The Na+-K+-ATPase (NKA) can affect intracellular Ca2+ concentration regulation via coupling to the Na+-Ca2+ exchanger and may be important in myogenic tone. We previously reported that in mice carrying a transgene for the NKA α2-isoform in smooth muscle (α2sm+), the α2-isoform protein as well as the α1-isoform (not contained in the transgene) increased to similar degrees (2–7-fold). Aortas from α2sm+ mice relaxed faster from a KCl-induced contraction, hypothesized to be related to more rapid Ca2+ clearance. To elucidate the mechanisms underlying this faster relaxation, we therefore measured the expression and distribution of proteins involved in Ca2+ clearance. Na+-Ca2+ exchanger, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and plasma membrane Ca2+-ATPase (PMCA) proteins were all elevated up to approximately fivefold, whereas actin, myosin light chain, and calponin proteins were not changed in smooth muscle from α2sm+ mice. Interestingly, the corresponding Ca2+ clearance mRNA levels were unchanged. Immunocytochemical data indicate that the Ca2+ clearance proteins are distributed similarly in wild-type and α2sm+ aorta cells. In studies measuring relaxation half-times from a KCl-induced contraction in the presence of pharmacological inhibitors of SERCA and PMCA, we estimated that together these proteins were responsible for ∼60–70% of relaxation in aorta. Moreover, the percent contribution of SERCA and PMCA to relaxation rates in α2sm+ aorta was not significantly different from that in wild-type aorta. The coordinate expressions of NKA and Ca2+ clearance proteins without change in the relative contributions of each individual protein to smooth muscle function suggest that NKA may be but one component of a larger functional Ca2+ clearance system.
- sodium-calcium exchanger
- plasma membrane calcium-adenosinetriphosphatase
- sarco(endo)plasmic reticulum calcium-adenosinetriphosphatase
- transgenic mice
the sodium-potassium-ATPase (NKA) is an enzyme that transports three Na+ ions out of the cell in exchange for two K+ ions into the cell with the hydrolysis of an ATP. By maintaining of the Na+ and K+ electrochemical gradients, the Na+ pump underlies numerous Na+-coupled transport processes. One of these Na+-coupled processes is Ca2+ exchange via the Na+-Ca2+ exchanger (NCX). Through this process, alterations in NKA activity can modulate myogenic tone and hence blood pressure by influencing intracellular Ca2+ concentration ([Ca2+]i) (8).
NKA is a heterodimer composed of a catalytic α-subunit and a β-subunit (3, 4, 23). There are four known isoforms of the α-subunit (α1, α2, α3, and α4) (3, 4, 10), two of which are found in smooth muscle [α1 and α2 (39)]. The α1-isoform is widely expressed in nearly all tissues and has a relatively uniform cellular distribution (18, 34, 39). Conversely, the α2-isoform is expressed in a more restricted set of tissue types and has a punctate cellular distribution (34, 39) that is colocalized with NCX in regions where the sarcoplasmic reticulum (SR) is adjacent to the plasma membrane (18, 29). Because of this specific localized distribution, it has been hypothesized that NKA can modulate not only [Ca2+]i but also Ca2+ loading of the SR (8, 29). However, the regulation of this interaction has not been completely elucidated.
Alterations in NKA activity, and specifically the α2-isoform, through either inhibition with cardiac glycosides (5) or by genetic manipulation in mouse models (12, 16) lead to changes in cell Ca2+ homeostasis and, in some cases, an enhanced loading of intracellular Ca2+ stores (5, 12). An explanation for the enhanced store loading is that the elevation in the intracellular Na+ concentration slows the clearance of Ca2+ from the cytosol via NCX and thereby allows the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) to sequester more Ca2+ into stores. The proposed selectivity for the loading of Ca2+ stores via inhibition of the α2-NKA is based on the close apposition of α2-isoform to the subsarcolemma SR. Interestingly, we recently reported that force in response to receptor-mediated stimulation but not to KCl was more sensitive in aorta from the α2-knockout than from wild-type (WT) (39) mice, suggesting that the activation via store release is enhanced in the α2-knockout mouse. From these data we predicted that the addition of the NKA α2-transgene would also cause significant effects leading to altered contractility.
We previously generated mice (34) carrying a transgene for the NKA α2-isoform coupled with the SMP8 smooth muscle specific promoter (α2sm+) (9). These mice display robust increases (2–7-fold) in α2-isoform protein (34). Surprisingly, the α1-isoform (not contained in the transgene) was similarly increased. This raises the possibility that other related proteins, for example, NCX, may also be altered. In the present study, we investigated our hypothesis that the decreased systolic blood pressure in α2sm+ mice observed in our previous study (34) was related to the altered expression of Ca2+ clearance proteins, leading to decreased contractile function in vascular smooth muscle. Our results show that with increased NKA, there is a similar upregulation of NCX, SERCA, and plasma membrane Ca2+-ATPase (PMCA), suggesting that these Ca2+ clearance proteins may be coordinately regulated in smooth muscle.
Mice on an FVB/n background carrying a transgene for the NKA α2-isoform (α2sm+ mice), specifically in smooth muscle using the α-actin smooth muscle specific promoter, SMP8 (9), were developed and identified as previously described (34). The treatment of animals followed experimental protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee. Mice were given normal rodent chow (Harlan Teklad, Madison, WI) and water ad libitum. Only sex-matched littermate pairs, 12–15 wk of age, were used.
