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(Circulation Research. 2007;101:1030.)
© 2007 American Heart Association, Inc.

Cellular Biology

Transient Receptor Potential Channel 6–Mediated, Localized Cytosolic [Na⁺] Transients Drive Na⁺/Ca²⁺ Exchanger–Mediated Ca²⁺ Entry in Purinergically Stimulated Aorta Smooth Muscle Cells

Damon Poburko, Chiu-Hsiang Liao, Virginia S. Lemos, Eric Lin, Yoshiaki Maruyama, William C. Cole, Cornelis van Breemen

From the Departments of Anesthesiology, Pharmacology & Therapeutics (D.P., C.-H.L., V.S.L., C.v.B.) and Cellular & Physiological Sciences (E.L.), University of British Columbia, Vancouver; Child & Family Research Institute (D.P., C.-H.L., V.S.L., C.v.B.), Vancouver; School of Kinesiology (E.L.), Simon Fraser University, Burnaby; and Smooth Muscle Research Group (Y.M., W.C.C.), Faculty of Medicine, University of Calgary, Alberta, Canada.

Correspondence to Cornelis van Breemen, Department of Anesthesiology, Pharmacology & Therapeutics, 2176 Health Sciences Mall, University of British Columbia, Vancouver, Canada, V6T 1Z1. E-mail breemen{at}interchange.ubc.ca

	Abstract

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The Na⁺/Ca²⁺ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca²⁺ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca²⁺ influx by the NCX (1) is the primary mechanism of Ca²⁺ entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na⁺ indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na⁺] ([Na⁺]_i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na⁺]_i elevations and localized [Na⁺]_i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 µmol/L) and 2-amino-4-phosphonobutyrate (75 µmol/L) reduced both global and localized [Na⁺]_i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca²⁺ entry with KB-R7943 (10 µmol/L) enhanced the LNats, whereas the global cytosolic [Na⁺] signal was unaffected. Inhibition of mitochondrial Na⁺ uptake with CGP-37157 (10 µmol/L) increased both LNats and global cytosolic [Na⁺] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca²⁺ entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6–containing channels.

Key Words: Na⁺/Ca²⁺ exchanger • localized [Na⁺] elevation • calcium • hypertension • mitochondria • CoroNa • ATP • TRPC

	Introduction

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The link between Na⁺ and smooth muscle activation was recognized in 1960 when Dodd and Daniel reported that removal of extracellular Na⁺ abolished agonist-induced constriction of trachea and aorta.¹ In 1978, we suggested that Na⁺/Ca²⁺ exchange (NCX) in the vascular smooth muscle (VSM) plasmalemma (PM) was linked to sarcoplasmic reticulum (SR) Ca²⁺ transport in areas where the PM and SR are in close apposition.² This idea was confirmed by the transfer of large quantities of Ca²⁺ in and out of smooth muscle by removal and restitution of extracellular Na⁺ without concomitant changes in contractile tension.³ We later proposed that spontaneous Ca²⁺ release from the superficial SR is linked to Ca²⁺ extrusion via NCX at junctions of the PM and SR (PM-SR junctions).^2,4,5 NCX-mediated Ca²⁺ entry is often termed "reverse-mode NCX" or "NCX reversal" because the plasmalemmal Na⁺ and Ca²⁺ gradients predict Ca²⁺ extrusion or "forward mode" at resting membrane potentials. Blaustein and colleagues proposed that during adrenergic stimulation, Na⁺ entry through nonspecific cation channels (NSCCs) could drive NCX-mediated Ca²⁺ entry (or reverse mode) in localized regions of the PM that are selectively populated by the -2 Na⁺/K⁺-ATPase (NKA2).⁶ This was confirmed using a fluorescent Ca²⁺ indicator fused to NKA2 that reported agonist-mediated [Ca²⁺]_i elevations in the presence of cytoplasmic EGTA, which were not detected with the probe fused to the diffusely distributed NKA1.⁷ Current evidence shows NCX-mediated Ca²⁺ entry to be physiologically relevant in many cell types, and the clinical relevance of smooth muscle Na⁺ transport and Ca²⁺ entry by NCX has been extensively documented in the development of essential hypertension.^8,9

Currently, we addressed 2 missing links in the proposed interactions between the NSCC and NCX in the PM-SR junctions: (1) the identity of the Na⁺-permeable channel and (2) the direct demonstration of the local [Na⁺]_i increases it mediates.

