini A. Biochem. Biophys. Res. Commun. 1994; 202: 1209-1214Crossref PubMed Scopus (24) Google Scholar). Cells were pretreated with imipramine for 15–20 min prior to Ang II (10−7 mol/liter) addition. In some experiments cells were exposed to benzamil (10−5mol/liter), a selective inhibitor of the Na+/Ca2+ exchanger, or to imipramine + benzamil. Each data point is the mean ± S.E. of four to seven experiments, with each experimental field comprising 7–26 cells. *,p < 0.05; **, p < 0.01versus Ang II counterpart.View Large Image Figure ViewerDownload (PPT) To examine whether Ang II influences [Mg2+]i by increasing [Ca2+]i, cells were exposed to BAPTA, which prevents [Ca2+]i elevation by clamping [Ca2+]i at 40–50 nmol/liter. Cells were also treated with thapsigargin, a potent endoplasmic reticular Ca2+-ATPase inhibitor, which depletes reticular Ca2+ stores by preventing Ca2+ reuptake. In the presence of BAPTA, the Ang II-induced increase in [Ca2+]i was attenuated by >80%, whereas the Ang II-mediated decrease in [Mg2+]i was unchanged (Fig. 8). Pre-exposure of cells to thapsigargin in Ca2+-free buffer increased basal [Ca2+]i and attenuated Ang II-stimulated [Ca2+]i responses compared with cells in the absence of thapsigargin (Fig. 8). These results indicate that Ca2+ stores were diminished and that Ang II was unable to mobilize intracellular Ca2+ and to significantly increase [Ca2+]i. Thapsigargin did not alter basal [Mg2+]i or Ang II-induced [Mg2+]i responses (Fig. 6). Taken together, these data suggest that Ang II modulates [Mg2+]i via mechanisms that are not dependent on [Ca2+]ielevations. The major findings of the present study indicate that 1) Ang II decreases [Mg2+]i and increases [Na+]i and [Ca2+]i in MDCK cells in a dose-dependent manner, 2) these effects are blocked by irbesartan but not by PD123319, 3) Ang II-mediated [Mg2+]i effects are abrogated by quinidine and imipramine and in Na+-free buffer, 4) Ang II elicits significant [Mg2+]i responses even when [Ca2+]i effects are blunted, and 5) benzamil inhibits Ang II-induced [Na+]i responses but does not influence [Mg2+]i. These data suggest that in MDCK cells Ang II modulates [Mg2+]i via Na+-dependent, Ca2+-independent mechanisms that are regulated exclusively by AT1 receptors. Furthermore, we demonstrate that the Na+/Ca2+exchanger is a major regulator of [Na+]i, but not of [Mg2+]i, in Ang II-stimulated cells. Although Mg2+ is the most abundant intracellular divalent cation, little is known about underlying mechanisms controlling [Mg2+]i, and there is much controversy whether cytosolic Mg2+ functions as a second messenger and whether it is regulated by hormonal signaling. A recent editorial by Murphy (41Murphy E. Circ. Res. 2000; 86: 245-248Crossref PubMed Scopus (86) Google Scholar) on the “mysteries of magnesium homeostasis” concluded that the challenge for future research will be to elucidate whether agonists induce physiologically significant alterations in Mg2+fluxes. We demonstrate here that Ang II, an important regulator of renal function (42Quan A. Baum M. Nephron. 2000; 84: 103-110Crossref PubMed Scopus (15) Google Scholar), modulates [Mg2+]i in kidney-derived epithelial cells. [Mg2+]i was measured by ratio-imaging microscopy and the indicator mag-fura-2AM, which fluoresces upon binding with Mg2+ (30Raju B. Murphy E. Levy L.A. Hall R.D. London R.E. Am. J. Physiol. 1989; 256: C540-C548Crossref PubMed Google Scholar). Although the affinity of mag-fura-2 for Ca2+ is greater than the affinity for Mg2+, physiological [Mg2+]i is 2–3 orders of magnitude greater than [Ca2+]i. Thus interference due to [Ca2+]i only becomes significant under extreme perturbations when [Ca2+]i is elevated to pharmacologically high levels. When [Ca2+]i is increased to 1 μmol/liter, the Ca2+-mag-fura-2 complex is <10% of the Mg2+-mag-fura-2 complex. In our previous and current studies, [Ca2+]i never reached levels as high as 1 μmol/liter, even when maximal doses of Ang II were used. Furthermore, we demonstrate here that mag-fura-2 fluorescence is unaffected by changes in [Ca2+]i, whereas it is increased by Mg2+ in a concentrationdependent manner. For these reasons, the fluorescence of cellular mag-fura-2 in the present study is considered to be closely and selectively associated with changes in [Mg2+]i. In unstimulated cells, [Mg2+]i was 0.51 ± 0.02 mol/liter, which is in agreement with previously reported values in MDCK cells (0.49 ± 0.03 mmol/liter) (40Quamme G.A. Dai L.