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(Hypertension. 1999;34:442-449.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Activation of the Na⁺-H⁺ Exchanger Modulates Angiotensin II–Stimulated Na⁺-Dependent Mg²⁺ Transport in Vascular Smooth Muscle Cells in Genetic Hypertension

Rhian M. Touyz; Ernesto L. Schiffrin

From the Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Quebec, Canada.

Correspondence to R.M. Touyz, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec, H2W1R7 Canada. E-mail touyzr{at}ircm.qc.ca

	Abstract

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Abstract—This study investigated the role of the Na⁺-H⁺ exchanger (NHE) on angiotensin II (Ang II)–induced activation of Na⁺-dependent Mg²⁺ transport in vascular smooth muscle cells (VSMCs) from Wistar-Kyoto rats (WKY; n=20) and spontaneously hypertensive rats (SHR; n=20). Intracellular free concentrations of Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) and intracellular pH (pH_i) were measured with the specific fluorescent probes mag–fura 2-AM, SBFI-AM, and BCECF-AM, respectively. Na⁺ dependency of Mg²⁺ transport was assessed in Na⁺-free buffer, and the role of the NHE was determined with the highly selective NHE blocker 5-(N-methyl-N-isobutyl) amiloride (MIA). Basal [Mg²⁺]_i was lower in SHR than WKY (0.59±0.01 versus 0.71±0.01 mmol/L, P<0.05). Basal pH_i and [Na⁺]_i were not different between the 2 groups. Ang II dose dependently increased [Na⁺]_i and pH_i and decreased [Mg²⁺]_i. Responses were significantly greater (P<0.05) in SHR versus WKY ([Na⁺]_i E_max=37.5±1.1 versus 33.7±1.9 mmol/L; pH_i E_max=7.35±0.04 versus 7.20±0.01; [Mg²⁺]_i E_min=0.28±0.09 versus 0.53±0.02 mmol/L, SHR versus WKY). In Na⁺-free buffer, Ang II–elicited [Mg²⁺]_i responses were inhibited. MIA (1 µmol/L) inhibited Ang II–stimulated responses in WKY and normalized responses in SHR ([Mg²⁺]_i E_min=0.49±0.02). Ang II–stimulated activation of NHE was significantly increased (P<0.05) in SHR (0.07±0.002 pH_i/s) compared with WKY (0.05±0.004 pH_i/s). These data demonstrate that in VSMCs [Mg²⁺]_i regulation is Na⁺ dependent, that activation of NHE modulates Na⁺-Mg²⁺ transport, and that increased activity of NHE may play a role in altered Na⁺-dependent regulation of [Mg²⁺]_i in SHR.

Key Words: Na⁺ • pH • signal transduction • vascular resistance • vasoconstriction • magnesium • rats, SHR

	Introduction

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Magnesium has been implicated as an important factor in vascular smooth muscle contraction and blood pressure regulation.^{1 2 3} Increased concentrations of extracellular Mg²⁺ cause vasodilation and attenuate agonist-induced constriction, whereas decreased concentrations cause contraction and potentiate agonist-evoked vasoconstriction.^{4 5 6} The cellular basis for the molecular action of Mg²⁺ in vasoconstriction is unknown, but Mg²⁺ probably influences intracellular free calcium concentration ([Ca²⁺]_i), which is a fundamental determinant of myocardial contraction and vascular smooth muscle constriction. Magnesium acts intracellularly as a Ca²⁺ antagonist^{7 8 9} or extracellularly by inhibiting transmembrane Ca²⁺ transport and Ca²⁺ entry, by decreasing contractile actions of vasoactive agents, and by enhancing relaxant effects of vasodilators.^{5 10 11}

For Mg²⁺ to significantly modulate intracellular events, Mg²⁺ itself must be regulated within the cell. Despite the fact that Mg²⁺ is the most abundant cytosolic divalent cation,¹² little is known about intracellular Mg²⁺ homeostasis, and the mechanisms that control [Mg²⁺]_i are poorly understood. Recent studies demonstrated that Mg²⁺ is an active ion that moves between compartments and across membranes.^{12 13 14} Mg²⁺ is mobilized from intracellular stores via an Na⁺-dependent mechanism,^{15 16} and it is extruded from cells via 2 major mechanisms: Na⁺-Mg²⁺ exchanger and Mg²⁺ ATPase.^{17 18} The Na⁺-Mg²⁺ exchanger, first demonstrated in the giant squid axon, induces Na⁺ influx and Mg²⁺ efflux.¹⁹ A similar mechanism has been demonstrated in many cell types, including vascular smooth muscle cells (VSMCs).^{14 16 20 21 22} Mechanisms that activate the exchanger are unclear, but protein kinase C (PKC) and activation of the Na⁺-H⁺ antiporter have been implicated.^{17 23 24}

