Na+ entry via store-operated channels modulates Ca2+ signaling in arterial myocytes

Assaf Arnon, John M. Hamlyn, and Mordecai P. Blaustein

Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In many nonexcitable cells, hormones and neurotransmitters activate Na+ influx and mobilize Ca2+ from intracellular stores. The stores are replenished by Ca2+ influx via "store-operated" Ca2+ channels (SOC). The main routes of Na+ entry in these cells are unresolved, and no role for Na+ in signaling has been recognized. We demonstrate that the SOC are a major Na+ entry route in arterial myocytes. Unloading of the Ca2+ stores with cyclopiazonic acid (a sarcoplasmic reticulum Ca2+ pump inhibitor) and caffeine induces a large external Na+-dependent rise in the cytosolic Na+ concentration. One component of this rise in cytosolic Na+ concentration is likely due to Na+/Ca2+ exchange; it depends on elevation of cytosolic Ca2+ and is insensitive to 10 mM Mg2+ and 10 µM La3+. Another component is inhibited by Mg2+ and La3+, blockers of SOC; this component persists in cells preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to buffer Ca2+ transients and prevent Na+/Ca2+ exchange-mediated Na+ entry. This Na+ entry apparently is mediated by SOC. The Na+ entry influences Na+ pump activity and Na+/Ca2+ exchange and has unexpectedly large effects on cell-wide Ca2+ signaling. The SOC pathway may be a general mechanism by which Na+ participates in signaling in many types of cells.

sodium-calcium exchange; magnesium; sodium pump; cytosolic sodium concentration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVATION OF MOST ANIMAL cells by hormones or other agents usually elevates the cytosolic Ca2+ concentration ([Ca2+]cyt), which, in turn, triggers a variety of biological responses (3, 8). Some of this "signal Ca2+" enters cells from the extracellular fluid, and some is released from intracellular Ca2+ stores in the sarcoplasmic (in muscle) or endoplasmic reticulum (S/ER) (3, 8). Emptying of the Ca2+ stores initiates a mechanism to refill the stores, termed "capacitative Ca2+ entry" (CCE) (36), that is mediated by "store-operated" channels (SOC) located in the plasmalemma (PL) (50). This widely distributed mechanism (10) has been reported to occur in vascular smooth muscle cells (31, 47), where it may play an important role in the regulation of tonic vascular tension ("tone") (11).

Several types of SOC have been proposed as mediators of S/ER Ca2+ store depletion-dependent Ca2+ entry. These include the Ca2+ release-activated Ca2+ (CRAC) channel (17, 23, 24) and various other channels (16, 27, 29), some of which are homologous to the transient receptor potential (TRP) and TRP-like (TRPL) channels of Drosophila photoreceptors (25, 35, 50, 51).

In mammals, recent observations suggest that most SOC are permeable to Na+ as well as Ca2+ (23, 35, 50), with permeability ratios (PCa/PNa) usually on the order of 10:1 under normal physiological conditions (35, 50), although relatively nonselective cation channels have also been described (25, 26, 51). Thus, with extracellular Ca2+ and Na+ concentrations ([Ca2+]o and [Na+]o) on the order of 1 and 150 mM, respectively, large amounts of Na+ may enter the cells through SOC. The physiological significance of this potentially important Na+ entry pathway has not attracted attention. Although many hormones and neurotransmitters mobilize sarcoplasmic reticulum (SR) Ca2+ and activate vascular smooth muscle cells, they also promote Na+ entry (6, 7) via unknown pathways. Vascular smooth muscle cells have few, if any, "classical" voltage-gated Na+ channels (44, 48) and do not exhibit Na+-dependent action potentials. This raises the possibility that the Na+ entry evoked by hormones and neurotransmitters in these cells may be mediated, in part, by SOC.

Here we use digital imaging methods to investigate SOC in mesenteric artery myocytes and to determine whether these channels influence Na+, as well as Ca2+, homeostasis. We show that SOC mediate the entry of Na+ and Ca2+ {measured as changes in cytosolic Na+ concentration ([Na+]cyt) and [Ca2+]cyt with Na+-binding benzofuran isophthalate (SBFI) and fura 2, respectively}.1 Furthermore, Na+ entry through the SOC apparently augments whole cell Ca2+ signaling by reducing the extrusion of Ca2+ in these cells; this effect is especially pronounced when the activity of a subset of Na+ pumps is suppressed. SOC also appear to be an important route of Na+ entry in arterial myocytes at rest.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Primary cell culture. Mesenteric artery myocytes were obtained from female Sprague-Dawley rats (150-200 g) and were grown in primary culture for 4-6 days, as described previously (40).

Measurement of [Ca2+]cyt and [Na+]cyt. The cells, on 25-mm coverslips, were loaded with fura 2 or SBFI by incubation with 3.3 µM fura 2-AM for 40 min or 10 µM SBFI-AM (TEFLABS, Austin, TX) for 1 h at 22-25°C. [Ca2+]cyt and [Na+]cyt were then studied with digital imaging methods (6, 13). The coverslips were mounted in a 1-ml chamber on the stage of an inverted fluorescence microscope. The cells were superfused at a rate of 5 ml/min (chamber washout half time ~30 s) with a physiological salt solution (PSS). Experiments were conducted at 32°C. Before the experimental protocols were started, cells were superfused for 40 min with PSS to wash away extracellular dye and permit the intracellular esterases to cleave the fura 2-AM or SBFI-AM.

