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Am J Physiol Heart Circ Physiol 291: H1226-H1235, 2006. First published April 14, 2006; doi:10.1152/ajpheart.00196.2006
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Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Hui Dong,1 Yanfen Jiang,1 Chris R. Triggle,2 Xiaofang Li,3 and Jonathan Lytton3

1Division of Gastroenterology, Department of Medicine, University of California, San Diego School of Medicine, San Diego, California; and 2Department of Pharmacology and Therapeutics and 3Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada

Submitted 22 February 2006 ; accepted in final form 4 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.

vascular tone; calcium homeostasis; rat aorta

CA2+ PLAYS A PIVOTAL ROLE in controlling vascular tone, thereby making significant contributions to the regulation of systemic blood pressure and the supply of blood to important organs in human body. Vascular contraction is induced by an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]cyt) or by an increase in the sensitivity to Ca2+ of the contractile elements in response to various signaling events (8, 23). The increase in [Ca2+]cyt required to initiate contraction comes from both extracellular and sequestered intracellular stores. Relaxation is then achieved by pumping Ca2+ back into the sarcoplasmic reticulum (SR) stores or out across the plasma membrane. Long-term vascular [Ca2+]cyt homeostasis, particularly the loading state of the SR stores, is controlled by the activity of ion channels and transporters on the plasma membrane. Among these proteins, plasma membrane Na+/Ca2+ exchange (NCX) has been demonstrated to be critical for controlling [Ca2+]cyt homeostasis (8, 23, 49, 66–68).

Two families of plasma membrane Na+/Ca2+ exchange proteins have been described in mammalian tissues, one in which Ca2+ movement is dependent only on sodium (NCX family, including NCX1–3) and one in which Ca2+ movement is also dependent on potassium (NCKX family, including NCKX1–6) (8, 12, 32, 39, 41, 59). Of note, both NCX and NCKX exchangers can operate either in a forward (Ca2+ exit) mode or in a reverse (Ca2+ entry) mode, depending on the Na+, Ca2+ (and K+) gradients and the potential across the plasma membrane (8, 41). NCX1 is abundantly expressed in heart, brain, kidney, and smooth muscle (45). However, NCX2 and NCX3 are expressed only in brain and skeletal muscle (40, 46). NCKX1 is expressed only in retinal rod photoreceptors, whereas NCKX2 expression is restricted to brain neurons and cone photoreceptors (41). NCKX3 and NCKX4 are expressed not only in brain but also in many other tissues, including aorta, uterus, and intestine, which are rich in smooth muscle cells (SMC) (12, 32, 39). NCKX5 has recently been demonstrated to be expressed in skin and retinal pigmented epithelium, where it is thought to present on the melanosome membrane and not the plasma membrane (33). NCKX6 has also been characterized; however, the physiological function of this protein remains controversial (12, 48). Studies examining the physiological role(s) for NCKX proteins have been restricted to NCKX1 in retinal rod photoreceptors and some recent reports describing NCKX function in brain, spermatozoa, mast cells, and platelet (1, 28–31, 37, 56, 58). Indeed, the latter studies could not discriminate among NCKX isoforms, and thus the function of NCKX3 and NCKX4 proteins has only been determined in recombinant systems (32, 39) and never in an endogenous setting.

Many functional studies have demonstrated the involvement of plasma membrane Na+/Ca2+ exchange in Ca2+ homeostasis of blood vessels (3, 4, 8, 50). The evidence for plasma membrane Na+/Ca2+ exchange in vessels is also supported by direct demonstration that NCX mRNA and protein are expressed in vascular smooth muscle cells (VSMC) and endothelial cells (22, 55). Immunocytochemical studies revealed that the exchanger in gastric SMC and VSMC appears to be restricted primarily to plasma membrane regions that are adjacent to junctional SR (22, 42), implying that a major role of the exchanger in smooth muscles is to modulate, indirectly, the Ca2+ content of SR stores and thereby influence Ca2+ signaling and tension development. Recently, increasing evidence has suggested that NCX can operate in reverse mode to bring extracellular Ca2+ into cells under physiological and pathological conditions and may play an important role in controlling Ca2+ homeostasis and vascular tone (2, 8, 20, 67, 68).

The studies discussed above that demonstrated a role for Na+/Ca2+ exchange in the control of smooth muscle Ca2+ homeostasis were performed using conditions that could not distinguish NCX from NCKX function. Thus, although NCKX3 and NCKX4 mRNA have been demonstrated to be expressed in rat smooth muscle (12, 32, 39, 41), nothing is known regarding their role in controlling vascular Ca2+ homeostasis or agonist-induced vascular contractility. Therefore, we used physiological and biochemical techniques to test whether NCKX family members contribute to [Ca2+]cyt signaling and the regulation of contractility in VSMC. Our results suggest that both NCKX3 and NCKX4 proteins are expressed in arterial smooth muscle and play a novel role in the regulation of vascular Ca2+ homeostasis and contractility. Some of these data have been published previously in abstract form (15).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of vascular tissues. Male Sprague-Dawley rats were killed by cervical dislocation after anesthesia with halothane at ~6 wk of age in accordance with the standards of the Canadian Council on Animal Care and approved by the local Animal Care Committee of the University of Calgary (Calgary, AB, Canada). Thoracic aorta, heart, and small intestine were rapidly removed and placed in ice-cold normal physiological salt solution (PSS) of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 12.5 NaHCO3, and 11.1 dextrose. The pH of the PSS after saturation with 95% O2-5% CO2 gas mixture was 7.4. Adherent connective tissue was removed from aorta, the proximal tail artery (500–800 µm ID), the second-order branches of the left coronary artery (200–250 µm ID), and the second-order branches of mesenteric artery (200–250 µm ID) under a surgical microscope. Endothelial cells were removed from all vessels by repeatedly passing stainless steel cannulas of appropriate size through the vessel lumen, and destruction of the endothelium was confirmed pharmacologically by loss of relaxation in response to acetylcholine (10 µM).

