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(Circulation. 1997;95:2567.)
© 1997 American Heart Association, Inc.

Articles

Extracellular Mg²⁺ Inhibits Capacitative Ca²⁺ Entry in Vascular Smooth Muscle Cells

Mitsuisa Yoshimura, MD; Tetsuya Oshima, MD; Hideo Matsuura, MD; Takafumi Ishida, MD; Masayuki Kambe, MD; Goro Kajiyama, MD

the First Department of Internal Medicine (M.Y., H.M., T.I., G.K.) and the Department of Clinical Laboratory Medicine (T.O., M.K.), Hiroshima (Japan) University School of Medicine.

Correspondence to Tetsuya Oshima, MD, Department of Clinical Laboratory Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan.

Abstract

Background Agonist-induced Ca²⁺ entry is thought to be mediated by capacitative Ca²⁺ entry other than L-type Ca²⁺ channels in vascular smooth muscle cells (VSMCs). The mechanism for capacitative Ca²⁺ entry has not been fully elucidated. Our objective was to examine the effect of external Mg²⁺ on capacitative Ca²⁺ entry in cultured rat aortic VSMCs.

Methods and Results Three doses of external Mg²⁺ concentration (nominally 0, 1, and 5 mmol/L) were used. After exposure to 1 µmol/L angiotensin II (Ang II) in Ca²⁺-free medium, addition of Ca²⁺ to the medium caused an increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), indicating Ang II–induced Ca²⁺ influx. This Ca²⁺ influx was attenuated in cells preincubated with high external Mg²⁺ concentrations or with 1 µmol/L nifedipine. After VSMCs in Ca²⁺-free medium were exposed to 1 µmol/L thapsigargin, which inhibits the sarcoplasmic reticulum Ca²⁺-ATPase and depletes Ca²⁺ stores, addition of Ca²⁺ to the medium induced an increase in [Ca²⁺]_i, indicating capacitative Ca²⁺ entry. This entry pathway was found to be independent of dihydropyridine-sensitive Ca²⁺ channels and inhibited by increased external Mg²⁺ concentration. External Mg²⁺ concentration did not influence Ca²⁺ efflux across the plasma membrane after stimulation with Ang II plus thapsigargin.

Conclusions Results suggest that in VSMCs, capacitative Ca²⁺ entry is reduced by external Mg²⁺. This mechanism may explain in part the inhibitory effect of external Mg²⁺ on Ca²⁺ handling.

Key Words: calcium • cells • muscle, smooth

Agonists such as Ang II that mobilize Ca²⁺ induce an initial release of Ca²⁺ from intracellular stores, followed by a sustained Ca²⁺ entry through the plasma membrane, and use Ca²⁺ as a second messenger.^{1 2 3} Increases in [Ca²⁺]_i are involved in many functions of VSMCs such as vasoconstriction,⁴ cell proliferation,⁵ and collagen synthesis.⁴

Magnesium deficiency has been implicated in the pathogenesis of cardiovascular disease. Magnesium supplementation has a beneficial effect on the vascular disease process.^{6 7} However, the basis for the molecular action of magnesium in the vascular system is not well known.

An increase in extracellular Mg²⁺ inhibits cell function by decreasing [Ca²⁺]_i.⁸ The inhibitory effect of external Mg²⁺ on Ca²⁺ handling is mediated primarily by a reduction of Ca²⁺ entry through the plasma membrane.⁸ In VSMCs, agonist-induced Ca²⁺ entry is mediated by VGCCs and non-VGCCs. The Ca²⁺ within intracellular Ca²⁺ stores is an important source of cytosolic Ca²⁺ signals and appears to be the primary determinant in the opening of plasma membrane Ca²⁺ channels. The mechanism by which Ca²⁺ entry is activated by the depletion of intracellular Ca²⁺ stores (capacitative Ca²⁺ entry) is thought to involve agonist-induced Ca²⁺ entry through non-VGCCs.^{9 10 11} This model received general acceptance when it was shown that agonist-independent depletion of intracellular Ca²⁺ stores with thapsigargin activated the Ca²⁺ entry pathway in VSMCs.^{9 10 11} Once a baseline in [Ca²⁺]_i measured with fura-2 is reestablished after peak response by the treatment of VSMCs with thapsigargin in the absence of extracellular Ca²⁺, the addition of Ca²⁺ into the medium resulted in an increase in [Ca²⁺]_i. This protocol was used to examine the capacitative Ca²⁺ entry. Although the inhibitory effect of external Mg²⁺ on the VGCCs is well established,¹² little is known about the effect of external Mg²⁺ on the capacitative Ca²⁺ entry pathway. The present study was designed to assess the effect of external Mg²⁺ on capacitative Ca²⁺ entry.