Fura 2 was calibrated as described previously (13). SBFI, which can detect 0.5 mM changes in [Na+]cyt (6), was calibrated after each experiment (32). Digital images of fields containing five to eight cells were acquired, background subtracted, and transformed to Ca2+ (or Na+) concentration images with a MetaFluor Imaging System (Universal Imaging, West Chester, PA). Fluorescence ratio data for each cell were obtained from a 5 × 5 pixel (1.5 × 1.5 µm) nonnuclear region (1/cell). To enhance the signal-to-noise ratio, 32 consecutive frames were averaged at video frame rate, except when agonists and/or ouabain were added; then only 4 frames were averaged to enhance temporal resolution. Images were acquired at a rate of two per minute, except at times of agonist application, when the rate was increased to two per second.

Solutions and reagents. The normal PSS contained (in mM) 140 NaCl, 5.0 KCl, 1.2 NaH2PO4, 5 NaHCO3, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 mM HEPES (titrated to pH 7.4 with NaOH). CaCl2 was omitted from the Ca2+-free PSS, and 0.2 mM EGTA was added. The Ca2+-free, 5 mM Na+ PSS (0 Ca2+-5 Na+ PSS) contained 5 mM NaCl and 140 mM N-methylglucamine. NaH2PO4 and NaHCO3 were omitted from PSS containing LaCl3. The osmolarity of all solutions was adjusted to 320 mosM with sucrose. In some experiments (see RESULTS), cells were preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) by incubation with 20 µM BAPTA-AM for 30 min at 22-25°C.

ANG II was purchased from Peninsula Laboratories (Belmont, CA). All other compounds were reagent grade or the highest grade available and were purchased from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

Data analysis and statistics. Data (ratios and calibrated values) were analyzed and plotted with Sigma Plot software (Jandel, San Rafael, CA). Significance of differences between means of different groups (means ± SE) was calculated by Student's paired t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characteristics of SOC in arterial myocytes: studies with fura 2. Some properties of SOC in rat mesenteric artery myocytes, as indicated by changes in [Ca2+]cyt, are illustrated in Figs. 1 and 2. The SOC are activated by unloading Ca2+ stores with 10 µM cyclopiazonic acid (CPA), which inhibits S/ER Ca2+-ATPase (SERCA) (18), and 10 mM caffeine (Caff), which induces Ca2+ release through ryanodine-sensitive S/ER channels (30) (Fig. 1, A and B). SR Ca2+ also is mobilized by neurotransmitters and hormones such as serotonin, vasopressin, phenylephrine (PE), and ANG II (31, 47; see below); this, too, opens SOC in vascular smooth muscle cells (31, 47; see below).


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Fig. 1.   Properties of store-operated channels (SOC) in arterial myocytes: effects of 10 mM Mg2+, 10 µM La3+, and extracellular Ca2+ concentration ([Ca2+]o) on external Ca2+-dependent elevations of cytosolic Ca2+ concentration ([Ca2+]cyt) induced after Ca2+ stores were unloaded by 10 µM cyclopiazonic acid (CPA) + 10 mM caffeine (Caff) in "0" Ca2+ physiological salt solution (PSS). SOC function and block by 10 mM Mg2+ (A) or 10 µM La3+ (B) are demonstrated in representative single cells. Solution changes are indicated by horizontal bars below records; all solutions contained 10 µM nifedipine to block L-type Ca2+ channels. C: data from several experiments similar to (and including) those illustrated in A and B. Bars indicate relative amplitudes of Ca2+ transients induced by reintroduction of 1.8 mM Ca2+ before rise in Mg2+ or addition of La3+ ("control" = 1.0), in presence of 10 mM Mg2+ or 10 µM La3+ (both ions significantly reduced transient: * P < 0.001), and during washout (dagger  P < 0.01 vs. 10 mM Mg2+; Dagger  P < 0.01 vs. 10 µM La3+); cell numbers are in parentheses. D: normalized amplitude of Ca2+ transient induced by external Ca2+ (concentration on abscissa) after incubation in 0 Ca2+ PSS containing Caff, CPA, and nifedipine (see 2nd Ca2+ transient in A for sample protocol at [Ca2+]o = 1.8 mM); Ca2+ transient amplitude = 1.0 at [Ca2+]o = 10 mM. Data (means ± SE; cell numbers in parentheses) fit following equation: NA = Amax × {[Ca2+]o/([Ca2+]o + K0.5)}, where NA is normalized amplitude, maximum Ca2+ transient amplitude (Amax) = 1.13, and apparent [Ca2+]o for half-maximal activation (K0.5) = 1.1 mM.



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Fig. 2.   Effects on [Ca2+]cyt of prolonged exposure (A) and repetitive ~2-min exposures, ~30 min apart (B), to PSS with 1.8 mM Ca2+ after Ca2+ stores were unloaded with CPA + Caff in 0 Ca2+ PSS. During maintained exposure to [Ca2+]o = 1.8 mM, [Ca2+]cyt rose to a peak and then declined by 13 ± 3% (A; n = 27 cells). During 5 repetitive exposures to Ca2+ (B; n = 24 cells), peak [Ca2+]cyt varied by an average of less than ±6%. Solution changes are indicated by horizontal bars below records.