Measurement of vascular tension. Isometric force development was recorded in segments of aortic rings (5 mm) by polygraph and in segments of tail arterial, coronary arterial, and mesenteric arterial rings (all 3 mm) with a wire myograph, as previously described (17) (14). Briefly, two tungsten wires (0.5-mm diameter for aorta, 40-µm diameter for tail artery, and 20-µm diameter for coronary artery and mesenteric artery) were inserted through the lumen of the vessel. One wire was then attached to a force transducer and the other connected to a micrometer. Aorta was placed in a 10-ml organ bath containing gassed PSS. Tail artery, coronary artery, and mesenteric artery were placed in a 5-ml myograph chamber containing gassed PSS. Vascular tissues were routinely allowed to equilibrate for 1 h before the start of the experiments. Isometric tension of aorta was recorded with a force displacement transducer (Grass FT03) coupled to a Grass polygraph (model 7E). Isometric tension of tail artery, coronary artery, and mesenteric artery was recorded at a sample interval of 0.2 s to a hard disk via an analog/digital converter (Axon Instruments, Foster City, CA) and analyzed using computer software (Axotape 2.0, Axon Instruments).

Isolation and culture of arterial myocytes and [Ca2+]cyt measurement. The thoracic aortas and mesenteric arteries isolated from 6-wk-old male Sprague-Dawley rats were placed in cold PSS bubbled with 95% O2-5% CO2. After adherent connective tissue was removed from arteries, they were cut longitudinally, and endothelial cells were also removed by mildly rubbing the internal sides with a cotton swab. The mesenteric arteries were incubated for 40 min (95% air-5% CO2, 37°C) in Hanks’ balanced salt solution containing collagenase IV (2 mg/ml) and elastase III (0.5 mg/ml) (Sigma). The dissociated cells were triturated with a Pasteur pipette and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. Explant tissue culture was used for the preparation of aortic SMC in the absence of enzymatic dissociation. The thoracic aortas were washed three times with fresh PSS and then cut with fine scissors into small pieces of 3 x 4 mm, which were seeded on P-100 plastic dishes (MatTek, Ashland, MA) and cultured in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin G, and 100 mg/ml streptomycin at 37°C in a humidified 5% CO2-95% air atmosphere. After culture for 4–5 days, the small pieces of aortic tissues were removed, and single cells were trypsinized and replated on 10-mm round glass coverslips that had been precoated with 1 mg/ml poly-D-lysine (Sigma). One day later, the cultured VSMC were loaded with 5 µM fluo-3 AM, 0.01% pluronic F-127 (Molecular Probes) in serum-free DMEM medium containing 25 mM Na-HEPES, pH 7.4, for 40–50 min at room temperature (22–24°C) and then washed with DMEM-HEPES solution for 10 min. The coverslips were mounted in a chamber (Warner Instruments) on the stage of a Zeiss Axiovert 135 microscope and perfused with a solution composed of (in mM) 135 NaCl, 10 KCl, 2 CaCl2, 10 HEPES-tetramethylammonium, and 10 D-glucose. Ca2+ entry mediated by reverse mode Na+/Ca2+ exchange activity was then initiated with a switch to a perfusion solution in which the NaCl was replaced with LiCl. Fluo-3 fluorescent intensity in a single VSMC was captured with a x20 Fluar objective and a Chroma filter set using the ImageMaster System and DeltaRAM rapid wavelength-switching illuminator and photometer from Photon Technology International.

RNA extraction and RT-PCR. Total RNA was isolated by guanidinium isothiocyanate extraction and CsCl centrifugation from various tissues of Sprague-Dawley rats, at 6 or 20 wk of age as described previously (39). Total brain RNA isolated from about 10-wk-old rats was used as a positive control.

Expression of NCX1, NCKX3, and NCKX4 mRNA was examined by using reverse transcription-coupled PCR (RT-PCR). Five micrograms of total RNA was reverse transcribed by Superscript II reverse transcriptase (Invitrogen) using an oligo-(dT) primer, and the cDNAs were amplified with the use of the High Fidelity PCR System from Roche Applied Science (Laval, QC, Canada) with specific pairs of primers used at 0.25 µM. Alternatively, RT-PCR was conducted with the SuperScript One-Step RT-PCR System (Invitrogen). Control amplification reactions were performed in the absence of reverse transcriptase. The primer pair for NCX1 was 5'-GCGATTGCTTGTCTCGGGTC-3' (sense) and 5'-CCACAGGTGTCCTCAAAGTCC-3' (antisense), which yield an amplified product of 231 bp. The primer pair for NCKX3 was 5'-GGTCGTGGCTCTTTCTTCCTG-3' (sense) and 5'-ATGTTCCCAGCACCTTTCGTC-3' (antisense), which yield an amplified product of 213 bp. The primer pair for NCKX4 was 5'-GGTGTGGCTGGTGACTATTATTG-3' (sense) and 5'-CGTTGCTTCCGATGGTGTTAGAG-3' (antisense), which yield an amplified product of 176 bp. Products were visualized on 1% agarose gels, gel purified and sequenced with the Amplitaq FS kit from Perkin Elmer. Fluorescently labeled sequencing reactions were analyzed at the core DNA facility of the University of Calgary. Nucleic acid sequence analysis was performed with the MacVector software package (Accelrys) and by connection to the National Center for Biotechnology Information at National Institutes of Health.