In the 1970s, we proposed the existence of receptor-operated channels and the functional linkage of the NCX to the regulation of SR Ca²⁺ content.² Unlike the voltage-gated Ca²⁺ channels (L and T type), the identity of the receptor-operated NSCC has not been fully resolved. Molecular identification of these NSCCs has been complicated by variation in their (1) mechanisms of activation, (2) cation selectivity, and 3) apparent molecular composition. Commensurate with such variability, a variety of the canonical transient receptor potential channel proteins (TRPCs) are proposed to fulfill the experimental criteria for receptor-operated channels.^10,11 TRPC expression is species and tissue specific. The rat aortic smooth muscle cells (RASMCs) used in our studies express TRPC1, TRPC4, and TRPC6.¹² Despite uncertainty of whether TRPC1 and TRPC4 are activated by SR Ca²⁺ depletion,¹³ TRPC6 appears to be activated by diacylglycerol and to form a NSCC (P_Na:P_Ca1:5).^14–16 TRPC6 is essential for agonist-mediated Ca^{2 +} entry in adrenergically stimulated rat prostate smooth muscle and rabbit portal vein.^14,16 The role of TRPC6 in Na⁺ entry and NCX-mediated Ca²⁺ entry has not been investigated, but the phenomenon of NCX-TRPC coupling was demonstrated for TRPC3 and NCX in HEK-293 cells and rat cardiomyocytes.^17,18 Recently, we found that sustained [Ca²⁺]_i elevation in RASMC is entirely dependent on NCX-mediated Ca²⁺ entry and that Ca²⁺ entry develops with kinetics similar to the agonist-mediated elevation of bulk cytosolic [Na⁺].^12,19

We now show that a dominant negative TRPC6 (TRPC6^dn) suppresses ATP-stimulated Na⁺ entry, which is responsible for peripherally localized [Na⁺]_i transient (LNat) elevation and significantly contributes to global [Na⁺]_i increases. We further show that LNats are modulated by mitochondrial Na⁺ uptake and linked to NCX-mediated Ca²⁺ entry.

	Materials and Methods

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Cultured rat aortic smooth muscle cells (RASMCs) were obtained from Dr Urs Ruegg (Faculty of Pharmacy, University of Geneva, Switzerland) and used between passages 8 to 13.^20,21 Experiments were performed at room temperature in physiological saline solution (in mmol/L): 145 NaCl, 5 KCl, 1 MgCl₂, 10 glucose, 5 HEPES, 1.2 CaCl₂ (pH 7.6). Generation of the dominant negative human TRPC6 (GenBank accession no. BC093660) and transfection of cells with Transfectin (Bio-Rad) have been described previously (transfection efficiency 30%).^22,23 Cytosolic [Na⁺] ([Na⁺]_i) was measured with CoroNa Green (Molecular Probes; 5 µmol/L, 45 minutes, 25°C, 100 to 200 µmol/L probenecid).^12,23 Mitochondrial [Na⁺] was measured with CoroNa Red (Molecular Probes; 0.2 µmol/L, 2.5 mmol/L TEMPOL, 20 minutes, 37°C). Images were acquired on an Ultraview Nipkow spinning-disc system, exciting CoroNa Green (488 nm excitation, 510 to 540 nm emission) and CoroNa Red (568 nm excitation, 590 to 650 nm emission) with an argon/krypton laser. Image acquisition, image analysis, statistical methods, and CoroNa Red/mitochondrial green fluorescent protein colocalization¹² are described in the online data supplement at http://circres.ahajournals. org. Details regarding CoroNa dyes are discussed in the online data supplement and elsewhere.^24–26