Methods

Reagents
Penicillin, streptomycin, L-glutamine, BSA, trypsin-EDTA, newborn calf serum, DMEM, and HBSS were obtained from GIBCO; collagenase (type I) was obtained from Worthington; and thapsigargin was purchased from LC Services. Fura 2-AM and mag-fura 2-AM were obtained from Molecular Probes. Stock solutions (1 mmol/L) of dyes were prepared in dimethyl sulfoxide. All other chemicals were from Sigma Chemical Co.

Cell Culture
Male Wistar rats 9 to 10 weeks of age were obtained from Charles River Japan (Atsugi, Kanagawa, Japan). Aortic VSMCs were isolated by collagenase digestion as described previously,¹³ with minor modifications. Briefly, the descending thoracic aorta was removed aseptically from rats anesthetized with ether, carefully cleaned, and opened longitudinally in 5 mL HBSS (138 mmol/L NaCl, 5 mmol/L KCl, 4 mmol/L NaHCO₃, 0.3 mmol/L KH₂PO₄, 0.3 mmol/L Na₂HPO₄, and 5.6 mmol/L D-glucose, pH 7.4) supplemented with 0.2 mmol/L CaCl₂ and 10 mmol/L HEPES. The intimal layer was gently scraped with a cotton-tipped applicator to remove endothelial cells, and the aorta was then incubated at 37°C for 30 minutes in collagenase solution (HBSS containing 0.2 mmol/L CaCl₂, 10 mmol/L HEPES, 1 mg/mL collagenase [type I, 194 U/mg], 0.125 mg/mL elastase [type III, 80 U/mg], 0.5 mg/mL trypsin inhibitor, and 2 mg/mL BSA). After the adventitia was removed, the aorta was minced and digested at 37°C for 2 hours in 15 mL collagenase solution. The preparation was passed through a 100-µm-mesh sieve to separate dispersed cells from undigested tissue fragments. After centrifugation at 200g for 5 minutes, the VSMCs were resuspended in 10 mL DMEM supplemented with 10% newborn calf serum, 2 mmol/L L-glutamine, 25 mmol/L HEPES, 100 U/mL penicillin, and 100 µg/mL streptomycin, pH 7.4. VSMCs were seeded into 100-mm culture dishes and cultured at 37°C under 95% air and 5% CO₂. Culture medium was changed after 12 hours and then every second day. At confluence (every week), cells were harvested for passage with a trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA) and were used after three to six passages. For each experimental protocol, four to six separate culture preparations were studied. The cells were identified as VSMCs by the typical "hill-and-valley" pattern at confluence and by immunocytochemical analysis with antibodies specific for smooth muscle -actin.

Measurement of [Ca²⁺]_i
VSMCs were allowed to attach to glass coverslips. Coverslips were washed twice with a PSS containing 145 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L glucose, and 10 mmol/L HEPES, pH 7.4, supplemented with 1 mmol/L CaCl₂ and 1 mmol/L MgSO₄. Cells were incubated for 60 minutes at 37°C in PSS containing 3 µmol/L fura 2-AM, 1 mmol/L CaCl₂, and 1 mmol/L MgSO₄. The coverslips were rinsed twice with the same medium without fura 2-AM to remove extracellular dye and stored in the dark at room temperature until use. Immediately before measurement, the coverslips were inserted into a cuvette containing either 2.5 mL Ca²⁺-containing PSS (PSS plus 1 mmol/L CaCl₂) or 2.5 mL Ca²⁺-free PSS (PSS plus 1 mmol/L EGTA). EGTA was added immediately before insertion of the coverslip. Fluorescence was monitored at 510 nm (excitation wavelengths, 340 and 380 nm) in a dual excitation wavelength spectrofluorometer (SPEX Fluorolog, SPEX Industries) equipped with a chamber thermostatically controlled at 37°C and a stirrer. Data were collected with dM3000 software (SPEX Industries). Integration time was 0.3 seconds at each wavelength, and the time increment was 1.0 seconds. After a stable basal value was obtained, cells were exposed to the test agent. Because the cuvette was not perfused, a stirrer system was used to achieve quickly a new steady state after the addition of drugs. Each coverslip was exposed to only one agent, and repetitive determinations were not made. The [Ca²⁺]_i was estimated by the ratio method as described previously.¹⁴ At the end of each experiment, the cells were permeabilized with 50 µmol/L digitonin in the presence of 1 mmol/L CaCl₂ to obtain the maximum fluorescence ratio. Subsequent incubation in 5 mmol/L EGTA, pH 8.3, allowed determination of the minimum fluorescent ratio.¹⁵ Autofluorescence from unloaded cells, test agents, and medium was subtracted from measured values.