Altered regulation of [Mg²⁺]_i may play a role in the pathogenesis of hypertension. The relationship between Mg²⁺ deficiency and hypertension has been extensively reported.^{1 25 26} At the cellular level, [Mg²⁺]_i is decreased in essential and experimental hypertension.^{27 28 29 30} The ability of Mg²⁺ to influence blood pressure could be related to the Ca²⁺-antagonistic actions of Mg²⁺. Underlying mechanisms for reduced [Mg²⁺]_i in hypertension are unclear, but increased activity of the Na⁺-Mg²⁺ exchanger could be important. Hypertension is characterized by increased peripheral resistance, which is caused in part by decreased lumen diameter and exaggerated vascular responsiveness to certain vasoconstrictor agents, especially angiotensin II (Ang II).^{31 32} Ang II induces greater [Ca²⁺]_i, pH_i, and [Mg²⁺]_i responses in VSMCs from spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto rats (WKY).^{32 33 34} Enhanced [Ca²⁺]_i and pH_i effects have been attributed to increased mobilization and Ca²⁺ influx and to activation of the Na⁺-H⁺ exchanger, respectively,^{35 36} but the mechanisms that underlie altered [Mg²⁺]_i responses in SHR are unknown. In hypertension, activation of the Na⁺-H⁺ exchanger is increased, and because Ang II activates this transporter, it may be possible that augmented Ang II–induced activation of the Na⁺-H⁺ exchanger contributes to altered regulation of [Mg²⁺]_i in hypertension. Mg²⁺ efflux is linked to an Na⁺-dependent transporter that is associated with the Na⁺-H⁺ exchanger.²² In the present study, we show that in VSMCs from SHR, increased Ang II–mediated activation of the Na⁺-H⁺ exchanger modulates Na⁺-dependent Mg²⁺ transport, which leads to decreased [Mg²⁺]_i and elevated [Na⁺]_i. These changes could inhibit the [Ca²⁺]_i antagonizing effects of [Mg²⁺]_i, which results in increased [Ca²⁺]_i, enhanced vascular contraction, and increased blood pressure.

	Methods

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Animal Experiments
The study was approved by the Animal Ethics Committee of the Clinical Research Institute of Montreal. Adult male (17-week-old) WKY (n=20) and SHR (n=20) (Taconic Farms Inc, Germantown, NY) were studied. Systolic blood pressure was recorded in prewarmed conscious rats by the tail-cuff method with a photoelectric pulse sensor and a polygraph (Grass Instruments Co) a few days before experimentation. Blood pressure was significantly higher (P<0.001) in SHR than in WKY (190±2.1 versus 112±1.0 mm Hg).

Cell Culture
VSMCs derived from mesenteric arteries, which contribute to peripheral resistance and consequently to blood pressure regulation, were isolated and characterized as described previously.^{22 27 32} Briefly, mesenteric arteries were cleaned, smooth muscle cells were dissociated by digestion, the tissue was filtered, and the cell suspension was centrifuged and resuspended in DMEM that contained fetal calf serum, L-glutamine, HEPES, penicillin, and streptomycin. VSMCs were grown on round glass coverslips and maintained at 37°C in a humidified incubator. Primary and first-passage cells were studied at subconfluence. Cells were rendered quiescent by serum deprivation for 30 hours before experimentation.

Measurement of Intracellular Free Mg²⁺ Concentration
The selective fluorescent probe mag–fura 2-AM was used to measure [Mg²⁺]_i.^{22 37} On complexation with Mg²⁺, mag–fura 2 exhibits fluorescent changes and undergoes a shift in excitation wavelength. Mag–fura 2 has a binding constant for Mg²⁺ of 1.5 mmol/L.³⁷ Although the affinity of mag–fura 2 for Ca²⁺ is greater than the affinity for Mg²⁺, physiological [Mg²⁺]_i is 2 to 3 orders of magnitude greater than [Ca²⁺]_i. Thus, interference caused by [Ca²⁺]_i becomes significant only under extreme perturbations when [Ca²⁺]_i is elevated to pharmacologically high levels. When [Ca²⁺]_i is increased to 1 µmol/L, the Ca²⁺–mag–fura 2 complex is <10% of the Mg²⁺–mag–fura 2 complex.³⁷ In our previous and current studies, [Ca²⁺]_i never reached levels as high as 1 µmol/L, even when maximal doses of Ang II were used.^{6 22 30 34 35} 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. Cells were washed with modified Hanks' buffered saline solution that contained (in mmol/L): NaCl 137, KCl 5.4, NaHCO₃ 4.2, Na₂HPO₄ 3, KH₂PO₄ 0.4, CaCl₂ 1.3, MgCl₂ 0.5, MgSO₄ 0.8, glucose 10, and HEPES 2, pH 7.4, and loaded with mag–fura 2-AM (5 µmol/L), which was dissolved in dimethyl sulfoxide with 0.02% pluronic acid. Cells were incubated for 30 minutes at 37°C in a humidified incubator (95% air/5% CO₂) and then washed with warmed buffer and incubated for another 15 minutes to ensure complete deesterification. Under these loading conditions, the ratiometric (343 and 380 nm) fluorescence cell images were homogeneous, indicating that there was no significant intracellular compartmentation of mag–fura 2. The coverslip that contained cells was placed in a stainless steel chamber and mounted on the stage of an inverted microscope (Axiovert 135) as previously described.³²