	Results

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Characterizing ATP-Mediated Na⁺ Responses
We have shown previously that RASMC stimulation with ATP (1 mmol/L) induces a monotonic increase in global [Na⁺]_i via activation of Na⁺-permeable plasmalemmal channels.¹² We now show that global [Na⁺]_i elevations are accompanied by localized, [Na⁺] elevations near the periphery of the cell (Figure 1; Movies 1 and 2 in the online data supplement). In a given experiment, monotonic [Na⁺]_i elevations and LNats were observed in 50% to 80% and 20% of cells, respectively. The time-to-onset of LNats following ATP was 10 to 30 seconds poststimulation. Hypothesizing that LNats underlie NCX-mediated Ca²⁺ entry, it is noteworthy that the time-to-onset of LNats was consistent with the delay for ATP-mediated Ca²⁺ entry in RASMCs (30 to 40 seconds), inferred from inhibition of [Ca²⁺]_i plateaus by KB-R7943 and TRPC6^dn.²³ The time-to-peak and decay half-life averaged 44±2 and 57±5 seconds, respectively (n111 transients; Table). Image acquisition at 10 Hz confirmed that each transient was a single event rather than the summation of many shorter events (not shown). The spatial and temporal characteristics of the LNats are illustrated in Figure 1A through 1C. The full-width half-maximal diameter (FWHM) of the LNats averaged 2.29±0.13 µm (n=199 transients, 26 experiments). The amplitude of LNats was 0.34±0.02 F/F_o, whereas the amplitude of global [Na⁺]_i elevations averaged 0.13±0.02 F/F_o. Although numerous factors limit the accuracy of subcellular calibration of single-wavelength indicators, previous calibration of CoroNa Green in situ showed ATP to increase [Na⁺]_i from 6 to 8 mmol/L at rest to 14 to 16 mmol/L.¹² Given that peripheral [Na⁺] transients exhibited F/F_o that was 100% greater than global F/F_o, we conservatively estimate localized and global [Na⁺]_i elevations at 30 mmol/L and 14 mmol/L (assuming linear fluorescence/[Na⁺] relationship from 3 to 120 mmol/L and essentially uniform [Na⁺]_i at rest).²⁷

View larger version (88K): [in this window] [in a new window]   Figure 1. ATP stimulates monotonic elevations in global [Na+]i and transient [Na+]i elevations in localized peripheral regions. A, Arrows illustrate the location of CoroNa Green transients occurring spontaneously (yellow) and in response to ATP (1 mmol/L) added at 140 seconds (green, early onset; red, delayed onset). B, Subregions of CoroNa Green–loaded cells (dotted boxes on grayscale images) are magnified at right (pseudocolor) with line scans at the far right. Dotted white lines in magnified images and line scans show positions of matching line scans and time points of the x-y image. ATP added at dotted red line. C, Traces from individual cells show onset and kinetics of global (black) and localized (colored) [Na+]i elevations from matching images below (white region, black trace). ATP present from black arrows. Gray and pseudocolor scales showing 12-bit fluorescence intensity with linear contrast stretch.

View this table: [in this window] [in a new window]   Table 1. Table. Spatiotemporal Characteristics of [Na+] Transients

TRPC6^dn Suppresses ATP-Mediated Na⁺ Entry
The steady-state [Ca²⁺]_i elevation stimulated by ATP in these RASMCs is almost entirely dependent on NCX-mediated Ca²⁺ entry.²³ We hypothesized that SKF-96365 and 2-APB indirectly inhibit the [Ca²⁺]_i elevation by suppressing TRPC6-mediated [Na⁺]_i elevations that underlie NCX-mediated Ca²⁺ entry. As predicted, 2-amino-4-phosphonobutyrate (2-APB) (75 µmol/L) and SKF-96365 (50 µmol/L) reduced the amplitude of the global [Na⁺]_i elevation by 60% (Figure 2). Moreover, these compounds reduced the proportion of cells exhibiting LNats by 80% (Figure 2). However, neither the amplitude nor FWHM of the few persisting LNats was affected by either inhibitor (Figure 2 and the Table). Inhibiting SR/endoplasmic reticulum Ca²⁺-ATPase (SERCA) with cyclopiazonic acid (30 µmol/L) in the absence of agonist depletes the SR Ca²⁺ stores,²¹ but this treatment did not cause an observable increase in [Na⁺]_i or prevent global and localized responses to ATP (data not shown), indicating that without receptor stimulation, Na⁺ entry was not activated in response to SR Ca²⁺ depletion. This suggests that TRPC1 and TRPC4, which are candidates for store-operated channels, were likely not responsible for the observed Na⁺ influx. Given the evidence for TRPC6 activation by diacylglycerol rather than SR Ca²⁺ depletion, we investigated the role of native TRPC6 in ATP-induced Na⁺ entry by transfecting RASMCs with TRPC6^dn.²² This pore mutant subunit combines with native subunits creating cation-impermeable channels. Thus, TRPC6^dn acts as an inhibitor of processes relying on TRPC6-mediated Na⁺ influx.²² In cell populations transfected with the TRPC6^dn, the amplitude of the global [Na⁺]_i plateau averaged across all cells on cover slips transfected with TRPC6^dn (n=76 cells) was reduced by 30% when compared with the average response from nontransfected cover slips (n=166 cells) (Figure 3i and 3ii). The proportion of cells per cover slip showing LNats was also decreased by 30% in cover slips transfected with TRPC6^dn (Figure 3iv). The correlation between the mean inhibition of both global and localized [Na⁺]_i elevations by TRPC6^dn with its transfection efficiency was also observed for inhibition of steady-state [Ca²⁺] elevations.²³ Similar to the effects of 2-APB and SKF-96365, the amplitude of the LNats was not significantly affected by expression of TRPC6^dn (Figure 3iii). Global and transient [Na⁺] responses to ATP from coverslips transfected with pcDNA3 vector showed no difference from untransfected coverslips (not shown).