In Ca2+-free ("0" Ca2+) medium, emptying the stores with CPA + Caff caused [Ca2+]cyt to rise rapidly and then decline below the original resting level (Fig. 1A). When extracellular Ca2+ was restored in the continued presence of CPA + Caff, to prevent store refilling (and with 10 µM nifedipine to block L-type Ca2+ channels), a second large Ca2+ transient, the "hallmark" of SOC activation and CCE (36), was observed. This transient depended on store Ca2+ depletion and on external Ca2+. The [Ca2+]o needed to evoke half-maximal Ca2+ transients was 1.1 mM (Fig. 1D), consistent with reported EC50 values for Ca2+ entry through SOC (~0.8-3.3 mM) (9, 17). These [Ca2+]o-dependent Ca2+ transients were blocked reversibly by 10 mM Mg2+ (Fig. 1, A and C) and 10 µM La3+ (Fig. 1, B and C), known blockers of SOC (12, 35, 46, 47). Neither Mg2+ nor La3+ inhibits agonist-evoked Ca2+ transients in normal medium (2, 38, 46; see below). Moreover, the CPA- and agonist-evoked Ca2+ transients were prevented by pretreatment with the Ca2+ chelator BAPTA (see below), which buffers intracellular Ca2+ (17, 28, 29).

To test for inactivation of the SOC (23, 24, 46), external Ca2+ was restored for 10-15 min in the presence of CPA + Caff. [Ca2+]cyt rapidly rose to a peak and then declined (by ~13%) to a plateau that was maintained for >= 10-15 min (Fig. 2A). This decline might reflect accelerated Ca2+ sequestration (e.g., in mitochondria) and/or extrusion (26), as well as some (limited) inactivation. There was, however, no long-term inactivation: the peak amplitudes of the Ca2+ transients remained constant when the cells were repetitively exposed (for ~2 min) to Ca2+-containing medium at ~30-min intervals (Fig. 2B).

Na+ entry mediated by the Na+/Ca2+ exchanger: studies with SBFI. Figures 3-5 present evidence that Ca2+ store unloading also activates Na+ entry. Figure 3 shows data from an experiment analogous to that shown in Fig. 1A, except Na+ entry was estimated by measuring changes in the cytosolic Na+ concentration ([Na+]cyt) with the Na+-sensitive dye SBFI (6). Here, SOC were blocked with 10 mM Mg2+ (Fig. 1, A and C) or 10 µM La3+ (Fig. 1, B and C) in the presence of the Ca2+ and Na+ channel blockers nifedipine (10 µM) and tetrodotoxin (50 µM). When the Ca2+ stores were unloaded with CPA + Caff in 0 Ca2+ PSS, there was an initial transient rise in [Na+]cyt as well as [Ca2+]cyt (not observed here, but see Fig. 1, A and B). This first evoked Na+ transient appeared to be the result of Na+/Ca2+ exchange, because it depended on external Na+ (Figs. 4A and 5B) and on the rise in [Ca2+]cyt. This Na+ transient was insensitive to 10 mM Mg2+ (Figs. 3 and 5B) or 10 µM La3+ (Fig. 5B), but it was eliminated when the Ca2+ released from the stores was buffered with BAPTA (Figs. 4, B and C, and 5B). The top ([Ca2+]cyt) record in Fig. 4C confirms that intracellular BAPTA abolished the evoked Ca2+ transient. In other words, this Na+ transient did not depend directly on the unloading of the SR Ca2+ stores but, rather, on the rise in [Ca2+]cyt.


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Fig. 3.   Effect of unloading sarcoplasmic reticulum (SR) Ca2+ stores with CPA (10 µM) + Caff (10 mM) on cytosolic Na+ concentration ([Na+]cyt) in a cell bathed in 0 Ca2+ PSS containing 10 or 1.4 mM external Mg2+ ([Mg2+]o; horizontal bar below record in A). Solution changes are indicated by horizontal bars below records. All solutions contained 10 µM nifedipine to block voltage-gated, L-type Ca2+ channels and 50 µM tetrodotoxin (TTX) to block voltage-gated Na+ channels. A: time course for data from B. B, a-e: "Na+ images" for times indicated by a-e in A. Unlabeled panel (top left) is an original fluorescent image (340-nm excitation) of 2 cells; horizontal bar, 5 µm. Small white boxes surround 5 × 5 pixel areas used for analysis (see A and Fig. 5). SFBI, Na+-binding benzofuran isophthalate.



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Fig. 4.   A: effect of extracellular Na+ concentration ([Na+]o) on rise of [Na+]cyt evoked by unloading Ca2+ stores in 0 Ca2+ PSS containing 10 mM Mg2+. B: effect of preincubation with 20 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on rise of [Na+]cyt evoked by unloading Ca2+ stores in 0 Ca2+ PSS at 10 and 1.4 mM Mg2+. C: effects of BAPTA preincubation on increases of [Na+]cyt (bottom record) and [Ca2+]cyt (top record, parallel experiment) evoked by unloading Ca2+ stores in PSS at 10 and 1.4 mM Mg2+. D: effect of [Na+]o on rise of [Na+]cyt evoked by unloading Ca2+ stores in 0 Ca2+ PSS containing 10 or 1.4 mM Mg2+ in BAPTA-pretreated cells. E: effects of 15 min of incubation in 0 Ca2+ PSS on increases of [Na+]cyt (bottom record) and [Ca2+]cyt (top record, parallel experiment) evoked by lowering [Mg2+]o to 1.4 mM and (for [Ca2+]cyt measurement only) adding back Ca2+ (= PSS). F: data from experiments similar to E, except cells were incubated in 0 Ca2+ for only 1 min before [Mg2+]o was lowered to 1.4 mM. Time course curves are data from single representative cells; summary data are presented in Fig. 5. All solutions contained 10 µM nifedipine to block voltage-gated, L-type Ca2+ channels and 50 µM TTX to block voltage-gated Na+ channels. Solution changes are indicated by horizontal bars below records.