Northern blot analysis. Ten-microgram samples of RNA, isolated from rat aorta by the guanidinium isothiocyanate/CsCl centrifugation method, were separated on 1% agarose-formaldehyde gels and transferred to nylon membranes by capillary diffusion overnight. The ultraviolet cross-linked membranes were hybridized at high stringency with a digoxigenin-labeled antisense riboprobes according to the instructions of the manufacturer (Roche Applied Science) as described previously (64). The probe for NCKX3 corresponded to nucleotides 259–1846, whereas that for NCKX4 corresponded to nucleotides 680–1398 of the respective mouse clones.

Vascular smooth muscle extraction and immunoblotting analysis. Postnuclear homogenates of vascular smooth muscles isolated from 6-wk-old rats were prepared as previously described with minor modifications (63). Briefly, frozen muscles (50–150 mg) were pulverized under liquid nitrogen in a mortar. The powder was weighed and homogenized by hand with a glass Dounce homogenizer in 1 ml of 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (pH 7.5) containing 250 mM sucrose, 0.2% NaN3, and 0.1 mM phenylmethysulfonyl fluoride (PMSF). The extract was further homogenized after the addition of 1 ml of 5 mM sodium phosphate buffer, pH 7.4 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 0.1 mM PMSF, and 100 U/ml aprotinin and subsequently incubated on ice for 20 min. After microcentrifugation at 10,000 g for 15 min, the supernatant was stored frozen at –80°C until assay.

Equal amounts of protein extract were combined with 2x Laemmli sample buffer and boiled for 5 min. Proteins were separated by electrophoresis on 7.5% SDS-PAGE and transblotted to nitrocellulose membranes. The protein-bound nitrocellulose sheets were first incubated overnight at 4°C in blocking buffer containing 5% nonfat dry milk in PBS. Nitrocellulose sheets were incubated with the NCX1 monoclonal antibody R3F1 (1:5,000), NCKX3 anti-peptide polyclonal antibody (1:2,000), or NCKX4 anti-peptide polyclonal antibody (1:5,000) diluted in blocking buffer for 1 h at room temperature and then rinsed for 1 h with a wash buffer containing 20 mM Tris, pH 7.5, 500 mM NaCl, and 0.2% Tween 20. Peptide preabsorption was conducted by mixing 1 µg of peptide per microliter of antibody serum together in 200 µl of PBS and incubated for 15 min at room temperature before dilution with blocking buffer and incubation with the membranes. The membranes were then incubated with horseradish peroxidase-conjugated donkey anti-mouse IgG antibody diluted in blocking buffer for 30 min at room temperature and washed for 1 h with agitation, with the wash buffer changed every 15 min. Protein bands were visualized with ECL Plus detection reagents (Pierce Chemical).

Immunocytochemistry. Rat mesenteric arterial SMC were isolated as described above for the culture of arterial myocytes. The cell suspensions were centrifuged at 500 g for 5 min at room temperature. The cells were fixed with 10% formalin in PBS for 15 min, washed with PBS three times for 5 min each, and then permeabilized with 0.5% Triton X-100 in PBS for 5 min. The cells were washed with PBS for 5 min and then with 3% BSA in PBS two times for 5 min each. The cells were incubated with NCX1 monoclonal antibody R3F1 (1:2,000), NCKX3 anti-peptide polyclonal antibody (1:200), or NCKX4 anti-peptide polyclonal antibody (1:500) in 3% BSA in PBS for 1 h at room temperature. The specificity of Na+/Ca2+ exchanger labeling was demonstrated with control experiments using second antibody only (in the case of the anti-NCX1 antibody R3F1) or using peptide preabsorption (as described above, except that 1 µg peptide and 1 µl of serum were incubated together for 15 min in 200 µl of 3% BSA in PBS, and then used directly) for the polyclonal antibodies against NCKX3 or NCKX4. Excess antibodies were removed by washing with PBS for 2 x 5 min and 3% BSA in PBS for 5 min. The cells were then incubated with tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgG (diluted 1:1,000), or CY3-conjugated goat anti-rabbit IgG (diluted 1:1,000), respectively, in 3% BSA in PBS for 1 h in the dark at room temperature. After being washed three times for 5 min each with PBS, the cells were mounted in 90% glycerol and examined with the use of a Zeiss Axioskop 2 microscope with a x63 PlanApoChromat (numerical aperture 1.4) oil-immersion objective and photographed with the use of a SPOT RT-slider camera (Diagnostic Imaging). The exposure settings and times used for acquisition of the control experiments (described above) were the same as those used to acquire the corresponding antibody stained images. Experimental and control photographs were then contrast adjusted in parallel using identical settings with Adobe Photoshop CS.