View larger version (32K): [in this window] [in a new window]   Figure 2. ATP-stimulated Na+ entry is in part mediated by nonselective cation channels. i, Averaged traces of ATP-mediated global [Na+]i elevation following pretreatment with SKF-96365 (50 µmol/L) (A) or 2-APB (75 µmol/L) (black traces) (B) vs untreated cells (gray traces). Effects of SKF-96365 and 2-APB are shown for amplitude of global [Na+]i elevations (ii), amplitude of LNats (iii), and the proportion of cells exhibiting LNats (iv). The data are from 8 independent experiments. Numbers on bars show number of cells (ii), transients (iii), and coverslips (iv) analyzed, respectively. This convention also applies to Figures 3 through 5.

View larger version (18K): [in this window] [in a new window]   Figure 3. TRPC6 contributes to global and localized elevations of [Na+]. i, Average trace of global [Na+]i response from all cells on nontransfected coverslips (gray) and all cells on TRPC6dn-transfected coverslips (black). TRPC6dn transfection reduced the amplitude of global [Na+]i response to ATP (ii) and the proportion of cells on a given cover slip exhibiting LNats (iv), but the amplitude of LNats was not altered (iii). The data are from 18 independent experiments.

Ca²⁺ Entry by the Plasmalemmal NCX Is Triggered by ATP-Mediated Na⁺ Entry
Selective inhibition of NCX-mediated Ca²⁺ entry with KB-R7943 (10 µmol/L) did not affect ATP-mediated, global [Na⁺]_i elevations, ruling out a widespread membrane effect (Figure 4i and 4ii). In contrast, KB-R7943 dramatically increased the proportion of cells exhibiting LNats by 170% (Figure 4iv), the amplitude of LNats by 70% (Figure 4iii), and the average number of LNats/cell by 60% (Table). Interestingly, the FWHM diameter and time-to-peak of LNats was unaffected by KB-R7943, but the LNats decayed slightly faster (Table). These results indicate that inhibiting NCX-mediated Na⁺ efflux increases the amplitude and occurrence of localized [Na⁺] transients and support the hypothesis of localized coupling of TRPC6-mediated Na⁺ entry with Na⁺ efflux through the NCX in VSM. Moreover, NCX-mediated Na⁺ efflux appears to mask the occurrence of transients in untreated cells.

View larger version (18K): [in this window] [in a new window]   Figure 4. NCX-mediated Na+ entry modulates localized but not global [Na+]i. Cells pretreated with KB-R7943 (KBR) (10 µmol/L) 5 minutes before the start of experiments. i and ii, The average trace (i) and mean amplitude (ii) of global [Na+]i response to ATP shows no difference between KB-R7943–treated (black) and control (gray) cells. KB-R7943 increased the amplitude of LNats (iii) and proportion of cells exhibiting LNats (iv). The data are from 16 independent experiments.