Measurement of [Mg²⁺]_i
The procedure for measuring [Mg²⁺]_i was similar to that for [Ca²⁺]_i. VSMCs on coverslips were loaded with 5 µmol/L mag-fura 2-AM in the same medium used for fura 2-AM loading for 60 minutes at 37°C. All fluorescent measurements were performed in Ca²⁺-free PSS with three different concentrations of MgSO₄ (nominally 0, 1, and 5 mmol/L). Fluorescence was monitored at 510 nm, with excitation wavelengths of 340 and 380 nm. [Mg²⁺]_i was determined as previously described.¹⁶ At the end of each experiment, cells were permeabilized with 50 µmol/L digitonin in the presence of 50 mmol/L MgSO₄ to obtain the maximum fluorescence ratio and 50 mmol/L EDTA plus 20 mmol/L Tris (hydroxymethyl) aminomethane, pH 8.5, to obtain the minimum fluorescence ratio.¹⁷

Determination of Ca²⁺ Entry
To assess Ca²⁺ entry through the plasma membrane, cells were exposed to 25 µL of 0.1 mmol/L Ang II at a final concentration of 1 µmol/L or 25 µL of 0.1 mmol/L thapsigargin at a final concentration of 1 µmol/L in 2.5 mL Ca²⁺-free PSS containing Ca²⁺ and/or nifedipine (1 µmol/L). [Ca²⁺]_i reached a peak value, corresponding to Ca²⁺ mobilizing agent–stimulated Ca²⁺ discharge or mobilization from intracellular Ca²⁺ stores, and then returned to the original baseline. Subsequently, 2 mmol/L CaCl₂ was added to the medium. The peak increase in [Ca²⁺]_i after the addition of Ca²⁺ to the medium was defined as the parameter of the agent-induced Ca²⁺ entry.

Determination of Recovery Kinetics
The rate of recovery from the agonist-induced peak [Ca²⁺]_i in Ca²⁺-free PSS was assessed by the collection of data points each second over the first 20 seconds and each 5 seconds thereafter. The data were fit to a two-compartment efflux model (StatView II [Macintosh] statistical package) described by the equation¹⁸ [Ca²⁺]_i=Ae^-k₁t, where A is compartment size, k₁ is the rate constant describing efflux, and t is time.

Statistical Analysis
Data are expressed as mean±SEM. ANOVA and Scheffé's test were used to compare the [Ca²⁺]_i levels among the experimental groups. ANOVA with repeated measures was used to compare the time course of [Mg²⁺]_i levels. A value of P<.05 was considered statistically significant.

Results

Effect of Extracellular Mg²⁺ on Ang II–Stimulated [Ca²⁺]_i
In Ca²⁺-containing PSS, external Mg²⁺ inhibited the [Ca²⁺]_i response to 1 µmol/L Ang II in a concentration-dependent manner (Fig 1. In the absence of extracellular Ca²⁺, however, changes in extracellular Mg²⁺ concentration did not affect [Ca²⁺]_i. The effect of extracellular Mg²⁺ in Ca²⁺-containing PSS was assumed to be due to inhibition of Ca²⁺ entry across the plasma membrane. The highest external Mg²⁺ concentration chosen (5 mmol/L) to investigate the effect of extracellular Mg²⁺ on Ca²⁺ entry was the lowest concentration that had maximal effect. In subsequent experiments, we used the three doses of external Mg²⁺ concentration (nominally 0, 1, and 5 mmol/L).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of external Mg2+ concentration on the difference between the basal and peak [Ca2+]i induced by 1 µmol/L Ang II. Measurements were taken in Ca2+-containing medium () or Ca2+-free medium (). Mg2+ was added before the addition of Ang II. Values are the mean±SEM of nine independent experiments. *P<.05 vs 1 mmol/L external Mg2+.