Chemicals. All chemicals were of analytical grade or better and were obtained from Fisher (Nepean, ON, Canada), BDH (Toronto, ON, Canada), or Sigma-Aldrich (St. Louis, MO) unless indicated otherwise. Cell culture reagents were from Life Technologies (Rockville, MD), and other chemicals were of analytical or molecular biology grade or better. Phenylephrine (PE), ouabain, and nifedipine were purchased from Sigma-Aldrich. KB-R7943 was from Tocris (Ellisville, MO). Fluo-3 AM was from Molecular Probes (Eugene, OR). R3F1 monoclonal antibody was from Taconic. Polyclonal anti-peptide antibodies specific for NCKX3 and NCKX4 were prepared at Affinity Bioreagents by using peptides coupled to KLH via a COOH-terminus-introduced cysteine residue. For NCKX3, the peptide NGTGPSSAPDRGVNG corresponding to amino acids 382–396, and for NCKX4, the peptide NPEDPQQNQEQQPPP corresponding to amino acids 366–380 were used both as immunogens and for affinity purification.

Data analysis. All data were expressed as means ± SE. Where appropriate, Student’s t-test for paired or unpaired data and one-way ANOVA were used and considered significant at P < 0.05. In all experiments, n equals the number of cells or arterial rings prepared from at least three rats.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of NCX1, NCKX3, and NCKX4 in vascular smooth muscle. Previously published Northern blotting data have demonstrated that NCX1, NCKX3, and NCKX4 mRNA are all expressed in rat tissues containing smooth muscle, including aorta (41). However, these experiments with whole tissues could not distinguish between expressions in endothelial cells versus SMC in these whole tissues. Therefore, the first aim of this study was to provide evidence at the transcript and protein levels for the expression of NCKX isoforms in vascular smooth muscle.

Specific primers for the various NCX and NCKX exchangers were used to perform RT-PCR on rat aortic smooth muscles. As shown in Fig. 1A, products amplified from NCX1 and NCKX3 transcripts were readily detected in the rat aorta from both 6- and 20-wk-old rats. NCKX4 products were also detected but only at low levels in the rat aorta from 6-wk-old rats and were barely visible in 20-wk-old rats, although this product was readily detected in the rat brain control sample. Analysis of transcript abundance for NCKX3 and NCKX4 by Northern blot (Fig. 1B) was largely consistent with the PCR result, revealing a clear band for NCKX3 but only a weaker signal for NCKX4.


Figure 1
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  		Fig. 1. Expression of K+-independent Na+/Ca2+ exchanger (NCX1) and K+-dependent exchangers (NCKX3 and NCKX4) in rat vascular smooth muscle. NCX1, NCKX3, and NCKX4 transcripts in rat aortic smooth muscle were analyzed by RT-PCR (A) and Northern blot (B). Total RNA was isolated from aortic smooth muscle of young (Y, 6 wk old) or old (O, 20 wk old) rats, and from whole brain of 10-wk-old rats. Samples equivalent to 0.5 µg were analyzed by RT-PCR with specific primer sets, and 10-µg samples were analyzed by Northern blot using specific riboprobes. Ctl indicates a sample of RNA from aorta of young rats processed in the absence of reverse transcriptase enzyme (a negative control). Arrowheads in B indicate sizes of major transcripts for NCKX3 (5.5 kb) and NCKX4 (9.5 and 4.5 kb). Western blot analysis of NCX1, NCKX3, and NCKX4 proteins in vascular smooth muscle is shown in C. Twenty-microgram samples of arterial myocyte homogenates prepared from aorta (Ao) or mesenteric artery (MA) were subjected to immunoblot analysis with the NCX1 monoclonal antibody R3F1 and anti-peptide antibodies against NCKX3 and NCKX4. Peptide competition reveals specific bands (arrowheads) for NCKX3 and NCKX4 (several bands, possibly corresponding to products from alternatively spliced transcripts) present both in aorta and mesenteric artery.

 
Protein lysates from 6-wk-old rat aortic and mesenteric arterial smooth muscle were prepared and were subjected to immunoblotting with an NCX1-specific monoclonal antibody, R3F1, and anti-peptide polyclonal antibodies against NCKX3 and NCKX4. As shown in Fig. 1C, R3F1 recognized a prominent band with molecular mass of 120 kDa, corresponding to previous reports of the native NCX1 protein (38, 53). Two other minor bands with molecular masses of 50–60 kDa, which might be proteolytic fragments of the 120-kDa protein, were also detected by R3F1. The anti-peptide polyclonal antibody against NCKX3 recognized a single band with molecular mass of 64 kDa in both aortic and mesenteric arterial smooth muscle, which was competed out by antigenic peptide preabsorption, and was consistent with the anticipated protein size predicted from the cDNA sequence. The NCKX4 antibody recognized a series of bands of similar size in both in aorta and mesenteric artery. Three of these were specifically competed out by antigenic peptide preabsorption and might correspond to protein products of different, alternatively spliced NCKX4 transcripts.

Freshly isolated arterial myocytes from 6-wk-old rat mesenteric arteries were analyzed by immunofluorescence using the different NCX and NCKX antibodies. As illustrated in Fig. 2, strong staining was evident in the vicinity of the plasma membrane of mesenteric arterial myocytes with both the NCX1-specific monoclonal antibody and the anti-peptide polyclonal antibodies against NCKX3 and NCKX4. Of note, NCX1 displayed a more evident punctate distribution than either NCKX3 or NCKX4. The specificity of labeling was demonstrated by control studies using second antibody only in the case of R3F1 antibody, and with competing peptide preabsorption, for the polyclonal antibodies against NCKX3 or NCKX4, which all failed to label similar structures at a comparable level. Thus our RT-PCR, Northern blotting, immunoblotting blotting, and immunofluorescence data provide strong evidence for the presence of NCX1, NCKX3, and NCKX4 molecules on the plasma membrane of VSMC.