Mitochondria and Na⁺ Influx
We recently hypothesized that ATP-mediated stimulation should enhance mitochondrial Na⁺ uptake. Following ATP stimulation, some Ca²⁺ enters VSM cells through the sarcolemmal NCX, and some of this Ca²⁺ is taken up by mitochondria. Increased mitochondrial NCX activity permits [Ca²⁺]_MT recovery to resting levels despite maintained Ca²⁺ uptake.¹² As predicted, inhibition of mitochondrial NCX with CGP-37157 (10 µmol/L) increased the global [Na⁺]_i plateau by 210%, similar to the 180% increase in the proportion of cells exhibiting LNats, whereas the mean number of LNats per cell increased by 30% (Figure 5 and the Table). CGP-37157 did not affect LNat amplitude, kinetics, or FWHM (Table). We confirmed that CGP-37157 inhibited mitochondrial Na⁺ uptake using the mitochondrial-selective Na⁺ dye CoroNa Red. The distribution of CoroNa Red was indistinguishable from mitochondria-targeted green fluorescent protein (Figure 6A). ATP (1 mmol/L) induced a monotonic increase in CoroNa Red fluorescence of 0.11±0.03 F/F_o (n=11 coverslips; supplemental Movie 3), which CGP-37157 (10 µmol/L) inhibited by 80% (0.02±0.02 F/F_o, n=14 coverslips) (Figure 6B and 6C). Importantly, these findings demonstrate that mitochondrial Na⁺ buffering masks the detection of LNats, thus partially explaining why LNats are normally detected in only 20% of cells.

View larger version (17K): [in this window] [in a new window]   Figure 5. Mitochondria buffer ATP-stimulated Na+ entry. Cells pretreated with CGP-37157 (10 µmol/L) 5 minutes before start of experiment. i, Average traces of ATP responses in CGP-37157–treated (black traces) or untreated (gray traces) cells. The effects of CGP-37157 on the amplitude of global [Na+]i elevations (ii), the amplitude of LNats (iii), and the proportion of cells exhibiting LNats (iv). The composite data are from 9 independent experiments.

View larger version (50K): [in this window] [in a new window]   Figure 6. CoroNa Red reveals ATP-mediated mitochondrial [Na+]elevations. A, Maximum intensity projection of confocal image stack (3x300 nm steps) from a cell (i) expressing mitochondrial green fluorescent protein (mito-GFP) (i) and loaded with CoroNa Red (ii) shows extensive overlap in the merged image (iii). The average trace (B) and composite data (C) from 11 independent experiments show inhibition of mitochondrial Na+ uptake by CGP-37157 (gray, control). D, Original images of a CoroNa Red–loaded cell illustrate mitochondrial morphology (grayscale) and signal intensities (pseudocolor) at the times matching numbers on graph in B. CoroNa Red fluorescence increased throughout the cell with greater relative increases in peripheral mitochondria.

	Discussion

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Contraction of VSM by phospholipase C–linked agonists is initiated by SR Ca²⁺ release and sustained by Ca²⁺ influx. Investigation of this Ca²⁺ influx has focused on identifying Ca²⁺-selective channels activated by SR Ca²⁺ depletion or second messengers such as diacylglycerol.²⁸ However, our laboratory and several others have shown that NCX-mediated Ca²⁺ entry is essential to purinergically and adrenergically stimulated Ca²⁺ influx in intact blood vessels and cultured aorta smooth muscle cells.^6,12,29–31 Global [Ca²⁺] and [Na⁺] gradients predict NCX-mediated Ca²⁺ extrusion at resting membrane potentials, but NCX-mediated Ca²⁺ entry can occur at localized sites of increased [Na⁺]_sub-PM in a cytosolic microdomain similar to the "fuzzy space" in cardiac myocytes^32,33 attributable to Na⁺ entry through NSCCs.^11,13 Agonist-stimulated, global [Na⁺]_i elevations in VSM were reported at least 14 years ago²⁷ and were partly attributed to Na⁺ entry via NCX and store-operated channels.²⁷ We show here that agonist stimulation also causes localized [Na⁺]_i elevation at the periphery of cells, in addition to global [Na⁺]_i elevations, and that NCX-mediated Ca²⁺ entry is coupled to these localized [Na⁺]_sub-PM elevations. Both the global and localized [Na⁺]_i elevations require functional, TRPC6-containing NSCCs. These findings highlight the spatial restriction of functional NCX-TRPC6 coupling, which we have shown to be essential for agonist-mediated Ca²⁺ entry in RASMCs.^12,23 We also show that mitochondrial Na⁺ uptake may contribute to the control of agonist-mediated, global [Na⁺]_i elevations. We discuss how this novel understanding of subcellular Na⁺ signaling relates to current views of localized Ca²⁺ signaling in VSM.

