Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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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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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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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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 × 106 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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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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DISCUSSION |
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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+]oNa+ 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 () subunit in these cells is the
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)
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
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"
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.
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 |
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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.
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FOOTNOTES |
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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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