Figure 2
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  		Fig. 2. Immunofluorescent labeling of NCX1, NCKX3, and NCKX4 proteins in rat mesenteric arterial myocytes. Freshly isolated mesenteric arterial myocytes were fixed with 10% formalin and labeled with NCX1 monoclonal antibody R3F1 or with anti-peptide antibodies against NCKX3 and NCKX4, followed by fluorescently conjugated second antibodies. Right-hand column shows differential-interference contrast (DIC) light images of myocytes, whereas left-hand column shows corresponding immunofluorescent images. Negative control images are shown beneath corresponding antibody-stained image [second antibody only is control for anti-NCX1 antibody R3F1; antibodies preabsorbed with antigenic peptide (+ peptide) are the controls for both anti-NCKX3 and anti-NCKX4 antibodies]. Scale bar = 20 µm. Insets: enlarged views of indicated regions. Exposure and contrast settings used for each control fluorescent image were set to match corresponding antibody-stained image. This figure is representative of several dozen myocytes observed from three separate experiments.

 
NCKX can function in reverse mode to transport Ca2+ into aortic VSMC. To test whether NCKX-type exchangers function in VSMC, [Ca2+]cyt changes in single primary cultured aortic SMC were measured with the Ca2+ indicator fluo-3 and the activity of NCKX-type exchangers in reverse (Ca2+ entry) mode was assessed. In previous studies using whole cell patch clamp, we have shown that NCX currents recorded from HEK-293 cells overexpressing recombinant NCX1 were independent of extracellular K+ but strongly inhibited with 10 µM KB-R7943. In contrast, NCKX currents were absolutely dependent on extracellular K+ (13, 16) but insensitive to 10 µM KB-R7943 (data not shown). These data are consistent with the findings from other laboratories (19, 60). Therefore, the contribution of NCX and NCKX activity can be distinguished as KB-R7943 sensitive and K+ insensitive versus KB-R7943 insensitive and K+ sensitive, respectively.

Nifedipine (10 µM), SKF-96365 (10 µM), and ouabain (1 mM) were present throughout the experiments to block voltage-gated Ca2+ channels, store-operated Ca2+ channels, and the Na+ pump, respectively. When the extracellular Na+ was replaced with Li+ in the presence of 10 mM K+ (10 K+, 0 Na+), a large increase in the [Ca2+]cyt signal was observed. However, when both the extracellular Na+ and K+ were replaced with Li+ (0 K+, 0Na+), this [Ca2+]cyt signal was reduced by one-half (Fig. 3A). Conversely, in the presence of 1 µM KB-R7943 to block NCX-type exchangers, no [Ca2+]cyt signal was observed when the extracellular Na+ was replaced with Li+ in the absence of K+ (0 K+, 0 Na+), confirming full inhibition of NCX activity by KB-R7943 (Fig. 3B). After NCX function was inhibited, NCKX function could be identified as an absolute requirement for extracellular K+ to observe an increase in the [Ca2+]cyt signal on Na+ replacement (10 K+, 0 Na+) (Fig. 3B). These data provide direct evidence at the cellular level to support our notion that not only the NCX-type but also the NCKX-type exchangers can function in reverse mode to transport Ca2+ into VSMC.


Figure 3
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  		Fig. 3. NCKX and NCX function is dissected by measuring cytoplasmic free Ca2+ ([Ca2+]cyt) in single aortic smooth muscle cells. A: fluorescent intensity in single smooth muscle cells preloaded with 5 µM fluo-3 AM was measured with a Zeiss Axiovert 135 inverted microscope equipped with a photometer. Function of reverse modes of NCKX and NCX exchangers was measured as an increase in fluorescence when bath solution was switched from one containing 135 mM NaCl, 10 mM KCl (Na) to 135 mM LiCl, 10 mM KCl (10K, 0Na) or 145 mM LiCl (0K, 0Na). B: NCX was blocked by treatment with 1 µM KB-R7943 (KBR), allowing NCKX function to be identified as an absolute requirement for extracellular K+. Nifedipine (10 µM), SKF-96365 (10 µM), and ouabain (1 mM) were present throughout the experiment. Top: illustrative traces. Bottom: summary of experiments. Values are expressed as means ± SE; n = 5–6. **P < 0.01 vs. control.

 
NCKX contributes to PE-induced vasoconstriction in arterial smooth muscle. It has been demonstrated that reverse-mode Na+/Ca2+ exchange activity was involved in PE-induced Ca2+ oscillation and tonic contraction in vessels (34, 35). This Ca2+ flux was attributed to the activity of the NCX1 exchanger known to be present in vascular smooth muscle. However, because these studies measuring Na+/Ca2+ exchange function in vessels included K+ in the perfusion solutions, the observed transport function may have been a sum of NCKX and NCX activity (25, 26, 34, 35). Therefore, in light of our data demonstrating the presence and function of NCKX proteins in VSMC, we tested the hypothesis that both NCKX and NCX contribute to PE-induced vasoconstriction in arterial smooth muscle.

As shown in Fig. 4, the {alpha}1-adrenergic agonist PE at 1 µM induced contraction of endothelium-denuded rat aortic rings that could not be reversed in response to 10 µM acetylcholine. PE-induced vasoconstriction was inhibited ~40% by prior treatment with 10 µM nifedipine. This result is consistent with previous reports (35), suggesting that ~60% of residual vasoconstriction is presumed to be via mechanisms other than Ca2+ influx through voltage-gated Ca2+ channels. In the subsequent experiments, we focused on this nifedipine-resistant arterial constriction. To prevent the involvement of the Na+ pump in the contraction (particularly upon K+ withdrawal), 1 mM ouabain was present throughout these experiments. As shown in Fig. 5, a significant fraction of the nifedipine-resistant, PE-induced constriction of aortic rings could be prevented by removal of extracellular K+. The magnitude of the response was not significantly influenced by the presence of 10 µM KB-R7943, either alone or in combination with K+ withdrawal. These data suggest that, in aortic myocytes, NCKX-type exchangers make a significant contribution to nifedipine-resistant, PE-induced Ca2+ entry and subsequent contraction.