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Fig. 5.   Summary of effects of Na+, Mg2+, La3+, and BAPTA on resting and transient peak [Na+]cyt during SOC-mediated Na+ entry (SOCNE) and Na+/Ca2+ exchange (NCX)-mediated Na+ entry (NCXNE) (see Figs. 3 and 4 for representative original experiments). Left-hand bar and thin horizontal line correspond to resting (control) [Na+]cyt (8.7 ± 0.1 mM, n = 385 cells). A: effects of 5 mM Na+, 10 mM Mg2+, 10 µM La3+, and pretreatment with 20 µM BAPTA on SOC-mediated Na+ transients (SOCNE) induced by 10 µM CPA + 10 mM Caff. Effects of Ca2+ depletion by 1 and 15 min of incubation in 0 Ca2+ PSS before reducing Mg2+ from 10 to 1.4 mM are also shown (2 right-hand bars). B: effects of 5 mM Na+, 10 mM Mg2+, 10 µM La3+, and pretreatment with 20 µM BAPTA on NCXNE induced by 10 µM CPA + 10 mM Caff. In all experiments, all solutions contained 10 µM nifedipine to block voltage-gated, L-type Ca2+ channels and 50 µM TTX to block voltage-gated Na+ channels. Solution changes are indicated by horizontal bars below records. Error bars, SE; numbers of cells observed for each condition are in parentheses. * Significantly different from control (P < 0.01). Bars labeled "control" in A and B represent control [Na+]cyt transients.

In most experiments, 0 Ca2+ PSS was employed (Figs. 3A and 5B) to rule out the participation of an external Ca2+-dependent mechanism, but a similar 10 mM Mg2+-insensitive Na+ transient was observed in PSS with 1.8 mM Ca2+ (not shown). Inhibition of Na+/Ca2+ exchange by removal of external Na+ or antisense knockdown of the exchanger greatly augments and prolongs the Ca2+ transients evoked by store unloading (38, 39), implying that much of the decline of [Ca2+]cyt is mediated by the Na+/Ca2+ exchanger. Neither 10 mM Mg2+ (unpublished data) nor 10 µM La3+ (38) interferes with Ca2+ extrusion via the Na+/Ca2+ exchanger. Therefore, the initial rise in [Na+]cyt after store depletion (Fig. 3) apparently results from Na+/Ca2+ exchange-mediated Na+ entry (NCXNE) that is promoted by the elevation of [Ca2+]cyt.

Na+ entry mediated by SOC: studies with SBFI. When the SOC were subsequently unblocked by reducing Mg2+ to 1.4 mM (Figs. 3 and 4, B-E) or removing La3+ (Fig. 5A), a second large transient rise in [Na+]cyt was induced. This Na+ transient also was abolished reversibly when [Na+]o was reduced to 5 mM (Figs. 4D and 5A), but, unlike the first Na+ transient in Fig. 3 (also see Fig. 5B), the second Na+ transient was blocked by 10 mM Mg2+ and by 10 µM La3+ (Fig. 5A). Furthermore, this second Na+ transient (Fig. 3) was observed when cytosolic Ca2+ was buffered with BAPTA (Figs. 4, B-D, and 5A; see top record in Fig. 4C); thus it depended on Ca2+ store depletion and not on the rise in [Ca2+]cyt. Also it did not require the reintroduction of external Ca2+ after the stores were unloaded (Figs. 4, B and D, and 5A). These results are consistent with the view that the second Na+ transient in Fig. 3 is the result of Na+ entry through SOC [i.e., SOC-mediated Na+ entry (SOCNE)].

SOCNE also was stimulated when the Ca2+ stores were depleted by nonpharmacological means. Incubation in 0 Ca2+ PSS (with 10 mM Mg2+, to block SOC) for 15 min to deplete the Ca2+ stores by slow "leakage" and extrusion of Ca2+ led to a slow decline in [Na+]cyt (Fig. 4E, bottom record). The subsequent lowering of extracellular Mg2+ concentration ([Mg2+]o) from 10 to 1.4 mM evoked a rise in [Na+]cyt (Figs. 4E, bottom record, and 5A). In parallel experiments on cells from the same cultures, the simultaneous reintroduction of external Ca2+ induced a Ca2+ transient, indicating that the SOC were activated (Fig. 4E, top record; Ca2+ was not restored for the [Na+]cyt measurement to prevent NCXNE). In contrast, when Mg2+ was lowered from 10 to 1.4 mM after only 1 min of incubation in 0 Ca2+ PSS, reintroduction of external Ca2+ had little effect on [Ca2+]cyt (Fig. 4F, top record) or [Na+]cyt (Fig. 4F, bottom record), indicating that the stores had not yet unloaded and most SOC were still closed, so that SOCNE was not stimulated. With continued incubation in 0 Ca2+ PSS, however, [Na+]cyt rose slowly (Fig. 4F, bottom record), reflecting progressive depletion of the Ca2+ stores and some stimulation of SOCNE and a consequent rise in [Na+]cyt.

