Angiotensin II Type I Receptor Modulates Intracellular Free Mg2+ in Renally Derived Cells via Na+-dependent Ca2+-independent Mechanisms*

Rhian M. TouyzDagger, Chantal Mercure, and Timothy L. Reudelhuber

From the Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec H2W 1R7, Canada

Received for publication, September 5, 2000, and in revised form, January 19, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 AT1 receptor blocker) but not by PD123319 (selective AT2 receptor 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.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-5). Mg2+ is an important modulator of intracellular free Ca2+ concentration ([Ca2+]i) and pHi, which are major determinants of cell contraction, secretion, and motility (6-8). Furthermore, increasing evidence suggests that Mg2+ plays a major role in cell differentiation and proliferation by regulating transphosphorylations, DNA synthesis, and intracellular signaling (4). 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 (9). 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 (10). Mg2+ enters cells along a concentration gradient and by facilitated diffusion (11) and is extruded from cells via a Na+/Mg2+ exchanger (11-13). The Na+/Mg2+ exchanger was first demonstrated in the squid giant axon where Na+ entry is coupled to Mg2+ moving out of the cell (14). 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 (15-19). 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 (20). Another Mg2+ transporter, the Mg2+/H+ exchanger, has recently been fully characterized in plant cells but not in mammalian cells (21).

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 (22-27). We recently reported that angiotensin II (Ang II),1 a potent vasoactive agent, modulates [Mg2+]i in vascular smooth muscle cells and in platelets (17, 28, 29). 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 (30). 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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 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).

Cell Culture-- 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 (31). At subconfluence, cells were rendered quiescent by serum deprivation for 30 h prior to experimentation.

Measurement of Intracellular Free Mg2+ and Ca2+ Concentrations-- 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 (31).

Cells were exposed to increasing concentrations of Ang II (10-12-10-6 mol/liter). In some experiments, cells were pretreated (15 min) with 10-5 mol/liter irbesartan (AT1 receptor 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 (30). 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)] × beta  (30), 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 beta  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 (30). 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 (31) 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 (32). The Kd for the fura-2-Ca2+ complex was assumed to be 224 nmol/liter (32).

Measurement of Intracellular Free Na+ Concentration-- [Na+]i was measured using the Na+-selective fluorescent probe SBFI-AM (33). 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+]i was measured using an emission wavelength of 20 nm and alternating excitatory wavelengths of 343 and 380 nm. [Na+]i was calibrated by equilibrating [Na+]i with the extracellular Na+ concentration using the monovalent cation ionophore gramicidin D (1 µmol/liter) (33). 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.

Determination of Na+-dependent Mg2+ Transport-- 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 (5, 10, 16, 17, 34-36). In some experiments, cells were pre-exposed to benzamil (10-5 mol/liter), which selectively inhibits the Na+/Ca2+ exchanger (37).

Determination of Ca2+-dependent Mg2+ Transport-- Intracellular free Ca2+ concentration was manipulated with 10-5 mol/liter thapsigargin (endoplasmic reticular Ca2+-ATPase inhibitor) (38) or BAPTA-AM (50 µmol/liter), a high capacity chelator that selectively binds Ca2+ about 105 over Mg2+ (39). 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.

Statistical Analysis-- 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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mag-fura Is a Mg2+-sensitive Fluoroprobe-- 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 (37). Bay K 8644 did not alter mag-fura-2 fluorescence but significantly increased fura-2 fluorescence (Fig. 1, B and C), indicating that elevations in [Ca2+]i do not influence mag-fura-2 fluorescence, at least within the range of concentrations tested.



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Fig. 1.   Mag-fura-2 is a Mg2+-sensitive fluoroprobe. A, line graphs demonstrate mag-fura-2 selectivity for Mg2+ in MDCK cells. Mag-fura-2-loaded cells were lysed in graded Mg2+ or Ca2+ buffers. Shown is the 343:380 nm fluorescence excitation ratio at different Mg2+ and Ca2+ concentrations. B, representative tracings of fluorescence signals for fura-2 and mag-fura-2 in cells exposed to the Ca2+ channel agonist Bay K 8644 (10-7 mol/liter). Arrows indicate time of Bay K 8644 addition. C, bar graphs demonstrate effects of Bay K 8644 on [Ca2+]i and [Mg2+]i in MDCK cells. Data were derived from fluorescence signals as demonstrated in B. Results are means ± S.E. of four experiments, with each experimental field comprising 15-26 cells. **, p < 0.01 versus basal counterpart.

Effects of Ang II on [Mg2+]i, [Na+]i, and [Ca2+]i in MDCK Cells-- 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 (40). 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.