Figure 4
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  		Fig. 4. Partial inhibition by nifedipine (Nif) of phenylephrine (PE)-induced contraction of rat aorta. A: endothelium-denuded aortic rings were mounted in an organ bath and contracted by 1 µM PE as indicated. Ouabain (1 mM) was added at the same time as PE and then remained present throughout the experiment. Acetylcholine (ACh, 10 µM) could not induce vasorelaxation, confirming the complete removal of endothelium. First PE-induced vasoconstriction was considered as control (Con). Aortic ring was then treated with 10 µM nifedipine for 20 min before the second PE-induced vasoconstriction. W, washout with normal physiological salt solution (PSS). B: summary of experiments, expressed as percentage of control constriction. Values are means ± SE; n = 8. **P < 0.01 vs. control.

 

Figure 5
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  		Fig. 5. NCKX contributes to PE-induced contraction of rat aorta. A: aortic rings denuded of endothelium were mounted in an organ bath filled with normal PSS (5 mM K+, 5K) and contracted by 1 µM PE as indicated. Ouabain (1 mM) and nifedipine (10 µM) were added 5 min before the first PE addition and then remained present throughout the experiment. First PE-induced vasoconstriction in 5 mM K+, normal PSS, was considered as control. Ring was subsequently pretreated with 10 µM KB-R7943 for 20 min (5K/KBR, top), K+-free solution (0K, middle), or K+-free solution plus 10 µM KB-R7943 (0K/KBR, bottom), before second PE constriction. In bottom two panels, a third PE-induced vasoconstriction in normal PSS, 5 mM K+ solution was performed to demonstrate reversibility of inhibition induced by treatment with 0K or 0K/KBR. B: summary of experiments, expressed as percentage of control constriction. Values are means ± SE; n = 7–12. **P < 0.01 vs. control.

 
The situation was somewhat different in mesenteric smooth muscle, even though the relative fraction of the nifedipine-resistant contraction was similar to that observed in aorta. A significant fraction of the nifedipine-resistant constriction in mesenteric artery could be inhibited by 10 µM KB-R7943 (Fig. 6), whereas removal of K+ had a negligible effect, particularly in combination with KB-R7943. These results suggest that, in mesenteric arterial myocyte, NCX-type exchangers are predominantly responsible for nifedipine-resistant, PE-induced Ca2+ entry and subsequent contraction.


Figure 6
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  		Fig. 6. NCX contributes to PE-induced contraction of rat mesenteric artery. A: rat mesenteric artery (second order) denuded of endothelium was mounted in a wire myograph filled with normal PSS (5 mM K+, 5K) and contracted by 1 µM PE. Ouabain (1 mM) and nifedipine (10 µM) were added 5 min before first PE addition and then remained present throughout the experiment. First PE-induced vasoconstriction in 5 mM K+ solution was considered as control. Second and third PE-induced vasoconstrictions were performed in samples pretreated with K+-free solution (0K) or in K+-free solution plus 10 µM KB-R7943 (0K/KBR). B: summary of experiments, expressed as percentage of control constriction. Values are means ± SE; n = 5. *P ≤ 0.05, **P < 0.01 vs. control.

 
Contribution of NCX vs. NCKX to PE-induced vasoconstriction in different types of arteries. Because it had been observed that a significantly larger fraction of nifedipine-resistant, PE-induced vasoconstriction was inhibited by KB-R7943 in mesenteric artery than in aorta (compare Fig. 5 with Fig. 6), we systematically compared the vasorelaxation of different types of arteries induced by KB-R7943. As shown in Fig. 7A, KB-R7943 began to induce vasorelaxation in PE-constricted mesenteric artery and coronary artery at 3 µM, with maximal effects seen at 10 µM. However, the threshold for KB-R7943-induced relaxation of PE-induced vasoconstriction of the tail artery and aorta was at 10 µM, with maximal effects seen at 60 and 100 µM, respectively. Fig. 7B illustrates the increase in fractional relaxation induced by 10 µM KB-R7943 with arteries of decreasing diameter at the following order: mesenteric artery = coronary artery > tail artery > aorta. These data suggest that the relative contribution of NCX-mediated Ca2+ entry in response to PE stimulation was different in different types of vessels.