Effect of Extracellular Mg²⁺ on [Mg²⁺]_i
In our experiments to investigate the effect of external Mg²⁺ on the Ca²⁺ movement across the plasma membrane, cells were incubated in Ca²⁺-free PSS containing a variety of external Mg²⁺ concentrations for 10 minutes. It is important whether the change in extracellular Mg²⁺ is accompanied by the change in [Mg²⁺]_i in the present study. To examine the effect of extracellular Mg²⁺ on [Mg²⁺]_i, mag-fura 2–loaded cells were incubated for 10 minutes in Ca²⁺-free PSS with either nominally 0 (low Mg²⁺), 1 (standard Mg²⁺), or 5 MgSO₄ (high Mg²⁺) mmol/L. Table 1 shows the time course of the effect of external Mg²⁺ concentration on [Mg²⁺]_i. [Mg²⁺]_i was not significantly affected by a change in external Mg²⁺ concentration during short-term (10-minute) incubation. The average [Mg²⁺]_i in standard Mg²⁺ PSS (318±42 µmol/L, n=6) resembled values reported previously.^{17 19} Ang II (1 µmol/L) or thapsigargin (1 µmol/L) had no significant effect on [Mg²⁺]_i in standard Mg²⁺ PSS (data not shown).

View this table: [in this window] [in a new window]   Table 1.

Effect of Extracellular Mg²⁺ and Nifedipine on Ang II–Induced Ca²⁺ Entry
Cells were exposed to Ang II (1 µmol/L) for 120 seconds, and then 2 mmol/L CaCl₂ was added to the Ca²⁺-free medium. Fig 2 shows typical records of [Ca²⁺]_i incubated in Ca²⁺-free PSS with various concentrations of external Mg²⁺ and stimulated with Ang II (1 µmol/L). The maximal response of [Ca²⁺]_i to Ang II was achieved within 5 seconds and returned to the original baseline within 100 seconds. No significant differences in the basal [Ca²⁺]_i, peak [Ca²⁺]_i, [Ca²⁺]_i before addition of CaCl₂ (pre-Ca²⁺ value of [Ca²⁺]_i), or k₁ were observed (Table 2). Ang II–evoked Ca²⁺ entry, the difference between pre-Ca²⁺ and post-Ca²⁺ values, was significantly greater in the presence of reduced external Mg²⁺ and reduced by elevated external Mg²⁺ concentration (Table 2). Blockade of L-type Ca²⁺ channels with nifedipine (1 µmol/L) also caused a significant reduction in Ca²⁺ entry, but concomitant treatment with both nifedipine (1 µmol/L) and a high Mg²⁺ concentration (5 mmol/L) did not reduce Ca²⁺ entry more than nifedipine alone (Table 2). Although high Mg²⁺ concentration appeared to reduce Ang II–evoked Ca²⁺ entry less than 1 µmol/L nifedipine, it was not statistically significant.

View larger version (12K): [in this window] [in a new window]   Figure 2. Typical traces showing the effect of external Mg2+ concentrations (nominally 0 [low Mg2+], 1 [standard Mg2+], and 5 [high Mg2+] mmol/L) on 1 µmol/L Ang II–induced Ca2+ entry. Cells in Ca2+-free medium were first stimulated with Ang II (1 µmol/L) for 120 seconds; then, Ca2+ (2 mmol/L) was added (as indicated by the arrow).

View this table: [in this window] [in a new window]   Table 2.

Ability of Ang II to Mobilize Ca²⁺ From Intracellular Stores
To evaluate the ability of Ang II to mobilize Ca²⁺ from intracellular stores, the effect of sequential additions of Ang II and thapsigargin on the changes in [Ca²⁺]_i was investigated in Ca²⁺-free medium. Cells were stimulated with 1 µmol/L Ang II in Ca²⁺-free PSS with standard Mg²⁺. After [Ca²⁺]_i had returned to baseline (80.2±7.3 nmol/L, n=6) from peak values (500.4±55.4 nmol/L), 1 µmol/L thapsigargin was added to the medium. We observed a slow increase in [Ca²⁺]_i, which reached a maximum concentration of 191.9±21.5 nmol/L within 120 seconds. Thapsigargin was found to mobilize a significant portion of Ca²⁺ from intracellular Ca²⁺ stores remaining after stimulation with 1 µmol/L Ang II.