[Mg²⁺]_i was measured in multiple cells simultaneously by a fluorescence digital imaging system (Attofluor Ratiovision) with an emission wavelength of 520 nm and alternating excitatory wavelengths of 343 and 380 nm.³⁷ The Attofluor system was calibrated by viewing mag–fura 2, tetrapotassium salt solutions that contained 0 Mg²⁺, 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 following equation: [Mg²⁺]_i (in mmol/L)=K_d[(R-R_min)/(R_max-R)]xß,³⁸ where R is the ratio of fluorescence at 343 and 380 nm; R_max and R_min are the ratios for mag-fura–free acid at 343 and 380 nm in the presence of saturating Mg²⁺ and 0 Mg²⁺, respectively; and ß is the ratio of fluorescence of mag–fura 2 at 380 nm in 0 and saturating magnesium. K_d, the dissociation constant of mag–fura 2 for Mg²⁺, is assumed to be 1.5 mmol/L.³⁷ Video images of fluorescence at 520-nm emission were obtained with an intensified charge-coupled device camera system with the output digitized to a resolution of 512x480 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.

Measurement of Intracellular Free Na⁺ Concentration
[Na⁺]_i was measured with the Na⁺-selective fluorescent probe SBFI-AM.³⁹ Cells were washed with modified Hanks' buffered solution and loaded with SBFI-AM (8 µmol/L) for 90 minutes at room temperature. The loaded cells were washed and incubated for another 15 minutes. [Na⁺]_i was measured by use of an emission wavelength of 520 nm and alternating excitatory wavelengths of 343 and 380 nm. [Na⁺]_i was calibrated by equilibrating [Na⁺]_i with the extracellular Na⁺ concentration by use of the monovalent cation ionophore gramicidin D (1 µmol/L).³⁹ Na⁺ calibration solutions were made from appropriate mixtures of high-Na⁺ and high-K⁺ solutions. The former contained 90 mmol/L sodium gluconate, 60 mmol/L NaCl, 1.2 mmol/L CaCl₂, 10 mmol/L 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 to 100 mmol/L Na⁺ or K⁺. Cells were exposed to buffers in which the extracellular Na⁺ concentration ([Na⁺]_e) was varied from 10 to 80 mmol/L. 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/L in WKY (r=0.98, n=5) and SHR (r=0.99, n=9) cells, was used to obtain [Na⁺]_i.

Determination of Na⁺-Dependent Mg²⁺ Transport
To determine the dependency of [Mg²⁺]_i on Na⁺, [Mg²⁺]_i responses were measured in Na⁺-free solution. Cells were preincubated in Na⁺-free Hanks' buffer (Na⁺ isosmotically replaced with N-methylglucamine) for 15 minutes before [Mg²⁺]_i measurement. To further assess the relationship between Mg²⁺ and Na⁺, [Mg²⁺]_i responses were determined in cells that had been preexposed to quinidine (0.5 mmol/L), an inhibitor of the Na⁺-Mg²⁺ exchanger.^{9 12 17 40}

Measurement of Intracellular pH
Intracellular pH (pH_i) was measured with the pH-sensitive dye BCECF-AM according to described methods.^{41 42} VSMCs were loaded with BCECF-AM (0.2 µmol/L) and incubated for 30 minutes at 37°C. The cells were washed and used after a 10-minute stabilization period. Alternating excitatory wavelengths of 488 and 460 nm and an emission wavelength of 520 nm were used to measure BCECF fluorescence. pH_i was calculated from a calibration curve obtained for each experiment by determining the fluorescence ratios at pH_i values of 7.4, 7.2, 7.0, and 6.8. pH_i was set by incubating the cells in K⁺-rich buffer in the presence of 10 µmol/L nigericin (an exogenous K⁺-H⁺ exchange ionophore).⁴¹