Figure 7
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  		Fig. 7. Different sensitivity to KB-R7943 of different types of arteries. A: different arterial rings, including the second-order branch of rat mesenteric artery (MA), coronary artery (CA), tail artery (TA), and aorta (AO), all denuded of endothelium, were mounted in a wire myograph and contracted by 1 µM PE or U46619 in 2.5 mM Ca2+ normal PSS. When vasoconstriction stabilized, KB-R7943 was added at increasing doses as indicated until full relaxation was reached. Nifedipine (10 µM) and ouabain (1 mM) were present throughout the experiment. B: comparison of vasoconstriction in the presence of 10 µM KB-R7943 in MA, CA, TA, and AO, compared with the initially induced constriction. Values are expressed as means ± SE; n = 6–7. *P < 0.05, **P < 0.01 vs. control.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that NCKX-type exchangers may play an important role in the regulation of vascular function. On the basis of the following observations, we have concluded that both NCKX- and NCX-type exchangers exist and function in vascular smooth muscle in reverse mode to control [Ca2+]cyt homeostasis and regulate vascular tone: 1) after arterial rings were denuded of endothelium, 60% of PE-induced vasoconstriction was resistant to the block of Ca2+ voltage-gated channels by 10 µM nifedipine; 2) a significant fraction of the PE-induced, but nifedipine- and ouabain-resistant, aortic constriction could be prevented by removal of extracellular K+; 3) about one-half of the [Ca2+]cyt signal in single aortic SMC induced by Na+/Ca2+ exchangers operating in reverse mode depended on the presence of extracellular K+; 4) a portion of the [Ca2+]cyt signal was sensitive to KB-R7943, but another portion of the [Ca2+]cyt signal was not; and 5) RT-PCR, Northern blotting, immunoblotting, and immunofluorescence analyses clearly showed that NCX1, NCKX3, and NCKX4 were all expressed in both aortic and mesenteric smooth muscle. In addition, we have found an increase in sensitivity to KB-R7943, a selective inhibitor of the reverse mode NCX, in arteries of decreasing diameter, suggesting that NCX activity might make different contributions to the regulation of vascular tone in different types of vessels.

Several issues need to be considered during functional studies on the role for NCX or NCKX in Ca2+ homeostasis in VSMC. The first consideration is Ca2+ movement via pathways other than Na+/Ca2+ exchangers. The use of 10 µM nifedipine is expected to effectively prevent Ca2+ movement through voltage-gated (including L- and T-type) Ca2+ channels. Furthermore, SKF-96365 should inhibit Ca2+ flux through receptor-operated Ca2+ channels (9, 18, 35). Recent evidence suggests that TRP channels, particularly the TRPC subfamily, play an important role in Ca2+ homeostasis and its regulation in vascular smooth muscle (5). Indeed, TRP channels have been implicated in the mechanism that leads to activation of Na+/Ca2+-exchanger-mediated Ca2+ entry (as discussed below). We do not believe that TRP channels are directly responsible for the Ca2+ flux underlying our observations, however, because neither KB-R7943 nor K+ removal is expected to influence TRP channel activity or activation (5, 52). It is possible that the residual PE-induced contraction observed in the presence of KB-R7943 and the absence of K+ (Figs. 5 and 6) is due to entry via TRP channels.

The second consideration is the use of extracellular K+ omission to define NCKX function. Removing K+ is expected to induce plasma membrane hyperpolarization, which would inhibit the activation of voltage-gated Ca2+ channels and which would also oppose Ca2+ entry via reverse-mode Na+/Ca2+-exchanger activity. We do not believe that the removal of K+ induces a reduction in [Ca2+]cyt, causing relaxation, however, for the following reasons. The use of nifedipine in our experiments rules out an effect via voltage-gated Ca2+ channels. Because we observed a significant effect of K+ removal even when reverse-mode NCX activity was blocked by KB-R7943 (Fig. 5), we think this mechanism unlikely also. It is possible, however, that the small influence of K+ removal observed in mesenteric artery in the absence of KB-R7943 (Fig. 6) may be due to inhibition of NCX-mediated Ca2+ entry, because this effect was not seen when KB-R7943 was present.

A final consideration is the possible induction of intracellular Ca2+ mobilization. We did not try to eliminate intracellular Ca2+ release from SR because it has been considered as an essential component for the operation of Na+/Ca2+ exchangers in reverse mode as discussed below (35, 36, 52). Therefore, we believe that our conditions allow accurate and precise dissection of the NCX vs. NCKX contribution to Ca2+ homeostasis and contraction in arterial smooth muscle.

The first functional evidence for vascular Na+/Ca2+ exchangers was described by Bohr et al. (10) in 1969. Since then, many functional studies have demonstrated that Na+/Ca2+ exchangers are implicated in Ca2+ homeostasis of blood vessels via operating either in a forward (Ca2+ exit) mode or in a reverse (Ca2+ entry) mode (35, 49, 57, 65–68). Extrusion of Ca2+ via the forward mode of Na+/Ca2+ exchangers is thought to dominate over extrusion via the plasma membrane Ca2+ ATPase pump. This is illustrated by data from arteries that are contracted in K+-rich media, in which the SR Ca2+ stores are presumably overloaded due to a large increase in [Ca2+]cyt derived from Ca2+ entry via plasma membrane channels. Under these circumstances, relaxation is markedly slowed by removing extracellular Na+ and greatly accelerated by adding back the Na+ (4). Furthermore, it was found that in the membrane fraction of bovine aortic smooth muscle, Na+/Ca2+ exchangers have 3- to 6-fold higher transporting capacity compared with that of the plasma membrane Ca2+ ATPase pump (54).