Some Na+ enters unstimulated myocytes through SOC. In resting cells incubated in PSS, the mean [Na+]cyt (8.7 ± 0.1 mM, n = 385 cells; Fig. 5A) was constant. Elevation of [Mg2+]o to 10 mM in standard PSS with Ca2+ (Figs. 4C and 6, A and B) or in 0 Ca2+ PSS (Fig. 4E) caused [Na+]cyt in the unstimulated cells to decline slowly to a new steady level. Over the course of 40-60 min, [Na+]cyt reversibly declined by ~50%, to 5.1 ± 0.4 mM when [Mg2+]o in the PSS was elevated from 1.4 to 10 mM Mg (Fig. 6, A and B). The final, steady [Na+]cyt was inversely dependent on [Mg2+]o, with apparent half-maximal decline of [Na+]cyt observed at [Mg2+]o of 3.0 mM (Fig. 6C). Comparable results were obtained when the SOC were inhibited with 10 µM La3+ (Fig. 6B). Indeed, when 10 µM La3+ was added to PSS that already contained 10 mM Mg2+, no further effect on [Na+]cyt was observed (Fig. 6B), which implies that the effects of these two cations on SOCNE are not additive. These results suggest that some SOC are open at rest and that SOCNE contributes to the resting [Na+]cyt in mesenteric artery myocytes under normal physiological conditions. We should not, however, ignore other possible routes of Na+ entry that might also be inhibited by La3+ and high [Mg2+]o, such as nonselective cation channels and Na+/Mg2+ exchange. Also, we have not ruled out the possibility that these polyvalent cations might affect the membrane potential and, thus, the driving force for Na+ entry. There is no evidence, however, that the myocytes possess nonselective cation channels (see Relative permeabilites of SOC to Ca2+ and Na+) or that La3+ inhibits Na+/Mg2+ exchange, and it seems unlikely that 10 mM Mg2+ and 10 µM La3+ will have comparable effects on the membrane potential (indeed, neither cation affected the evoked Ca2+ transients; see Possible role of SOC in the augmentation of Ca2+ transients by low-dose ouabain).


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Fig. 6.   Effect of [Mg2+]o on resting [Na+]cyt. A: time course of changes in [Na+]cyt when [Mg2+]o was raised from 1.4 to 10 mM, 10 µM La3+ was added to medium, and La3+ was washed out and [Mg2+]o was reduced to 1.4 mM; representative data from 1 cell are shown. B: summary data (means ± SE) showing resting [Na+]cyt in control PSS (1.4 mM Mg2+) and in PSS with 0.3 and 10 mM Mg2+, 10 µM La3+, and 10 mM Mg2+ + 10 µM La3+. Numbers of cells are shown in parentheses. C: relationship between resting [Na+]cyt and [Mg2+]o. Values are means ± SE from 32 cells. Data fit following equation: [Na+]cyt (mM) = (9.4 - 5.1)/{1 + ([Mg2+]o/IC50)nH} + 5.1, where IC50 = 3.0 mM Mg2+, Hill's coefficient (nH) = 3, and 2 constants (9.4 and 5.1) are maximal and minimal [Na+]cyt in PSS with 0.33 and 20 mM [Mg2+]o, respectively. All solutions contained 10 µM nifedipine to block voltage-gated, L-type Ca2+ channels and 50 µM TTX to block voltage-gated Na+ channels. Solution changes are indicated by horizontal bars below records.

Relative permeabilities of SOC to Ca2+ and Na+. The data in Figs. 3-5 demonstrate that depletion of SR Ca2+ stores induces a rise in [Na+]cyt by a process with the same properties as those of SOC. Therefore, the rising phases of the Ca2+ and Na+ transients evoked by Ca2+ store depletion were used to estimate PCa/PNa. The rates of rise of [Ca2+]cyt (Fig. 1A) and [Na+]cyt (Fig. 7B) in PSS, during the 2nd min after [Mg2+]o was lowered from 10 to 1.4 mM, were used to estimate PCa (6.48 ± 0.80 × 10-6 cm/s, n = 43 cells) and PNa (0.79 ± 0.23 × 10-6 cm/s, n = 37 cells). The [Na+]cyt data were obtained in cells preloaded with BAPTA (to eliminate NCXNE) with 1 mM ouabain present (to avoid underestimating SOCNE because of Na+ pump-mediated Na+ extrusion); we did not, however, compensate for possible Ca2+ efflux via the PL Ca2+ pump, which may have caused us to underestimate PCa. [Inhibition of virtually all the Na+ pumps with 1 mM ouabain (4) in the absence of CPA and Caff induced comparable elevation of [Na+]cyt (not shown, but see Ref. 6).] Cell surface-to-volume ratios were assumed to be constant. The permeabilities (P) were calculated as follows: P = -J/C (cm/s), where J is molar flux (mol · cm-2 · s-1) and C is the difference between extracellular and cytosolic ion concentration (15).


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Fig. 7.   Effects of 1 mM ouabain on rising (A) and falling (B) phases of SOCNE transient in arterial myocytes treated with 20 µM BAPTA. Dotted lines, changes of [Na+]cyt expected in absence of ouabain (see Fig. 4C). Dotted line in A is rising phase of SOCNE transient from B; dotted line in B is from an experiment similar to Fig. 4C. C: mean maximal rates of change of [Na+]cyt (Delta [Na+]cyt) from experiments similar to and including those in A and B (cell numbers in parentheses). Open bars, no ouabain; solid bars, 1 mM ouabain; hatched bars, ouabain-sensitive Na+ pump-mediated Na+ transport. All solutions contained 10 µM nifedipine to block voltage-gated, L-type Ca2+ channels and 50 µM TTX to block voltage-gated Na+ channels. Solution changes are indicated by horizontal bars below records.

The calculated PCa/PNa of 8.2 ± 1.0 in PSS containing 1.8 mM Ca2+ is comparable to values obtained in electrophysiological studies on TRP/TRPL channels, ~8-10:1 (35, 46). Moreover, measurements of the maximum rate of rise of [Na+]cyt in BAPTA-loaded cells (i.e., in the absence of NCXNE; Fig. 8) as a function of [Ca2+]o indicated that PNa was reduced nearly sixfold as [Ca2+]o was increased from 0 to 10 mM. This is consistent with the low monovalent cation permeability of CRAC channels measured in high-Ca2+ medium (17).