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Fig. 2.   A, representative fluorescence tracings of MDCK cells loaded with mag-fura-2AM, SBFI-AM, or fura-2AM. Ang II (10-7 mol/liter) was added at the time indicated by the arrows. The y axis is the 343:380 nm fluorescence excitation ratio. B, line graphs demonstrate effects of increasing concentrations of Ang II on [Mg2+]i, [Na+]i, and [Ca2+]i in MDCK cells. Each data point is the mean ± S.E. of five to seven experiments, with each experimental field comprising 8-17 cells. Data were derived from minimum (for [Mg2+]i) and maximum (for [Na+]i and [Ca2+]i) fluorescence signals from tracings similar to those shown in A.

Mg2+ Effects of Ang II Are Mediated via AT1 Receptors-- 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.



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Fig. 3.   Bar graphs demonstrate Ang II (10-7 mol/liter) effects on [Mg2+]i in MDCK cells in the absence or presence of irbesartan (Irb) (AT1 receptor antagonist) or PD123319 (PD12) (AT2 receptor blocker). Cells were pre-exposed to either antagonist for 15 min prior to Ang II addition. Results are means ± S.E. of four experiments, with each experimental field comprising 6-11 cells.*, p < 0.01.

Ang II Modulation of [Mg2+]i Is Na+-dependent-- 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.



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Fig. 4.   Representative fluorescence tracings of MDCK cells loaded with mag-fura-2AM, SBFI-AM, or fura-2AM. Cells were stimulated with Ang II (10-7 mol/liter) in the presence (+Na+) or absence (-Na+) of extracellular Na+. The arrows indicate time of Ang II addition.

As shown in Figs. 4 and 5, exposing cells to Na+-free buffer inhibited the Ang II-induced [Mg2+]i response. [Na+]i effects 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.



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Fig. 5.   Bar 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+ with N-methylglucamine. Results are means ± S.E. of four to six experiments, with each experimental field comprising many cells. +, p < 0.05; **, p < 0.01 versus basal counterpart.

As demonstrated in Figs. 6 and 7, quinidine and imipramine abrogated Ang II-elicited [Mg2+]i effects. [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.



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Fig. 6.   Line graphs demonstrate effects of Ang II on [Mg2+]i and [Na+]i in MDCK cells in the absence and presence of quinidine (5 × 10-4 mol/liter), a putative inhibitor of the Na+/Mg2+ exchanger. Cells were pretreated with quinidine for 15-20 min prior to Ang II (10-7 mol/liter) addition. Each data point is the mean ± S.E. of four experiments, with each experimental field comprising 7-11 cells. *, p < 0.05; **, p < 0.01 versus Ang II counterpart.



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Fig. 7.   Line graphs demonstrate effects of Ang II on [Mg2+]i and [Na+]i in MDCK cells in the absence and presence of imipramine (5 × 10-4 mol/liter), the most effective inhibitor of the Na+/Mg2+ exchanger (36). 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 (10-5 mol/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 < 0.01 versus Ang II counterpart.

Ang II Modulation of [Mg2+]i in MDCK Cells Is Ca2+-independent-- 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+]i elevations.



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Fig. 8.   Bar graphs demonstrate effects of Ang II (10-7 mol/liter) on [Mg2+]i and [Ca2+]i in MDCK cells in the absence and presence of thapsigargin (Thaps) (10-5 mol/liter) or BAPTA-AM (50 µmol/liter). Results are means ± S.E. of four experiments, with each experimental field comprising many cells. *, p < 0.05; **, p < 0.01.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (41) 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 (42), 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+ (30). 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) (40). Basal [Mg2+]i is maintained within a narrow range by intracellular buffering processes and by specific and highly regulated Mg2+ influx pathways (40).

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 (17), but to our knowledge, these are the first data demonstrating that AT1 receptors 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 (13, 15, 16, 43-47). Gunther (13) 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 (37).

Some studies failed to show that intracellular Mg2+ homeostasis is regulated by Na+-dependent mechanisms and suggested that changes in [Mg2+]i are due primarily to changes in Ca2+ (22). 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 (22, 48). In sublingual acini, muscarinic-induced Mg2+ mobilization is both Na+- and Ca2+-dependent (34). In MDCK cells, Quamme and Dai (40) 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+]i responses 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 (17).

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.


    FOOTNOTES

* This study was supported by Grant MT15420 and by a group grant to the Multidisciplinary Research Group on Hypertension from the Canadian Institute of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger A scholar of the Canadian Hypertension Society/Canadian Institute of Health Research. To whom correspondence should be addressed: Clinical Research Inst. of Montreal, 110 Pine Ave. West, Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5747; Fax: 514-987-5585; E-mail: touyzr@ircm.qc.ca.

Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M008101200


    ABBREVIATIONS

The abbreviations used are: Ang II, angiotensin II; MDCK, Madin-Darby canine kidney.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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