Increasing evidence has suggested that not only forward-mode but also reverse-mode Na+/Ca2+ exchangers contribute to Ca2+ homeostasis in blood vessels. Reduction of the Na+ gradient across the plasma membrane, which promotes Ca2+ entry, augmented agonist-evoked contractions and even induced tonic contractions in unstimulated smooth muscles (6, 11). On the basis of their experimental data, Blaustein (2, 7) and van Breemen (35, 43, 44, 61) have recently proposed a working model for Ca2+ entry via reverse mode Na+/Ca2+ exchangers during PE-induced Ca2+ oscillations and tonic contraction in vessels. Briefly, on {alpha}1-adrenergic receptor stimulation, the opening of inositol 1,4,5-trisphosphate receptor channels induces emptying of Ca2+ from the SR store. This leads to opening of the putative store-operated nonselective cationic channel, which is believed to be much more permeable to Na+ than Ca2+. Under physiological conditions, opening of this nonselective cationic channel should result mainly in Na+ influx into the restricted plasma membrane-SR junctional space. This inward cationic current causes membrane depolarization. Both the increase in Na+ and depolarization drive Na+/Ca2+ exchangers into its reverse mode of operation, bringing Ca2+ into the cell. Using NCX1 knockout mice, Wakimoto et al. (62) observed that NCX1 protein content in aorta was decreased by ~50% and tension development of the aortic ring in Na+-free solution was markedly impaired in heterozygous NCX1-deficient mice, also suggesting that reverse mode NCX1 plays a critical role in Na+-dependent Ca2+ handling in the aorta. Recent studies (20) using selective Na+/Ca2+ exchanger inhibitor and genetically engineered mice provide compelling evidence that salt-sensitive hypertension is triggered by Ca2+ entry through NCX1 in arterial smooth muscle. Our results provide further evidence to support Blaustein’s and van Breemen’s proposed model for Ca2+ entry via reverse mode of vascular Na+/Ca2+ exchangers.

Na+/Ca2+ exchanger activity in VSMC has been assumed to be due to NCX1 protein expression. However, it has been also reported that noradrenalin-induced 45Ca2+ uptake in VSMC was dependent on K+ (24, 27). It thus seems likely that {alpha}1-adrenoceptor agonists stimulate or modulate both vascular NCKX and NCX function. This report, to the best of our knowledge, is the first to provide convincing evidence that NCKX proteins are expressed and function in vascular smooth muscle. Our data demonstrate that NCKX, in addition to NCX, working in the reverse mode, plays a role in PE-stimulated vascular contraction via controlling Ca2+ homeostasis in VSMC. NCKX activity in forward mode has been demonstrated to be the principle component of Ca2+ extrusion from brain synaptic preparations (37). Although it would be interesting to determine whether the same were true in VSMC, the resolution of this issue requires the application of techniques beyond the scope of this study.

Interestingly, we have also found that of the contribution of NCKX to PE-induced vasoconstriction was much larger in aorta than in mesenteric artery, although the expression levels of NCKX vs. NCX proteins are similar in both arteries. This result suggests that regulatory factors and/or spatial considerations may modulate the physiological function of these exchangers in different ways in different vessels. It is possible that only a subset of the Na+/Ca2+-exchanger molecules are coupled to the agonist-induced contractile response pathway. In this regard, it may be important to note the discrete punctate distribution of NCX1 in comparison to the more uniform distributions for NCKX3 and NCKX4, evident in Fig. 2. Of further interest, we observed a decrease in the level of mRNA transcripts encoding NCKX4, but not NCKX3 or NCX1, in aorta of increasing age. Whereas this suggests a selective alteration in gene expression, further work will be required to unravel the significance of this observation.

After systematically comparing the vasorelaxation of different types of arteries induced by KB-R7943, we demonstrated an increase in sensitivity to KB-R7943 with decreasing vessel size (Fig. 7), suggesting an increase in the contribution of NCX activity to PE-induced contractility in arteries of decreasing diameter. The reason for this difference between different types/sizes of arteries is unclear at the moment. We have not determined the contribution of NCKX activity to contractility in coronary or tail artery, although the data from aorta and mesenteric artery suggest an inverse relationship between the NCX and NCKX contributions. We suspect that only the fraction of vasorelaxation observed at 10 µM KB-R7943 or lower corresponds to inhibition of NCX1 activity, on the basis of earlier studies on the potency and specificity of this compound (21, 53). Further relaxation at higher doses of KB-R7943 is likely due to nonspecific effects of the drug at these concentrations(47, 51). In our previous study (68), we found that NCX rather than NCKX proteins are functionally expressed in cultured human pulmonary arterial SMC and that Ca2+ entry via the reverse mode NCX contributes to store depletion-mediated increase in [Ca2+]cyt. Taken together, our previous and present findings suggest that NCX vs. NCKX activity might contribute in a tissue-dependent manner to the regulation of vascular tone.

In summary, our data demonstrate that, in addition to NCX1, NCKX-type exchangers encoded by both NCKX3 and NCKX4 molecules exist and control [Ca2+]cyt homeostasis in VSMC, leading to the regulation of vascular tone. Our data represent both the first documented functional role for the NCKX3 and NCKX4 molecules in their endogenous setting and also the first evidence that NCKX-type exchangers make an important contribution to the regulation of Ca2+ homeostasis in vascular smooth muscle. Further studies, beyond the scope of the current paper, are needed to elucidate physiological implications of our findings. This information will also have the potential to illuminate mechanisms involved in vascular disease, such as hypertension and hypoxic vasoconstriction.


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 ABSTRACT
 METHODS
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This work was supported by a grant from American Heart Association (to H. Dong) and by grants from Canadian Institutes for Health Research (to J. Lytton and C. R. Triggle).


   ACKNOWLEDGMENTS
 
We thank Kathy Zhang for technical help during Western blot analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Dong, Division of Gastroenterology, Dept. of Medicine, University Center 303, 9500 Gilman Dr., La Jolla, CA 92093-0063 (e-mail: h2dong{at}ucsd.edu)

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. Section 1734 solely to indicate this fact.


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