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Fig. 8.   Effect of [Ca2+]o on rate of rise of [Na+]cyt evoked by reducing [Mg2+]o to 1.4 mM after unloading Ca2+ stores in BAPTA-pretreated cells. A: representative original records. At time indicated by horizontal bar at bottom, [Ca2+]o was changed to value shown beside each record. B: summarized data (number of cells in parentheses). All solutions contained 10 µM nifedipine to block voltage-gated, L-type Ca2+ channels and 50 µM TTX to block voltage-gated Na+ channels. Solution changes are indicated by horizontal bars below records.

Rise in [Na+]cyt mediated by SOCNE stimulates the Na+ pump. The transiency of the SOCNE-mediated rise in [Na+]cyt (Fig. 3) was unexpected, because SOC exhibit little inactivation (Fig. 2, A and B). To determine whether the decline in [Na+]cyt during SOCNE (Figs. 3 and 4, A and C-E) might reflect accelerated Na+ extrusion by PL Na+ pumps, we used 1 mM ouabain to inhibit all Na+ pumps (4). With NCXNE blocked by BAPTA, activation of SOCNE by reducing [Mg2+]o from 10 to 1.4 mM caused [Na+]cyt to rise at a rate of 0.25 mM/min, whereas in the presence of 1 mM ouabain, this rate doubled to 0.51 mM/min (Fig. 7, A and C). The difference between these two rates (-0.26 mM/min) is, by definition, the ouabain-sensitive rate of Na+ extrusion (i.e., reduction of [Na+]cyt) via Na+ pumps (Fig. 7C). When 1 mM ouabain was added during the falling phase of the SOCNE transient (-0.42 mM/min), [Na+]cyt began to rise (0.32 mM/min; Fig. 7, B and C); the difference between these two rates (-0.74 mM/min) also reflects ouabain-sensitive Na+ pump activity (Fig. 7C). Thus the Na+ pump rate increased almost threefold (from -0.26 to -0.74 mM/min) during the course of the SOCNE transient. This may reflect modulation of Na+ pump activity (4).

Possible role of SOC in the augmentation of Ca2+ transients by low-dose ouabain. The Ca2+ transients evoked by a 30-s exposure to 10 nM ANG II (Fig. 9, A and D) and 100 nM PE (Fig. 9D) were significantly augmented by the simultaneous application (i.e., for 30 s) of 100 nM ouabain. There was, however, no measurable increase in whole cell [Na+]cyt during these brief exposures to the low concentrations of agonists and ouabain (not shown). Nevertheless, the augmentation of the Ca2+ transients by ouabain depended on external Na+, but not Ca2+ (Fig. 9, B and C). This augmentation may be mediated by SOC, because it was abolished by 10 mM Mg2+ and 10 µM La3+. In contrast, the responses to ANG II or PE, alone (i.e., the "controls"), were unaffected by these cations (Fig. 9, A and D).


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Fig. 9.   Augmentation by 100 nM ouabain of 10 nM ANG II- and 100 nM phenylephrine (PE)-evoked Ca2+ transients in arterial myocytes. In all experiments, myocytes were exposed to vasoconstrictor for 30 s. In some instances, as indicated by bars below records, 100 nM ouabain was added to vasoconstrictor-containing solution. A: effect of elevating [Mg2+]o from 1.4 to 10 mM on augmentation, by ouabain, of ANG II-evoked Ca2+ transient. B: ANG II-evoked Ca2+ transients, without and with 100 nM ouabain, in 0 Ca2+ PSS. C: ANG II-evoked Ca2+ transients, without and with 100 nM ouabain, in 0 Ca2+-5 mM Na+ medium. D: summarized data from experiments similar to those in A-C, including effects of 10 mM Mg2+ and 10 µM La3+ in absence of ouabain and of 10 mM Mg2+, 10 µM La3+, and 100 nM PE in presence of 100 nM ouabain. Unlabeled solid bar, effect of ouabain on response to ANG II in PSS. * Significantly greater than control (P < 0.01) but not significantly different from each other. dagger  Significantly greater than control and 0 Ca2+ (P < 0.01) and 5 mM Na+ (P < 0.05). Dagger  Not different from controls or from each other (P > 0.05). § Significantly greater than with 10 µM La3+ (P < 0.05). All solutions contained 10 µM nifedipine to block voltage-gated, L-type Ca2+ channels and 50 µM TTX to block voltage-gated Na+ channels. Solution changes are indicated by horizontal bars below records.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SOC and Ca2+ entry in arterial myocytes. SOC, which apparently serve as a mechanism for replenishing depleted S/ER Ca2+ stores, have been studied in many types of cells (50), including vascular smooth muscle cells (31, 47). We examined the properties of SOC in primary cultured rat mesenteric artery myocytes by measuring changes in [Ca2+]cyt with fura 2. When SR Ca2+ was unloaded with CPA + Caff in cells bathed in Ca2+-free media, a brief rise in [Ca2+]cyt, a rapid recovery, and then a decline below the original resting level occurred. The restoration of extracellular Ca2+ evoked a large rise in [Ca2+]cyt, indicative of Ca2+ entry through SOC (36). This external Ca2+-dependent rise in [Ca2+]cyt was half-maximally activated by [Ca2+]o of 1.1 mM and was prevented by pretreating the cells with BAPTA to buffer the cytosolic Ca2+. Furthermore, the rise in [Ca2+]cyt was reversibly blocked by 10 mM Mg2+ or 10 µM La3+, known blockers of SOC (12, 35, 46, 47). Thus the general properties of the SOC in the arterial myocytes seem comparable to those in many other types of cells (10).