Mechanism of Global [Na⁺]_i Elevations
Purinergic stimulation, like vasopressin,²⁷ increases global [Na⁺]_i in RASMCs, which we have linked to opening of a Na⁺-permeable channel.¹² We propose that TRPC6-containing channels mediate this Na⁺ entry. To the best of our knowledge, this is the first report that 2-APB and SKF-96365, widely shown to block receptor-operated Ca²⁺ entry, also inhibit agonist-induced Na⁺ entry. TRPC-containing channels, typically forming NSCCs, are increasingly reported to mediate agonist-stimulated Ca²⁺ entry in smooth muscle. The RASMCs used here express TRPC1, TRPC4, and TRPC6.²⁰ SKF-96365 can inhibit TRPC1,³⁴ which like TRPC4, has been implicated in store-operated Ca²⁺ entry.^13,28 Blaustein and colleagues observed that SR Ca²⁺ depletion with caffeine and cyclopiazonic acid increased [Na⁺]_i in mesenteric artery myocytes⁶; however store depletion did not increase [Na⁺]_i in our RASMCs. This discrepancy could be attributable to tissue-specific differences in the organization of PM-SR junctions as the function, extent, and coupling of ion transporters in PM-SR junctions likely vary between smooth muscle types and vascular beds. Alternatively, the SR Ca²⁺ released by caffeine in the protocols of Blaustein and colleagues could have enhanced NCX-mediated Ca²⁺ extrusion and Na⁺ entry. Thus the 20% to 25% of the global [Na⁺]_i elevation resistant to SKF-96365 and 2-APB could represent NCX-mediated Ca²⁺ extrusion of ATP-induced SR Ca²⁺ release. The insensitivity of RASMC [Na⁺] to cyclopiazonic acid suggests that 2-APB acted on PM channels, rather than 1',4',5'-triphosphate receptors, that were likely not store-operated channels given evidence from prostate smooth muscle cells.¹⁴ The physiological importance of these findings is reinforced by direct measurements of [Ca²⁺]_ER showing that agonist stimulation only modestly depleted [Ca²⁺]_ER in HeLa and endothelial cells.^35,36 At present the relative contributions of store-operated versus receptor-operated Ca²⁺ entry in "physiologically" stimulated VSM have not been resolved.

Current evidence indicates that TRPC6-containing NSCCs are activated by diacylglycerol, rather than store depletion, and inhibited by 2-APB and SKF-96365.^14–16 In aorta and prostate smooth muscle cells, TRPC6 is essential to agonist-mediated Ca²⁺ entry.^14,23,37 Here we further demonstrate the role for TRPC6 in receptor-operated Na⁺ entry using the pore mutant TRPC6^dn construct. Our findings corroborate reports that TRPC6 forms the -adrenergically stimulated NSCCs in rabbit portal vein¹⁶ and that TRPC6^dn suppresses vasopressin-evoked whole cell NSCC currents in A7r5 RASMCs.²² These reports and the currently demonstrated inhibition of ATP-mediated Na⁺ elevations by SKF-96365, 2-APB, and TRPC6^dn suggest that Na⁺ entry via TRPC6-containing channels is a common response to a variety of phospholipase C–coupled receptors in VSM.