-J. Am. J. Physiol. 1990; 259: C521-C525Crossref PubMed Google Scholar). Basal [Mg2+]i is maintained within a narrow range by intracellular buffering processes and by specific and highly regulated Mg2+ influx pathways (40Quamme G.A. Dai L.-J. Am. J. Physiol. 1990; 259: C521-C525Crossref PubMed Google Scholar). Ang II decreased [Mg2+]i in a dose-dependent fashion. These effects were blocked by irbesartan, but not by PD123319, indicating the exclusive role of AT1 receptors in these processes. Similar findings have been reported in vascular smooth muscle cells (17Touyz R.M. Schiffrin E.L. J. Biol. Chem. 1996; 271: 24353-24358Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), but to our knowledge, these are the first data demonstrating that AT1receptors regulate [Mg2+]i in MDCK cells. Mg2+ exchanges, or is cotransported, with other ions, and it uses the electrochemical gradient of other ions to cross the plasma membrane. Na+ may be the counterion exchanged with Mg2+. The Na+/Mg2+ exchanger, inhibitable by imipramine and quinidine, has been functionally demonstrated in sublingual mucous acini, erythrocytes, hepatocytes, thymocytes, cardiac cells, and vascular smooth muscle cells (13Gunther T. Mag. Bull. 1996; 18: 1-6Google Scholar, 15Gunther T. Vormann J. FEBS Lett. 1992; 297: 132-134Crossref PubMed Scopus (71) Google Scholar,16Feray J.C. Garay R. Biochim. Biophys. Acta. 1986; 856: 76-84Crossref PubMed Scopus (93) Google Scholar, 43Zhang G.H. Melvin J.E. FEBS Lett. 1995; 371: 52-56Crossref PubMed Scopus (24) Google Scholar, 44Picado M.J. de la Sierra A. Aguilera M.T. Coca A. Urbano-Márquez A. Hypertension. 1994; 23: 987-991Crossref PubMed Google Scholar, 45Dai L.J. Ritchie G. Bapty B. Quamme G.A. Am. J. Physiol. 1998; 274: F336-F341PubMed Google Scholar, 46Wolf F.I. Di Francesco A. Covacci V. Corda D. Cittadini A. Arch. Biochem. Biophys. 1996; 331: 194-200Crossref PubMed Scopus (26) Google Scholar, 47Cefaratti C. Romani A. Scarpa A. J. Biol. Chem. 2000; 275: 3772-3780Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Gunther (13Gunther T. Mag. Bull. 1996; 18: 1-6Google Scholar) reported that the membrane-spanning Mg2+ transporter is bidirectional and that reversing the Na+ gradient results in net Mg2+ influx. In our experimental conditions, [Mg2+]i responses were temporally associated with [Na+]i changes and were completely abolished in Na+-free medium and by quinidine and imipramine, one of the most effective inhibitors of the antiporter, suggesting that Mg2+ modulation by Ang II is dependent on extracellular Na+ and that the Na+/Mg2+ exchanger is the major regulator of [Mg2+]i, exchanging extracellular Na+for intracellular Mg2+. The stoichiometry of this Ang II-activated exchanger in MDCK cells appears to be complex, and the exact coupling awaits clarification. Inhibitors of the Na+/Mg2+ antiporter abrogated [Mg2+]i responses but only partially decreased [Na+]i effects. These findings suggest that whereas the Na+/Mg2+ exchanger is the primary transporter for Mg2+, it contributes only partially to [Na+]i regulation by Ang II and that other mechanisms also control [Na+]i. To this end we investigated the role of the Na+/Ca2+ exchanger by exposing cells to benzamil, a selective inhibitor of the transporter. Benzamil significantly decreased [Na+]i responses but did not influence [Mg2+]i. Inhibition of both antiporters with imipramine and benzamil blocked Ang II-stimulated [Na+]i responses. These findings demonstrate that the Na+/Ca2+ exchanger does not control transmembrane Mg2+ fluxes in MDCK cells but that it plays an important role in Na+ transport. Similar findings for [Na+]i have been demonstrated in cardiomyocytes and vascular smooth muscle cells (37Bkaily G. Jaalouk D. Sader S. Shbaklo H. Pothier P. Jacques D. D'Orleans-Juste P. Cragoe E.J. Bose R. Mol. Cell. Biochem. 