The molecular nature of the channels responsible for SOC activity is not known. Several candidate channels have, however, been identified and characterized by molecular genetics and/or electrophysiological methods. These candidate channels include the CRAC channels of mast cells, leukocytes, and leukemia cells (17, 23, 24) and a variety of TRP and TRPL channels (25, 35, 46, 50, 51). Particularly important for the present investigation is the recent observation that many of these channels are permeable to Na+ as well as Ca2+ (23, 35, 46). Indeed, CRAC and TRP/TRPL channels have sometimes been characterized by measuring the Na+ currents that they conduct, simply because these currents are larger than the respective Ca2+ currents under comparable conditions (23, 24, 46).

Two modes of Na+ entry in arterial myocytes. Many studies indicate that vascular smooth muscle cells express few, if any, classical tetrodotoxin-sensitive, voltage-gated Na+ channels. Indeed, rat mesenteric artery myocytes do not exhibit a depolarization-activated inward current (48). Moreover, in the present study we were unable to detect any effects of the Na+ channel opener veratridine (44) or the blocker tetrodotoxin on [Na+]cyt. Through what pathway(s), then, do Na+ enter these cells? Our results reveal two mechanistically distinct pathways through which Na+ can enter the myocytes after cell activation and show how these pathways can influence cell function.

One Na+ entry pathway depends on the rise in [Ca2+]cyt and is abolished when cytosolic Ca2+ is buffered by BAPTA. This Na+ entry mechanism is unaffected by 10 mM Mg2+ or 10 µM La3+. Ca2+ transients induced by unloading the SR Ca2+ stores in these cells are greatly prolonged when external Na+ is removed and Ca2+ extrusion via Na+/Ca2+ exchange is prevented (38, 39). Thus it appears that one Na+ entry pathway (which can account for the first Na+ transient in Fig. 3A) is via the Na+/Ca2+ exchanger operating in the Na+ entry/Ca2+ exit mode (i.e., NCXNE).

A second Na+ entry pathway depends on the unloading of the SR Ca2+ stores, but not on the rise in [Ca2+]cyt, inasmuch as it is not inhibited by buffering the cytosolic Ca2+ with BAPTA. This pathway is inhibited by 10 mM Mg2+ and 10 µM La3+. These properties are consistent with the idea that the SOC are responsible for this Na+ entry. Our data also indicate that elevating external Ca2+ inhibits Na+ entry and that these channels have a PCa/PNa of ~8, which is in the range of published values for some SOC channels (35, 46). Because many SOC are permeable to Na+, [Na+]o >> [Ca2+]o, and arterial myocytes possess these channels, we might expect the SOC to be a significant Na+ entry pathway in these cells. Indeed, vasoconstrictors such as serotonin and vasopressin, which mobilize SR Ca2+ and evoke Ca2+ transients, also induce Na+ entry and elevate [Na+]cyt (6, 7). Therefore, it seems surprising that the role of SOC in Na+ entry and Na+ homeostasis has not been appreciated.

Na+ entry through SOC and resting [Na+]cyt. SOC are normally activated by unloading the SR Ca2+ stores; nevertheless, some of these channels are likely to be spontaneously active under normal resting conditions when the Ca2+ stores are replete. In resting cells, we observed that [Na+]cyt was markedly influenced by [Mg2+]o: lowering [Mg2+]o from 1.4 to 0.33 mM elevated [Na+]cyt from 8.7 to 9.4 mM, whereas raising [Mg2+]o to 10 or 20 mM (or adding 10 µM La3+) for long periods caused [Na+]cyt to decline to 5.1 mM. Mg2+ and La3+ may not be selective inhibitors of SOC channels, so we cannot be certain that the resting Na+ entry inhibited by Mg2+ is mediated solely by SOC. Nevertheless, the 10 mM Mg2+-induced decline in [Na+]cyt raises the possibility that SOC may contribute as much as 40-50% of the entering Na+ (i.e., the Mg2+-inhibitable component) in resting mesenteric artery myocytes. These results reveal that external Mg2+ is an important regulator of PNa in these cells.

A prolonged rise in [Na+]cyt stimulates Na+ pump activity. Another unanticipated finding was that prolonged elevation of [Na+]cyt increases the rate of extrusion of Na+ via the ouabain-sensitive Na+ pump (Fig. 7). This increase in Na+ pump activity occurred with a delay of 5-10 min. It seems most unlikely that this delay was a direct result of an increase in pump "substrate" (i.e., internal Na+), because the rise in [Na+]cyt preceded the increase in Na+ pump activity. The delay suggests that a secondary event, such as a phosphorylation of the Na+ pump (4), may be responsible. This may serve as a "physiological safety valve," so that adequate Na+ can be extruded under conditions of chronic stimulation in which [Na+]cyt may rise above normal.