Two probable mechanisms link Na⁺ entry via NSCCs to Ca²⁺ entry: NCX-mediated Ca²⁺ influx and activation of voltage-gated Ca²⁺ channels.^4,5,12,38 The relative contribution of these 2 mechanisms might vary between cell types and tissues. Although SKF-96365, KB-R7943, and CGP-37157 can inhibit voltage-gated Ca²⁺ channel, such an effect would not be consistent with the sum of our current findings. In addition, voltage-gated Ca²⁺ channels do not appreciably contribute to ATP-mediated Ca²⁺ entry in these RASMCs (see the expanded Discussion section in the online data supplement). Rather, the sustained, ATP-mediated [Ca²⁺]_i elevation requires NCX-mediated Ca²⁺ entry that develops with kinetics similar to the elevation of bulk [Na⁺]_i.^12,19 This NCX-mediated Ca²⁺ entry requires functional interaction between the NCX and endogenous TRPC6 as described for NCX and TRPC3 in cardiac myocytes and HEK293.^17,18 Such close functional coupling is thought to require the clustering of specific ion transporters at specialized junctions between the PM and SR. The molecular compositions and functions of these junctions may vary within single cells and between different tissues. For example, under Ca²⁺-free conditions, functional coupling of ryanodine receptors with the NCX facilitates localized SR Ca²⁺ unloading in these cells. However, the inhibition of ATP-mediated LNats by SKF-96365 suggests that the LNats were not the consequence of localized extrusion of SR Ca²⁺ release.

Local [Na⁺]_i Transients and PM-SR Junctions
The PM-SR junctions described above regulate ionic signaling by creating diffusionally restricted cytosolic microdomains, around which specific transporters and channels cluster to generate locally elevated ion concentrations.^4,11,39,40 The high density and close apposition of transporters and enzymes in these junctions mediate "linked ion transport," permitting purpose-specific, localized interactions of ion transporters without significant ionic diffusion into the bulk cytosol.⁴ Blaustein and colleagues proposed that activation of NSCCs in such junctions could generate the [Na⁺] required to mediate NCX-mediated Ca²⁺ entry.⁶ We have shown that this mechanism efficiently refills the SR following 1',4',5'-triphosphate receptor–mediated Ca²⁺ release.⁴¹ Functional and molecular evidence corroborates the junctional clustering of Ca²⁺ and Na⁺ transporters that would support agonist-induced NCX-mediated Ca²⁺ entry. For example, Ca²⁺ signals reported by a molecular Ca²⁺ probe fused to NKA2, but not diffusely localized NKA1, supported localization of Ca²⁺ signals to PM-SR junctions.⁷ Furthermore, NKA2 coimmunoprecipitates with NCX and TRPC6 in brain homogenates.^38,42 TRPC5 and TRPC6 are binding partners of the NKA,⁴² whereas NCX and TRPC3 interact and coimmunoprecipitate in HEK-293 cells.¹⁷

Our observation of LNats provides further support for functional linkage of receptor-operated NSCCs and NCX at PM-SR junctions. The inhibition of localized [Na⁺]_sub-PM transients by SKF-96365, 2-APB, and TRPC6^dn, combined with the increased occurrence and amplitude of LNats on addition of KB-R7943, demonstrates the localized coupling of the NSCCs and NCX. Failure of KB-R7943 to increase global [Na⁺]_i rules out a diffuse plasmalemmal effect and directly supports the linkage of TPRC6 and NCX in diffusionally restricted microdomains with elevated [Na⁺]_sub-PM. Our estimation that [Na⁺]_sub-PM increases to 30 mmol/L is likely conservative, but the accuracy of subcellular calibrations of single-wavelength probes is limited by variations in cell thickness, heterogeneous dye distribution, and dye sensitivity in varying microenvironments,^27,43 Because LNats likely measure some degree of Na⁺ spillage from PM-SR junctions, our estimate of LNat [Na⁺]_sub-PM may underestimate [Na⁺]_sub-PM immediately in the PM-SR junction. However, it is consistent with the estimates of Isenberg and colleagues of [Na⁺]_sub-PM ranging from 24 to 40 mmol/L measured by fluorescence microscopy and X-ray microprobe.^12,44,45 Such [Na⁺]_sub-PM elevations should suffice to "reverse" the NCX assuming modest depolarization to –30 mV, [Ca²⁺]_sub-PM0.5 to 1.0 µmol/L, [Ca²⁺]_o=1.2 mmol/L, and [Na⁺]_o=145.¹² In addition, the space constant for the subplasmalemmal [Na]_total gradient in ventricular myocytes is 28 nm,⁴⁵ which is highly consistent with the intermembrane separation in PM-SR junctions in smooth muscle (20 nm).⁴