1998; 188: 187-197Crossref PubMed Scopus (35) Google Scholar). Some studies failed to show that intracellular Mg2+homeostasis is regulated by Na+-dependent mechanisms and suggested that changes in [Mg2+]iare due primarily to changes in Ca2+ (22Murphy E. Freudenrich C.C. Levy L.A. London R.E. Lieberman M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2981-2984Crossref PubMed Scopus (92) Google Scholar). Because Ca2+ and Mg2+ bind to many common intracellular sites, an increase in [Ca2+]i could displace Mg2+ from mutual binding sites, thereby altering [Mg2+]i. In cardiomyocytes and isolated hearts, [Mg2+]i regulation is not due to Na+/Mg2+ exchange but appears to be secondary to changes in [Ca2+]i, which is influenced by the Na+/Ca2+ exchanger (22Murphy E. Freudenrich C.C. Levy L.A. London R.E. Lieberman M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2981-2984Crossref PubMed Scopus (92) Google Scholar, 48Buri A. McGuigan J.A. Exp. Physiol. 1990; 75: 751-761Crossref PubMed Scopus (42) Google Scholar). In sublingual acini, muscarinic-induced Mg2+ mobilization is both Na+- and Ca2+-dependent (34Zhang G.H. Melvin J.E. J. Biol. Chem. 1996; 271: 29067-29072Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In MDCK cells, Quamme and Dai (40Quamme G.A. Dai L.-J. Am. J. Physiol. 1990; 259: C521-C525Crossref PubMed Google Scholar) reported that Mg2+ refill into Mg2+-depleted cells is regulated by a verapamil-dependent pathway. In the present study we explored the role of [Ca2+]i on [Mg2+]i regulation, by depleting endoplasmic reticular Ca2+ stores with the Ca2+-ATPase inhibitor thapsigargin and by clamping [Ca2+]i at about 50 mmol/liter with the Ca2+ chelator BAPTA-AM. Under these conditions, Ang II-induced [Ca2+]iresponses were blunted, but [Mg2+]i transients were unaltered, indicating that in MDCK cells [Mg2+]i regulation by Ang II is not dependent on intracellular Ca2+ mobilization or on increased [Ca2+]i. Possible Ca2+-insensitive signaling cascades whereby Ang II influences [Mg2+]i could be via protein kinase C and the Na+/H+ exchanger, which we previously demonstrated to be functionally linked to the Na+/Mg2+ exchanger (17Touyz R.M. Schiffrin E.L. J. Biol. Chem. 1996; 271: 24353-24358Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In conclusion, the present study demonstrates that Ang II mobilizes intracellular Mg2+ in renally derived cells through AT1 receptor-mediated pathways. Ang II decreases [Mg2+]i by activating the Na+/Mg2+ exchanger to promote transmembrane Mg2+ transport in a Ca2+-independent fashion. These processes may play an important role in [Mg2+]i homeostasis and in Mg2+-dependent intracellular signaling pathways that do not require changes in [Ca2+]i. Moreover, we demonstrate that the Na+/Ca2+ exchanger is a major regulator of [Na+]i, but not of [Mg2+]i, in Ang II-stimulated cells. Our results support the notion that rapid modulation of [Mg2+]i is not simply a result of Mg2+ redistribution from intracellular buffering sites by Ca2+ and provide evidence for the existence of a Na+-dependent, hormonally regulated transporter for Mg2+ in renal epithelial cells. angiotensin II Madin-Darby canine kidney" @default.
• W1541252089 created "2016-06-24" @default.
• W1541252089 creator A5019575527 @default.
• W1541252089 creator A5020577597 @default.
• W1541252089 creator A5063192986 @default.
• W1541252089 date "2001-04-01" @default.
• W1541252089 modified "2023-10-15" @default.
• W1541252089 title "Angiotensin II Type I Receptor Modulates Intracellular Free Mg2+ in Renally Derived Cells via Na+-dependent Ca2+-independent Mechanisms" @default.
• W1541252089 cites W1501616315 @default.
• W1541252089 cites W1543226940 @default.
• W1541252089 cites W1577239482 @default.
• W1541252089 cites W1967356330 @default.
• W1541252089 cites W1974631822 @default.
• W1541252089 cites W1980542457 @default.
• W1541252089 cites W1981051540 @default.
• W1541252089 cites W1984929409 @default.