Is SOC the route of Na+ entry for modulating Ca2+ signaling by ouabain? As illustrated in Fig. 9, brief (30-s) exposure of the arterial myocytes to 100 nM ouabain augmented the Ca2+ transients evoked by vasoconstrictors (e.g., ANG II and PE) that mobilize SR Ca2+; this augmentation depended on external Na+. Inasmuch as the exposure to this low concentration of ouabain also was brief, it is unlikely to have raised "bulk" [Na+]cyt, because the most prevalent Na+ pump catalytic (alpha ) subunit in these cells is the alpha 1-subtype (21), which has a low affinity for ouabain (in rat, IC50 ~ 100 µM) (4). Indeed, our unpublished results reveal that a 10-min exposure to 100 nM ouabain does not raise [Na+]cyt in these myocytes. Thus the augmentation by low-dose ouabain must be mediated by the ouabain-sensitive (IC50 < 50 nM) alpha 3-isoform of the Na+ pump (4), which is restricted to PL microdomains that overlie the myocyte "junctional" (subplasmalemmal) SR (jSR) (21). Interestingly, SOC (19, 25) and the Na+/Ca2+ exchangers (20, 33) are likely to be clustered with the high ouabain affinity Na+ pumps in these same PL microdomains. Therefore, we propose the following hypothesis to account for the results in Fig. 9: Normally, at rest, the Na+ that enters via SOCNE (Fig. 6) is very rapidly extruded by the nearby alpha 3-type Na+ pumps (21). When these pumps are inhibited by 100 nM ouabain, applied along with the vasoconstrictors, the local [Na+]cyt within the tiny diffusion-restricted volume of cytosol (estimated to be 10-19-10-18 liters) between the PL and adjacent jSR (separated by 10-15 nm) (39, 41) rises within seconds, i.e., before the peak of the agonist-evoked Ca2+ transient. At this low concentration of ouabain, the uniformly distributed, "housekeeping" alpha 1-type Na+ pumps are not affected, so that the bulk [Na+]cyt does not change. However, the reduced Na+ gradient across the PL microdomains impairs Ca2+ extrusion locally, because the Na+/Ca2+ exchangers are located here (20, 33). The rise in local Ca2+ concentration should enhance Ca2+-induced Ca2+ release from more central regions of the SR and, thereby, rapidly amplify the "global" Ca2+ transients. Additional factors that may contribute to the augmented signaling include increased release of "trigger Ca2+" from the jSR because of sensitization of the inositol trisphosphate receptors and/or increased Ca2+ storage in the jSR and, possibly, increased Ca2+ entry across the PL; these possibilities are, however, too speculative to warrant further discussion here.

The aforementioned sequence of events not only may explain the vasotonic actions of low-dose cardiotonic steroids (42) but also may reveal a more general mechanism. Via this mechanism, isoform-specific changes in Na+ pump activity, mediated by protein kinases (4) or endogenous inhibitors (14), may regulate Ca2+ signaling and cell responsiveness in many other types of cells.

Implications of Na+ entry through SOC for the modulation of vascular tone. The mechanism described in Is SOC the route of Na+ entry for modulating Ca2+ signaling by ouabain? may also provide a role for SOCNE in the tonic modulation of vascular contractility. Others have suggested that Ca2+ entry via SOC may help regulate smooth muscle tone (11). The present results now suggest that interplay between the rate of SOCNE and the rate of Na+ pump-mediated Na+ extrusion, which may be altered by neurotransmitters such as dopamine (5) and inhibitors such as ouabain, may affect arterial tone by altering the rate of Ca2+ extrusion via Na+/Ca2+ exchange.

Our findings may also help elucidate the "calcium paradox" phenomenon in vascular smooth muscle (49) and the antihypertensive action of Mg2+ (45). Withdrawal of extracellular Ca2+ and Mg2+ promotes an Na+ influx in arterial myocytes that is unaffected by L-type Ca2+ channel blockers, whereas restoration of Ca2+ then induces contraction [the calcium paradox (49)]. Under these conditions, we suggest that Na+ entry may occur through SOC, inasmuch as the entry is inhibited by divalent cations such as Mg2+, Mn2+, and Ni2+ (49), all of which block SOC (17, 46, 47, 52). When Ca2+ is restored, the elevated [Na+]cyt may induce a contraction by promoting Ca2+ entry via Na+/Ca2+ exchange, although Ca2+ entry through SOC and through L-type Ca2+ channels (activated by depolarization) and receptor-operated channels may also contribute (2, 11).

The influence of Mg2+ on blood pressure has been the subject of numerous studies (1, 45). Epidemiological data reveal an inverse relationship between dietary Mg2+ and blood pressure (45). Although clinical trials of dietary Mg2+ supplementation in patients with essential hypertension have yielded inconsistent results (22), intravenous Mg2+ (as MgSO4) has long been used to lower blood pressure in the toxemias of pregnancy (37). Our results now suggest that these effects may be due, at least in part, to the ability of Mg2+ to block SOC and thereby diminish Na+ and Ca2+ entry and inhibit vasoconstriction.

In summary, our results demonstrate that Ca2+ signaling in arterial myocytes is critically influenced by the coentry of Na+ and Ca2+ through the SOC pathway. Under resting conditions, some Na+ enters cells via the SOC, and mobilization of stored Ca2+ opens these channels and greatly increases Na+ entry. In turn, the entering Na+ influences Ca2+ storage and release and, thereby, plays a key role in modulating cell signaling. The magnitude of this effect is determined by the amount of Na+ that enters and by the activities of the Na+ pumps and the Na+/Ca2+ exchangers colocalized with SOC in junctional microdomains of the PL. This role of dual-ion permeation in Ca2+ signaling may be applicable to numerous types of cells in which SOC have been reported (10).


    ACKNOWLEDGEMENTS

We thank K. Strauss for cell cultures, V. A. Golovina and M. Juhaszova for help with imaging methods, Y. Arnon for assistance with statistics, B. K. Krueger and D. Weinreich for comments on a preliminary version of the manuscript, and TEFLABS for a gift of fluorescent dyes.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-45215 (to M. P. Blaustein).

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. §1734 solely to indicate this fact.

1 Cation (M) concentrations in "bulk" cytosol and in the extracellular fluid are denoted by [M]cyt and [M]o, respectively, where M is Na+, K+, Ca2+, or Mg2+.

Address for reprint requests and other correspondence: M. P. Blaustein, Dept. of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201.

Received 15 June 1999; accepted in final form 21 September 1999.


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DISCUSSION
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