Measurement of Na⁺-H⁺ Exchanger Activity
Activity of the Na⁺-H⁺ exchanger was measured in cells after acidification by use of the NH₄Cl prepulse technique.^{43 44} After determination of basal pH_i, cells were exposed to Hanks' buffer solution that contained 30 mmol/L NH₄Cl for 5 minutes, followed by Na⁺-free solution that contained (in mmol/L) N-methyl-D-glucamine 140, KCl 4.7, KH₂PO₄ 1.2, MgCl₂ 1.2, CaCl₂ 2.0, glucose 10, and HEPES 20, pH=7.4. In the absence of Na⁺, there was no recovery from this acid load. pH_i recovered when the Na⁺-free solution was replaced with Na⁺-containing Hanks' buffer solution. This Na⁺-dependent recovery was inhibited by the Na⁺-H⁺ exchange blocker 5-(N-methyl-N-isobutyl) amiloride (MIA) (10^-5 mol/L)^{42 45} and was operationally defined as Na⁺-H⁺ exchange activity.^{43 44} To quantify the rate of pH_i recovery, a straight line was fitted to the initial 60 seconds after the onset of recovery, and the respective slopes were compared.

Protocols for Reagent Applications
[Mg²⁺]_i, [Na⁺]_i, and pH_i were measured in cells exposed to increasing concentrations of Ang II (10^-12 to 10^-5 mol/L). Many cells (10 to 25 per experimental field) were studied simultaneously. Cells were used for single experiments, and no repetitive determinations were performed. MIA⁴⁵ was used to study the Na⁺-H⁺ exchanger. Cells were preincubated with MIA for 15 to 20 minutes

Statistical Analysis
Each experiment was repeated 3 times with different cell preparations. Data were calculated as the mean response per experiment and then as the mean of multiple experiments. Results are presented as mean±SEM and compared by Student's t test or by ANOVA when appropriate. Tukey-Kramer's correction was used to compensate for multiple testing procedures. The Ang II concentration that elicited 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 E_max (or E_min). Sensitivity to Ang II was expressed as pD₂=-log[EC₅₀] (or pI₂=-log IC₅₀). P<0.05 was significant.

	Results

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Effects of Ang II on [Mg²⁺]_i and [Na⁺]_i in Cells From WKY and SHR
Basal [Mg²⁺]_i in SHR was significantly lower (P<0.01) than basal [Mg²⁺]_i in WKY rats (0.59±0.01 versus 0.71± 0.01 mmol/L). Ang II decreased [Mg²⁺]_i rapidly (within 40 seconds of stimulation) (Figure 1, top). Ang II induced a dose-dependent reduction in [Mg²⁺]_i in WKY and SHR (Figure 1, bottom). Responses were significantly augmented (P<0.05) in cells from SHR (E_min=0.28±0.09 mmol/L) compared with WKY (E_min=0.53±0.02 mmol/L).

View larger version (25K): [in this window] [in a new window]   Figure 1. Top, Time course for [Mg2+]i and [Na+]i in response to Ang II. Bottom, Effects of increasing concentrations of Ang II on VSMC [Mg2+]i and [Na+]i responses in cells from WKY and SHR. Data are presented as Ang II–induced change in [Mg2+]i and [Na+]i, determined as difference between stimulated and basal values. Each data point is mean±SEM of 3 to 7 experiments, with each experiment comprising 10 to 25 cells. A indicates time of Ang II (10-7 mol/L) addition. *P<0.05, **P<0.01 vs WKY counterpart.

Basal [Na⁺]_i was not significantly different between SHR (23.5±2.0 mmol/L) and WKY (20.3±0.5 mmol/L). Ang II stimulation resulted in a rapid and sustained elevation in [Na⁺]_i, with maximal responses occurring within 60 seconds (Figure 1, top). Ang II increased [Na⁺]_i in a dose-dependent manner in WKY and SHR, with responses greater (P<0.05) in SHR (E_max=37.5±1.1 mmol/L) than WKY (E_max= 33.7±1.9 mmol/L) (Figure 1, bottom).