Effect of Extracellular Mg²⁺ and Nifedipine on Elevated Potassium-Induced Ca²⁺ Entry
To depolarize the cell membrane and initiate Ca²⁺ entry through L-type VGCCs, cells were exposed to 100 mmol/L KCl solutions prepared by equimolar substitution of NaCl in Ca²⁺-free PSS with KCl for 30 seconds.¹⁰ In the absence of extracellular Ca²⁺, KCl had no significant effect on [Ca²⁺]_i (standard Mg²⁺, 85.7±8.8 nmol/L; n=6). There was no significant difference in basal [Ca²⁺]_i between the cells pretreated with different Mg²⁺ concentrations or nifedipine. Then, addition of 2 mmol/L CaCl₂ to the medium caused the sustained elevation of [Ca²⁺]_i. KCl-induced [Ca²⁺]_i elevation was significantly less in the high Mg²⁺ buffer than in the standard Mg²⁺ buffer (52.6±9.8 nmol/L [n=6] versus 391.7±56.5 nmol/L [n=6], P=.001). Nifedipine (1 µmol/L) also caused a significant reduction in Ca²⁺ entry compared with standard Mg²⁺ (36.5±6.5 nmol/L, n=6, P=.001). However, no further reduction was attained by combined treatment with both nifedipine and high Mg²⁺ concentration (37.6±6.1 nmol/L; n=6). Although high Mg²⁺ concentration appeared to reduce KCl-induced Ca²⁺ entry less than 1 µmol/L nifedipine, it was not statistically significant.

Effect of Extracellular Mg²⁺ and Nifedipine on Thapsigargin-Induced Ca²⁺ Entry
Intracellular Ca²⁺ stores were depleted by thapsigargin, a selective inhibitor of the sarcoplasmic reticulum Ca²⁺-ATPase²⁰ used to investigate capacitative Ca²⁺ entry.^{10 21 22} Exposure of VSMCs to 1 µmol/L thapsigargin induced an increase in [Ca²⁺]_i in Ca²⁺-free PSS. The peak [Ca²⁺]_i response was observed 120 seconds after introduction of a drug. Values returned to baseline after 10 minutes (Fig 3). The intracellular Ca²⁺ stores were thoroughly depleted by this procedure because subsequent addition of 1 µmol/L ionomycin and 20 mmol/L caffeine failed to evoke further detectable Ca²⁺ mobilization (data not shown). Addition of CaCl₂ to the medium after values had returned to baseline caused substantial capacitative Ca²⁺ entry^{10 21 22} (Fig 3). Ca²⁺-induced Ca²⁺ release from the internal Ca²⁺ stores presumably was not a factor because the Ca²⁺ stores had already been depleted. There was no significant difference in the basal [Ca²⁺]_i or Ca²⁺ transient induced by thapsigargin (peak and the following baseline) in the absence of extracellular Ca²⁺ between the cells pretreated with different Mg²⁺ concentrations and nifedipine (Table 3). The magnitude of the capacitative Ca²⁺ entry, the difference between pre-Ca²⁺ and post-Ca²⁺ values, was significantly increased in buffer with low external Mg²⁺ concentration and was decreased by high external Mg²⁺ concentrations (Table 3). Preincubation with nifedipine did not affect capacitative entry (Table 3). In standard Mg²⁺ buffer, Ca²⁺ entry was greater after treatment with thapsigargin than after treatment with Ang II (n=9, P<.05).

View larger version (12K): [in this window] [in a new window]   Figure 3. Typical traces showing the effect of external Mg2+ concentrations (nominally 0 [low Mg2+], 1 [standard Mg2+], and 5 [high Mg2+] mmol/L) on 1 µmol/L thapsigargin–induced Ca2+ entry (capacitative Ca2+ entry). Cells in Ca2+-free medium were first stimulated with thapsigargin (1 µmol/L) for 10 minutes; then Ca2+ (2 mmol/L) was added (as indicated by the arrow). The shutter was closed, protecting the cells from the light source, for 8 minutes between the time of the peak response and the time before the addition of Ca2+.

View this table: [in this window] [in a new window]   Table 3.

Effect of Extracellular Mg²⁺ on Ca²⁺ Decrease After Stimulation With Ang II and Thapsigargin
To evaluate the effect of external Mg²⁺ on Ca²⁺ efflux in Ca²⁺-free buffer, the rate of decay of the [Ca²⁺]_i transient after the concomitant addition of 1 µmol/L Ang II and 1 µmol/L thapsigargin was examined. Thapsigargin was added with Ang II to exclude the action of sarcoplasmic reticulum Ca²⁺-ATPase. The recovery rates were similar in buffer containing low, standard, and high Mg²⁺ concentrations (Table 4).