• W1541252089 cites W1998689607 @default.
• W1541252089 cites W2008907279 @default.
• W1541252089 cites W2010518771 @default.
• W1541252089 cites W2020707740 @default.
• W1541252089 cites W2021560824 @default.
• W1541252089 cites W2023081656 @default.
• W1541252089 cites W2024702250 @default.
• W1541252089 cites W2024916451 @default.
• W1541252089 cites W2025281987 @default.
• W1541252089 cites W2030426732 @default.
• W1541252089 cites W2031171517 @default.
• W1541252089 cites W2037704418 @default.
• W1541252089 cites W2047260669 @default.
• W1541252089 cites W2049389091 @default.
• W1541252089 cites W2050284940 @default.
• W1541252089 cites W2052793452 @default.
• W1541252089 cites W2060810600 @default.
• W1541252089 cites W2060904575 @default.
• W1541252089 cites W2073666694 @default.
• W1541252089 cites W2073755152 @default.
• W1541252089 cites W2077348695 @default.
• W1541252089 cites W2083631218 @default.
• W1541252089 cites W2098849350 @default.
• W1541252089 cites W2099750404 @default.
• W1541252089 cites W2103209108 @default.
• W1541252089 cites W2113923747 @default.
• W1541252089 cites W2129429762 @default.
• W1541252089 cites W2134824066 @default.
• W1541252089 cites W2141260882 @default.
• W1541252089 cites W2144684172 @default.
• W1541252089 cites W2159106985 @default.
• W1541252089 cites W2184699306 @default.
• W1541252089 cites W2395866306 @default.
• W1541252089 cites W2460070944 @default.
• W1541252089 cites W4242846507 @default.
• W1541252089 doi "https://doi.org/10.1074/jbc.m008101200" @default.
• W1541252089 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11278387" @default.
• W1541252089 hasPublicationYear "2001" @default.
• W1541252089 type Work @default.
• W1541252089 sameAs 1541252089 @default.
• W1541252089 citedByCount "26" @default.
• W1541252089 countsByYear W15412520892012 @default.
• W1541252089 countsByYear W15412520892013 @default.
• W1541252089 countsByYear W15412520892016 @default.
• W1541252089 countsByYear W15412520892017 @default.
• W1541252089 countsByYear W15412520892021 @default.
• W1541252089 countsByYear W15412520892022 @default.
• W1541252089 crossrefType "journal-article" @default.
• W1541252089 hasAuthorship W1541252089A5019575527 @default.
• W1541252089 hasAuthorship W1541252089A5020577597 @default.
• W1541252089 hasAuthorship W1541252089A5063192986 @default.
• W1541252089 hasBestOaLocation W15412520891 @default.
• W1541252089 hasConcept C12554922 @default.
• W1541252089 hasConcept C126322002 @default.
• W1541252089 hasConcept C134018914 @default.
• W1541252089 hasConcept C170493617 @default.
• W1541252089 hasConcept C185592680 @default.
• W1541252089 hasConcept C198710026 @default.
• W1541252089 hasConcept C2908929049 @default.
• W1541252089 hasConcept C55493867 @default.
• W1541252089 hasConcept C71924100 @default.
• W1541252089 hasConcept C79879829 @default.
• W1541252089 hasConcept C84393581 @default.
• W1541252089 hasConcept C86803240 @default.
• W1541252089 hasConcept C95444343 @default.
• W1541252089 hasConcept C98274493 @default.
• W1541252089 hasConceptScore W1541252089C12554922 @default.
• W1541252089 hasConceptScore W1541252089C126322002 @default.
• W1541252089 hasConceptScore W1541252089C134018914 @default.
• W1541252089 hasConceptScore W1541252089C170493617 @default.
• W1541252089 hasConceptScore W1541252089C185592680 @default.
• W1541252089 hasConceptScore W1541252089C198710026 @default.
• W1541252089 hasConceptScore W1541252089C2908929049 @default.
• W1541252089 hasConceptScore W1541252089C55493867 @default.
• W1541252089 hasConceptScore W1541252089C71924100 @default.
• W1541252089 hasConceptScore W1541252089C79879829 @default.
• W1541252089 hasConceptScore W1541252089C84393581 @default.
• W1541252089 hasConceptScore W1541252089C86803240 @default.
• W1541252089 hasConceptScore W1541252089C95444343 @default.
• W1541252089 hasConceptScore W1541252089C98274493 @default.
• W1541252089 hasIssue "17" @default.
• W1541252089 hasLocation W15412520891 @default.

• next