Ang II-Mediated [Mg²⁺]_i Transients Are Na⁺-Dependent
In Na⁺-free medium, basal [Mg²⁺]_i increased in WKY (0.76±0.02 mmol/L) and SHR (0.62±0.02 mmol/L). Ang II–induced [Mg²⁺]_i effects were completely inhibited in Na⁺-free buffer in both rat strains (Figure 2). To further clarify the role of the Na⁺-dependent Mg²⁺ transporter, Ang II responses were measured in the presence of quinidine, which has been used extensively to investigate the Na⁺-Mg²⁺ exchanger.^{9 12 14 16 17 21 40} Quinidine abolished Ang II–mediated [Mg²⁺]_i responses in both rat groups (Figure 2). These results, together with those obtained in Na⁺-free conditions, demonstrate that Ang II–stimulated [Mg²⁺]_i responses are dependent on extracellular Na⁺ and that transmembrane Na⁺ transport may contribute to altered [Mg²⁺]_i in SHR. Basal [Na⁺]_i and Ang II–induced [Na⁺]_i effects were significantly reduced in the presence of Na⁺-free buffer and quinidine (Figure 3). In Na⁺-free conditions, Ang II–stimulated [Na⁺]_i responses were similar in cells from SHR and WKY (Figure 3).

View larger version (39K): [in this window] [in a new window]   Figure 2. Effects of Ang II on [Mg2+]i in Na+-containing and Na+-free buffer (top) and in the presence of quinidine (0.5 mmol/L) in VSMCs from WKY and SHR (bottom). Each bar represents mean±SEM of 5 to 7 experiments, with each experiment comprising 10 to 20 cells. *P<0.05, **P<0.01 vs WKY counterpart; #P<0.05, ##P<0.01 vs Na+-containing buffer counterpart; +P<0.05, ++P<0.01 vs basal counterpart.

View larger version (34K): [in this window] [in a new window]   Figure 3. [Na+]i effects of Ang II in Na+-containing and Na+-free buffer (top) and in presence of quinidine (0.5 mmol/L) in VSMCs from WKY and SHR (bottom). Each bar represents mean±SEM of 3 to 5 experiments, with each experiment comprising 12 to 22 cells. *P<0.05, **P<0.01 vs WKY counterpart; #P<0.05 vs Na+-containing buffer counterpart; +P<0.05, ++P<0.01 vs basal counterpart.

Na⁺-H⁺ Exchanger Modulates Na⁺-Dependent Mg²⁺ Transport
MIA was used to determine whether the Na⁺-H⁺ exchanger contributes to altered Ang II–mediated [Mg²⁺]_i effects. MIA did not alter basal [Mg²⁺]_i in WKY (0.75±0.02 mmol/L) or SHR (0.62±0.02 mmol/L). At a concentration of 10^-6 mol/L, MIA abolished Ang II–induced responses in WKY but not in SHR (Figure 4), whereas 10^-5 mol/L MIA completely inhibited Ang II–induced effects in SHR (Figure 4). Basal [Na⁺]_i was significantly increased in the presence of MIA in SHR (Figure 4). MIA significantly decreased but did not abolish Ang II–stimulated [Na⁺]_i responses in WKY and SHR (Figure 4), suggesting that the Na⁺-H⁺ exchanger only partially contributes to Ang II–elicited [Na⁺]_i effects.

View larger version (33K): [in this window] [in a new window]   Figure 4. Top, [Mg2+]i effects of Ang II in VSMCs from WKY and SHR in absence and presence of selective Na+-H+ exchange blocker MIA. Each data point is mean±SEM of 4 to 6 experiments, with each experiment comprising 12 to 20 cells. Bottom, Effects of Ang II on [Na+]i in absence and presence of MIA in VSMCs from WKY and SHR. Each bar represents mean±SEM of 3 to 4 experiments, with each experiment comprising 11 to 18 cells. *P<0.05, **P<0.01 vs WKY counterpart; #P<0.05 vs counterpart in absence of MIA; +P<0.05, ++P<0.01 vs basal counterpart; P<0.05 vs MIA 10-5 mol/L.

Effects of MIA on Ang II–Induced pH_i Responses
To determine whether Ang II activates the Na⁺-H⁺ exchanger in VSMCs and to assess whether activity of the antiporter is altered in SHR, pH_i responses and activity of the Na⁺-H⁺ exchanger in response to Ang II were determined. Basal pH_i was similar in WKY (7.03±0.21) and SHR (7.06±0.01). Ang II dose dependently increased pH_i (Figure 5). At concentrations >10^-9 mol/L, Ang II–induced responses were significantly greater (P<0.05) in SHR (E_max=7.35±0.04) than WKY (E_max=7.20±0.01) (Figure 5). Preexposure of cells to 10^-6 mol/L MIA completely abolished Ang II–elicited alkalinization in WKY and only partially inhibited effects in SHR (Figure 5). However, higher concentrations of MIA (10^-5 mol/L) completely inhibited Ang II-stimulated alkalinization in SHR (Figure 5).