View this table: [in this window] [in a new window]   Table 4.

Discussion

The major findings of this study were that Ang II–evoked Ca²⁺ entry was inhibited by preincubation with high external Mg²⁺ or nifedipine and that capacitative Ca²⁺ entry induced by thapsigargin was found to be insensitive to nifedipine and inhibited by increased external Mg²⁺ concentration. To the best of our knowledge, this is the first report to demonstrate the inhibitory role of external Mg²⁺ in capacitative Ca²⁺ entry.

In the present study, extracellular Mg²⁺ inhibited the Ang II–induced increases in [Ca²⁺]_i in Ca²⁺-containing PSS in a concentration-dependent manner. However, the details of the mechanism linking external Mg²⁺ and cellular Ca²⁺ handling are not completely understood. Similar to other Ca²⁺-mobilizing ligands, Ang II first induces Ca²⁺ release from intracellular stores through inositol 1,4,5-trisphosphate production and then sustains [Ca²⁺]_i at a suprabasal level by enhancing Ca²⁺ influx from the extracellular space.^{2 3} In the absence of external Ca²⁺, Ang II–induced peak [Ca²⁺]_i was not affected by changes in external Mg²⁺ concentration. This finding suggested that external Ca²⁺ did not influence agonist-receptor interactions or Ca²⁺ release from intracellular stores. Therefore, the effect of external Mg²⁺ concentration on Ca²⁺ handling was restricted to movement of Ca²⁺ through the plasma membrane.

Increased extracellular Mg²⁺ concentration may affect Ca²⁺ handling secondary to changes in [Mg²⁺]_i. Short-term incubation of VSMCs in Ca²⁺-free medium containing extracellular Mg²⁺ concentrations ranging from nominally 0 to 5 mmol/L did not induce changes in [Mg²⁺]_i. Previous studies have demonstrated the extremely low permeability of the cell membrane to Mg²⁺.^{8 23 24 25} Therefore, the effect of external Mg²⁺ concentration on Ca²⁺ entry was mediated by its direct action from the extracellular side but not its indirect action from the intracellular side.

Previous studies have proposed a non–voltage-dependent Ca²⁺ entry mechanism activated by Ca²⁺-mobilizing agonists occupying receptors on VSMCs.³ In the present study, nifedipine did not completely inhibit the Ca²⁺ influx after the addition of Ang II. Non–voltage-dependent mechanisms dependent on and independent of IP₄ have been reported. The IP₄-dependent pathway has been described in endothelial cells,²⁶ but the evidence remains controversial. The IP₄-independent pathway, activated by depletion of intracellular Ca²⁺ stores, has been called capacitative Ca²⁺ entry.^{9 10 11} There is a growing body of evidence that decreased Ca²⁺ in intracellular stores provides important signals not only for capacitative Ca²⁺ entry but also for stimulated release of secretory proteins²⁷ and control of cell growth.²⁸

Activation of capacitative Ca²⁺ entry by depleted stores requires neither continued receptor occupation nor elevated levels of inositol phosphates.²⁹ Ca²⁺ entry remains activated as long as the stores remain depleted of Ca²⁺.²⁹ A small molecule called the "Ca²⁺ influx factor" may activate Ca²⁺ influx in several cell lines.³⁰ In basophils³¹ and lacrimal acinar cells,³² a small GTP binding protein is required for Ca²⁺ influx. In VSMCs, however, the second messenger has not been identified. In the present study, to examine the capacitative Ca²⁺ entry, CaCl₂ was added to cells after the intracellular stores were completely emptied by 1 µmol/L thapsigargin in Ca²⁺-free medium.^{10 21 22} Thapsigargin mobilizes intracellular Ca²⁺ in the absence of receptor activation and inositol 1,4,5-trisphosphate formation.²⁹ Therefore, the rapid increase in [Ca²⁺]_i caused by the addition of CaCl₂ to cells pretreated with thapsigargin was thought to be capacitative Ca²⁺ entry. Several investigators have characterized capacitative Ca²⁺ entry indirectly by measuring Ca²⁺-activated K⁺ currents or using fura 2 quenching by Mn²⁺, which enters the cells by the same influx pathway in some cell types.³³ Because capacitative Ca²⁺ entry is highly selective for Ca²⁺ over other divalent cations, this pathway should be investigated directly,^{22 33} as in this study. Because there was no difference in the thapsigargin-evoked peak [Ca²⁺]_i or recovery rate among cells exposed to different external concentrations of Mg²⁺ or nifedipine, the contents of intracellular Ca²⁺ stores were depleted to the same extent. Therefore, subsequent comparisons of the effect of pretreatment conditions on capacitative Ca²⁺ entry proceeded from the same baseline conditions. Capacitative Ca²⁺ entry was inhibited by increased external Mg²⁺ and insensitive to dihydropyridine calcium channel blockers.^{10 34}