The LNats are remarkably similar in size to Ca²⁺ sparks (ie, 2 to 3 µm FWHM), which are also thought to occur often at PM-SR junctions.⁴⁶ Given the spatial resolution in our experiments (480 nm/pixel), the size of the [Na⁺]_sub-PM transients was comparable to the mean diameter of PM-SR junctions in VSM cells (400 nm).^4,39,47 This spatial characteristic of the localized [Na⁺]_sub-PM transients is consistent with their generation at and spillage from the edges of PM-SR junctions. We recently reported that disruption of PM-SR junctions with calyculin A increased ATP-mediated [Na⁺]_i elevations and inhibited [Ca²⁺]_i elevations, indicating the role of junctions in permitting NCX-mediated Ca²⁺ entry.²³ The lack of effect of KB-R7943 and CGP-37157 on FWHM (Table) indirectly suggests some ultrastructural basis underlying the LNats. In contrast to Ca²⁺ sparks lasting hundreds of milliseconds, the [Na⁺]_sub-PM transients last tens of seconds to minutes. Although these slow kinetics may be related to the temperature sensitivity of the Na⁺/K⁺-ATPase,⁴⁸ our experiments underline the need to better understand the ultrastructural and molecular nature of the barriers creating localized [Na⁺] gradients (reviewed elsewhere⁴³).

Mitochondria Regulate Local [Na]_i and [Ca]_i
Mitochondria can rapidly buffer local [Ca²⁺]_sub-PM elevations to (1) prevent Ca²⁺ influx from diffusing into the bulk cytosol and (2) modulate neighboring Ca²⁺-sensitive targets.^12,49,50 The rapidly buffered Ca²⁺ is slowly extruded via mitochondrial NCX or H⁺/Ca²⁺ exchange, subsequently helping to refill near by ER/SR or being extruded from the cell.^51,52 Following agonist stimulation, increased mitochondrial NCX activity may also prevent mitochondrial Ca²⁺ overload.^12,51,53 Our current findings suggest an additional mitochondrial role of modulating local [Na⁺]_sub-PM elevations. We propose that mitochondria neighboring the NSCC-containing PM-SR junctions (ie, within 100 nm) buffer Na⁺ spilling from the junctions, in part, because of increased mitochondrial NCX activity. This hypothesis is supported by 3 effects of mitochondrial NCX inhibition: (1) CGP-37157 inhibited ATP-mediated [Na⁺]_mito elevations; (2) CGP-37157 increased bulk [Na⁺]_i elevations; and (3) CGP-37157 increased the occurrence of LNats. Consistent with this proposal, Bernardinelli et al reported that CGP-37157 inhibited mitochondrial uptake of glutamate-coupled Na⁺ entry in astrocytes.²⁵ Mitochondrial modulation of localized Na⁺ gradients may be an intriguing aspect of ionic homeostasis deserving further investigation.

In conclusion, we show 2 types of agonist-stimulated [Na⁺]_i responses. Both global monotonic and localized transient [Na⁺]_i elevations were TRPC6-dependent. We show directly that agonist-induced, localized Na⁺ entry stimulates NCX reversal, which is gaining recognition as a physiologically relevant Ca²⁺ entry mechanism. We propose that Na⁺ transients are generated in PM-SR junctions and are responsible for NCX-mediated Ca²⁺ entry in agonist-stimulated smooth muscle. Global Na⁺ responses are not functionally linked to NCX "reversal," but are likely attributable to Na⁺ spillage from the PM-SR junction, Na⁺ entry through nonjunctional NSCCs, and Ca²⁺ extrusion by NCX not coupled to TRPC6. Finally, we demonstrate that Na⁺ buffering by mitochondria appears to limit Na⁺ diffusion into the bulk cytosol. Together, these findings demonstrate a role for TRPC6-NCX coupling during physiological stimulation of VSM and support the theory that the etiology of salt-sensitive hypertension, in which plasma [Na⁺] is typically elevated, involves augmentation of NCX-mediated Ca²⁺ entry.^8,9

	Acknowledgments

 
Sources of Funding

This research was supported by Canadian Institutes of Health Research grant 20R90431 (to C.v.B.). V.S.L. thanks CNPq/Brazil (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico) for financial support. D.P. thanks the Michael Smith Foundation for Health Research and the Natural Sciences and Engineering Research Council of Canada for financial support.

Disclosures

None.

	Footnotes

 
Original received December 18, 2006; resubmission received May 7, 2007; revised resubmission received August 13, 2007; accepted September 5, 2007.

	References

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