View larger version (25K): [in this window] [in a new window]   Figure 5. Effects of Ang II on pHi in VSMCs from WKY and SHR in absence and presence of MIA. Each data point is mean±SEM of 6 to 8 experiments, with each experiment comprising 10 to 23 cells. *P<0.05 vs WKY counterpart; **P<0.01 vs counterpart in absence of MIA; +P<0.05 vs SHR in absence of MIA.

Ang II Activates the Na⁺-H⁺ Exchanger, Which Is Increased in SHR
Addition of 30 mmol/L NH₄Cl to VSMCs caused rapid alkalinization (Figure 6a) as NH₃ diffused into the cells and titrated intracellular H⁺. Removal of NH₄ from the external medium caused a rapid decrease in pH_i (Figure 6a). The cells were unable to recover from this acid load in Na⁺-free buffer. Reintroduction of Na⁺ (with Na⁺-containing Hanks' buffer solution) resulted in rapid recovery of pH_i that approached resting values (Figure 6a and 6b). This Na⁺-dependent recovery was completely inhibited in the presence of the Na⁺-H⁺ exchanger inhibitor MIA (10^-5 mol/L) (data not shown). Exposure of cells to Ang II increased Na⁺-dependent recovery of pH_i from an acid load (Figure 6c). In WKY cells, Ang II caused a 2-fold increase in Na⁺-dependent recovery of pH_i, whereas in SHR, Ang II elicited a 2.8-fold increase in pH_i recovery rate (Figure 6c and 6d). Ang II (10^-6 mol/L)–induced recovery rate was significantly greater in SHR cells compared with WKY cells (Figure 6d). These data indicate that Ang II increases activity of the Na⁺-H⁺ exchanger and that agonist-induced activation is greater in SHR compared with WKY.

View larger version (13K): [in this window] [in a new window]   Figure 6. Demonstration that Na+-H+ exchanger is activated by Ang II and responses are greater in SHR than WKY. a, pHi was measured in VSMCs with BCECF. Basal pHi was measured in Hanks' solution. Cells were then acid loaded by removing Hanks' buffer and adding NH4CL (30 mmol/L) for 4 to 5 minutes, followed by Na+-free Hanks' buffer. Cells were then returned to Na+-containing solution. Rapid removal of NH4Cl caused immediate reduction in pHi. Recovery of pHi did not occur until cells were exposed to Na+-containing solution. Expanded time courses (b and c) show pHi responses during recovery from acid load in VSMCs from WKY and SHR. b, pHi recovery in control conditions where recovery rate was determined in response to Na+-containing Hanks' buffer. c, pHi recovery in cells exposed to Ang II (10-6 mol/L). Ang II was present during Na+-free treatment and recovery in Na+-containing solution. d, Cumulative data from 3 or 4 experiments demonstrate rate of pHi change in response to Ang II in VSMCs from WKY and SHR. +P<0.05 vs WKY counterpart; **P<0.01 vs control counterpart.

	Discussion

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This study describes a novel transport abnormality in genetically hypertensive rats. In VSMCs from SHR, augmented Ang II–mediated intracellular signaling is associated with increased activation of the Na⁺-H⁺ exchanger, which could modulate [Mg²⁺]_i via an Na⁺-dependent Mg²⁺ transporter. This is related to intracellular alkalinization, increased [Na⁺]_i, and decreased [Mg²⁺]_i. Previously, we reported that Ang II increases Mg²⁺ efflux via a PKC-mediated Na⁺-dependent Mg²⁺ transporter, but this is the first study to show that the Na⁺-Mg²⁺ exchanger underlies altered Ang II–elicited Mg²⁺ responses in SHR. These effects may be linked to increased activation of the Na⁺-H⁺ exchanger. The present study was performed in cells obtained from small mesenteric arteries that contribute to peripheral resistance and consequently to blood pressure regulation. Only primary and first-passage cells that had undergone minimal phenotypic change were used. Although experimental conditions were optimally controlled so that cells from WKY and SHR could be compared, our data are derived from in vitro studies that cannot fully account for the multiple interacting factors and local milieu that cells are exposed to in vivo. Nevertheless, under controlled conditions, Ang II–stimulated responses are altered in cells from SHR, which may be indicative of more complex events in vivo.