The inhibitory effect of external Mg²⁺ was not additive to the effect of nifedipine on Ang II–induced Ca²⁺ influx. This finding suggests that Mg²⁺ and nifedipine work through a similar mechanism, ie, inhibition of VGCCs in Ang II–stimulated cells. This hypothesis would be supported by the fact that the inhibitory effect of external Mg²⁺ was not additive to the effect of nifedipine on elevated potassium-induced Ca²⁺ entry. Intracellular Ca²⁺ stores were not fully depleted by 1 µmol/L Ang II because 1 µmol/L thapsigargin induced a significant increase in [Ca²⁺]_i after stimulation with Ang II. Therefore, capacitative Ca²⁺ entry may not operate effectively in the entry pathway linked to Ang II receptors. Moreover, because concurrent exposure to nifedipine and a high concentration of external Mg²⁺ did not completely abolish Ang II–induced Ca²⁺ influx, other Ca²⁺ entry pathways that are resistant to external Mg²⁺ and nifedipine may exist in VSMCs.^{3 21 35}

In the present study, the Ca²⁺ influx induced by thapsigargin (capacitative Ca²⁺ entry) exceeded that induced by Ang II. This difference may be due to suppression of refilling of intracellular Ca²⁺ stores in thapsigargin-treated cells by the inhibition of the sarcoplasmic reticulum Ca²⁺-ATPase. It is also possible that Ca²⁺ extrusion across the plasma membrane was inhibited in the thapsigargin-treated cells because thapsigargin does not produce protein kinase C to activate plasma membrane Ca²⁺-ATPase.³⁶

Net Ca²⁺ movement through the plasma membrane is the balance between Ca²⁺ influx and efflux. To examine the effect of external Mg²⁺ on Ca²⁺ efflux through the plasma membrane, the recovery rate was assessed by measuring the decline of [Ca²⁺]_i from the peak level evoked by the concomitant addition of Ang II and thapsigargin in Ca²⁺-free medium. In this study protocol, thapsigargin and Ca²⁺-free medium were used to exclude the influence of uptake of Ca²⁺ into the stores and Ca²⁺ influx. There was no difference in recovery rate among different external Mg²⁺ concentrations. Thus, extracellular Mg²⁺ may not influence Ca²⁺ efflux. Although Mg²⁺ in the cytosol is required for Ca²⁺-ATPase activity,²⁴ [Mg²⁺]_i was not affected by extracellular Mg²⁺ because of low permeability to Mg²⁺.^{8 23 25} The acceleration of Ca²⁺ efflux does not appear to participate in the inhibitory effect of external Mg²⁺ on Ca²⁺ handling.

The disadvantages of using cell cultures are that the artificial extracellular environment used may differ from the native one and that phenotypic changes can occur. This could result in discrepancies between the original VSMCs and the cell cultures. Therefore, our data may have only limited relevance to the intact artery.³⁷

Results suggest that in VSMCs, the inhibitory effect of external Mg²⁺ on capacitative Ca²⁺ entry may make an important contribution to the inhibitory effect of external Mg²⁺ on Ca²⁺ handling. Additional study is necessary to elucidate the details of this mechanism.

Selected Abbreviations and Acronyms

AM = acetoxymethyl ester Ang II = angiotensin II [Ca2+]i = cytosolic free Ca2+ concentration HBSS = Hanks' balanced salt solution IP4 = inositol 1,3,4,5-tetrakisphosphate [Mg2+]i = cytosolic free Mg2+ concentration PSS = physiological salt solution VGCC = voltage-gated Ca2+ channel VSMC = vascular smooth muscle cell

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research (Nos. 06304028, 07407065, and 08457639) from the Ministry of Education, Science and Culture of Japan.

Received October 14, 1996; revision received December 16, 1996; accepted January 1, 1997.

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