Aberrations in Mg²⁺ metabolism have been implicated in the pathogenesis of essential and experimental hypertension.^{1 2 3 46} Decreased [Mg²⁺]_i has been reported in many cell types in SHR,^{27 28 29 30} and we show here that the magnitude of Ang II–elicited Mg²⁺ response is greater in SHR than WKY. This could be part of the upregulation of the Ang II signaling pathway in hypertension.^{32 47} Underlying mechanisms for reduced [Mg²⁺]_i in hypertension are unclear, but alterations in intracellular Mg²⁺ regulation may be important. To determine the role of the Na⁺-dependent Mg²⁺ transporter in SHR, effects of Ang II were determined in the absence of extracellular Na⁺ and in the presence of quinidine. Although quinidine is not a selective blocker of the Na⁺-Mg²⁺ exchanger, it has been extensively used to study this transporter in various cell types.^{12 14 16 17 21 40} Effects of quinidine appear to be independent of Ca²⁺ transporters, because quinidine did nor alter [Ca²⁺]_i in VSMCs from WKY or SHR (data not shown). In Na⁺-free conditions and in the presence of quinidine, Ang II–stimulated [Mg²⁺]_i responses were inhibited, indicating that the Na⁺-Mg²⁺ exchanger plays a major regulatory role in Mg²⁺ transport and that in SHR it may contribute to altered intracellular Mg²⁺ homeostasis in both the basal and stimulated states. Our results are in agreement with and extend those of others^{46 48} who demonstrated decreased [Mg²⁺]_i and increased activity of the Na⁺-Mg²⁺ transporter in erythrocytes from hypertensive patients. The ability of [Mg²⁺]_i to influence blood pressure may be related to its effect on intracellular Ca²⁺ and Na⁺ metabolism. Decreased [Mg²⁺]_i reduces Ca²⁺-ATPase–mediated Ca²⁺ efflux, enhances intracellular Ca²⁺ mobilization, inhibits Na⁺ and Ca²⁺ pumps, and increases [Ca²⁺]_i, resulting in increased excitation-contraction coupling and increased vascular tone and reactivity^{9 49} (Figure 7). Furthermore, enhanced activity of the Na⁺-Mg²⁺ exchanger may contribute to increased [Na⁺]_i in hypertension.

View larger version (22K): [in this window] [in a new window]   Figure 7. Hypothetical scheme demonstrating possible mechanisms that regulate Ang II–stimulated [Mg2+]i. Ang II receptor (AT rec) binding leads to activation of phospholipase C (PLC), resulting in phosphatidylinositol hydrolysis and formation of IP3 and DAG accumulation. IP3 mobilizes Ca2+ from reticular stores, and DAG activates PKC, which in turn activates Na+-H+ exchanger. These events result in increased [Ca2+]i, intracellular alkalinization, and increased [Na+]i, which in turn activate Na+-Mg2+ exchanger, leading to increased Mg2+ efflux and reduced [Mg2+]i. + indicates stimulation; dashed lines, indirect effect.

Activation of the Na⁺-Mg²⁺ antiporter results in net Na⁺ influx and net Mg²⁺ efflux.²¹ To assess whether Ang II–mediated [Mg²⁺]_i effects are associated with reciprocal [Na⁺]_i responses, [Na⁺]_i was determined under conditions similar to those for [Mg²⁺]_i. Ang II increased [Na⁺]_i in a dose-dependent manner, with responses greater in SHR than WKY. In Na⁺-free medium, [Na⁺]_i effects were inhibited and only partially reduced by quinidine, which suggested that in addition to the Na⁺-Mg²⁺ exchanger, other mechanisms mediate Ang II–elicited [Na⁺]_i actions. Okada et al⁵⁰ demonstrated that in SHR aortic cells, AVP-stimulated [Na⁺]_i responses are increased through enhancement of the mobilization of Ca²⁺ and activity of the Na⁺-H⁺ exchanger. Na⁺ is an important intracellular cation in VSMCs. It influences cell volume and shape and has a direct effect on cell growth.⁵¹ Na⁺ induces cellular hypertrophy in the absence of hemodynamic factors, and in hypertension, increased [Na⁺]_i may contribute to vascular hypertrophy and remodeling.⁵² [Na⁺]_i is also an important regulator of cellular AT₂ receptor expression, which may be increased in cardiovascular diseases.⁵³

Exact mechanisms that regulate the Na⁺-Mg²⁺ exchanger are unclear, but phosphorylation of the Na⁺-Mg²⁺ exchanger seems to be a prerequisite for Mg²⁺ binding to the regulator site.¹⁷ This is analogous to phosphorylation of Na⁺-H⁺ and Na⁺-Ca²⁺ antiporters, which increases the affinity of an intracellular modifier site for H⁺ and Ca²⁺. The Na⁺-Mg²⁺ transporter shares many properties with the Na⁺-H⁺ antiporter, and although it has not yet been unambiguously demonstrated, it is possible that part of the Na⁺-H⁺ exchanger functions as an Na⁺-Mg²⁺ antiporter.¹⁷ This is supported by our findings here and those of others that showed that Na⁺-H⁺ exchange blockers inhibit Mg²⁺ transport and that alkalinization is associated with increased Mg²⁺ efflux and decreased [Mg²⁺]_i.^{12 14 54 55} MIA, at 10^-6 mol/L, abrogated Ang II–stimulated [Mg²⁺]_i responses in WKY and only partially reduced effects in SHR. Responses in SHR were completely abolished when the concentration of MIA was increased to 10^-5 mol/L. These data suggest that the Na⁺-H⁺ exchanger regulates Ang II–stimulated Mg²⁺ efflux and that in SHR modifications of the exchanger may play a role in altered [Mg²⁺]_i responsiveness. It may also be possible that the Na⁺-H⁺ exchange blockers could directly inhibit the Na⁺-Mg²⁺ exchanger or that other Na⁺ transport mechanisms may play a role in SHR. These issues await clarification. Figure 7 is a hypothetical scheme that demonstrates the possible mechanisms that regulate Ang II–stimulated [Mg²⁺]_i. Ang II receptor binding leads to activation of phospholipase C, resulting in phosphatidylinositol hydrolysis and formation of inositol trisphosphate (IP₃) and diacylglycerol (DAG) accumulation. IP₃ mobilizes Ca²⁺ from reticular stores, and DAG activates PKC, which in turn activates the Na⁺-H⁺ exchanger. These events result in increased [Ca²⁺]_i, intracellular alkalinization, and increased [Na⁺]_i, which in turn activate the Na⁺-Mg²⁺ exchanger, leading to increased Mg²⁺ efflux and reduced [Mg²⁺]_i. We reported previously that PKC-dependent pathways regulate Ang II–stimulated [Mg²⁺]_i responses.²²

To determine whether Ang II activates the Na⁺-H⁺ exchanger and to assess whether activity of the antiporter is altered in VSMCs from SHR, we measured pH_i and activity of the Na⁺-H⁺ exchanger in response to Ang II. Ang II increased pH_i in a dose-dependent manner, with responses significantly higher in SHR than WKY. MIA, at 10^-6 and 10^-5 mol/L, completely inhibited Ang II–stimulated alkalinization in WKY and SHR, respectively. Na⁺-H⁺ exchanger activity in unstimulated cells was higher in SHR compared with WKY, but this was not statistically significant. However, Ang II–induced activation of the exchanger was significantly greater in SHR than WKY. These findings are in agreement with other studies that have demonstrated that Na⁺-H⁺ exchanger activity is increased in essential and experimental hypertension.^{56 57 58} Hyperactivation of the exchanger has been attributed to posttranslational regulation in SHR.⁵⁹ The Na⁺-H⁺ exchanger has important effects on vascular smooth muscle growth and contractility, and increased activity of the exchanger has been implicated to play an important role in the development and maintenance of the hypertensive state. The cellular mechanisms through which the antiporter mediates its actions relate to its effects on pH_i and [Na⁺]_i and, as demonstrated in the present study, on [Mg²⁺]_i regulation.

In summary, the results of the present study demonstrate that Ang II–stimulated activation of Na⁺-dependent Mg²⁺ transport significantly reduces [Mg²⁺]_i in VSMCs from SHR. Modulation of the Na⁺-Mg²⁺ exchanger may be associated with increased activation of the Na⁺-H⁺ exchanger in hypertension. Decreased VSMC [Mg²⁺]_i may inhibit the Ca²⁺-antagonizing effects of Mg²⁺, resulting in increased [Ca²⁺]_i and increased vascular contraction. Previously, we demonstrated that Ang II–induced [Ca²⁺]_i responses and contractility are increased in SHR and that manipulation of Mg²⁺ concentrations inversely influence [Ca²⁺]_i.³² Our data provide evidence for a novel ion transport abnormality in SHR. Future studies will characterize the kinetic properties of the Na⁺-Mg²⁺ exchanger and its exact relationship to the Na⁺-H⁺ antiporter in VSMCs from this genetic model of hypertension.

	Acknowledgments

 
This study was supported by a grant from the Heart and Stroke Foundation of Quebec and the Medical Research Council of Canada. R.M.T. was supported by scholarships from the Canadian Hypertension Society/Medical Research Council of Canada and the Fonds de la Recherche en Santé du Québec.

Received February 8, 1999; first decision March 15, 1999; accepted May 14, 1999.

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