Mice were euthanized in a precharged CO2 chamber. The thoracic aorta (from aortic arch to diaphragm, ∼2 mg wet wt) and gastric antrum (posterior portion of stomach, ∼120 mg wet wt) were immediately removed and rinsed with cold physiological saline solution (PSS). All associated perivascular fat and adventitial connective tissue were dissected free from the aorta, and the endothelium was removed. The gastric antrum was dissected from connective tissue and rinsed well of any digestive debris, and the mucosa was removed. Tissues were immediately frozen in liquid N2. The PSS contained (in mM) 118 NaCl, 4.7 KCl, 25 NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 0.026 EDTA, and 11 glucose.
Aortas were homogenized using a dental amalgamator (Caulk, Milford, DE) and antrum with a PowerGen 125 homogenizer (Fisher Scientific, Pittsburgh, PA) in buffer containing 50 mM Tris·HCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 500 mM NaCl, and 10 mM MgCl2. The buffer was supplemented with the following inhibitors used at a dilution of 1:100: phosphatase inhibitor cocktails I and II and protease inhibitor cocktail (Sigma, St. Louis, MO). Crude protein homogenates were assayed to determine the concentration using BSA as a standard (protein assay, Bio-Rad, Hercules, CA). Sufficient homogenate to run each sample in at least duplicate for each Western blot required aorta pooled from at least seven mice and antrum from three.
Western blot analysis.
Protein (20–40 μg) from aorta samples and 2.5–20 μg protein from antrum samples were incubated at 37°C for 30 min in Laemmli buffer supplemented with 5% β-mercaptoethanol. Proteins were separated by SDS-PAGE at 100 V for 3 h in Genemate 4–20% Tris-glycine gels (ISC BioExpress, Kaysville, UT). Proteins were electrotransferred at 220 mA overnight at 4°C to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated in 1% blocking reagent (Roche Diagnostics, Indianapolis, IN) in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 h at room temperature. Membranes were incubated with one of the following antibodies in TBST for 2 h at room temperature: mouse monoclonal anti-PMCA antibody clone 5F10 (recognizes all PMCA isoforms, Affinity Bioreagents, Golden, CO), monoclonal anti-NCX antibody (R3F1, recognizes primarily NCX1, Swant, Bellinzona, Switzerland), monoclonal anti-SERCA2 antibody (Affinity Bioreagents), monoclonal anti-actin C4 antibody (gift from Dr. James Lessard), rabbit polyclonal anti-myosin light chain 2 antibody (Cell Signaling Technology, Danvers, MA), and calponin (Sigma). Peroxidase-conjugated goat anti-mouse (Bio-Rad) or anti-rabbit (Calbiochem, La Jolla, CA) secondary antibodies were used. Blots were developed using an ECL kit (Amersham Biosciences, Piscataway, NJ) and exposed with Blue Basic Autorad Film (ISC BioExpress). Multiple exposures were taken to ensure the linearity of the signal. Protein loading was verified using anti-actin C4 antibody, Ponceau S staining of the actin band, or Coomassie blue staining. The films were scanned and quantitated by densitometry using ImageQuant 5.2 software (Molecular Dynamics, Amersham Biosciences). Transgenic density was normalized to WT mice on the same blot. At least three different pooled protein samples for the antrum and aorta per genotype were used.
Aortic smooth muscle cell isolation.
Aortas were placed in 35-mm culture dishes in PBS supplemented with 0.1% BSA and 6 mg/ml collagenase B. Aortas were minced in this solution and placed in an incubator equilibrated with 5% CO2-95% room air at 37°C for 40 min. The aortas were then triturated in 5 ml PBS and centrifuged at ∼1,000 g for 3 min. The supernatant was discarded, and the cell pellet was resuspended in 300 μl DMEM supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin. The 300-μl cell suspension was plated as 50-μl aliquots onto the center of six glass coverslips. The cells were placed into a 37°C incubator equilibrated with 5% CO2-95% room air for ∼16 h. DMEM (5 ml) was then added to each well, and the cells were fixed between 3 and 6 days after isolation.
The cells on coverslips were fixed with 3% paraformaldehyde, rinsed with 25 mM glycine, and permeabilized with 0.1% Triton X-100 (25). The cells were incubated with the primary antibodies against NCX (R3F1, Swant), PMCA (Research Diagnostics, Flanders, NJ), and/or SERCA2b (Affinity Bioreagents). After incubation with primary antibodies for 2 h at 25°C, the cells were washed three times (10 min each) in PBS to remove unbound primary antibody and then incubated with a secondary anti-rabbit IgG labeled with Texas red (Invitrogen) or anti-mouse IgG labeled with Alexa 530 (Invitrogen) for 45 min at 25°C. Secondary antibody-only controls were run for all combinations. All coverslips were mounted onto glass slides using a 50% glycerol-saline solution containing the anti-bleach agent paraphenylendiamine (0.1%).
For standard wide-field imaging, the slides were mounted onto the stage of an Olympus IX-70 microscope equipped with a ×60 1.4 numerical aperature objective. Illumination was provided by a 100-W Hg lamp, and images were acquired using a liquid-cooled CCD camera (Roper Scientific), equipped with a Kodak CCD array (KAF1401E).
Total RNA was extracted from antral tissue using TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. RNA concentrations and purity were determined spectrophotometrically. RNA (2 μg) isolated from each antrum sample was treated with RQ1 RNase-free DNaseI (Promega, Madison, WI) and then reverse transcribed to cDNA using the SuperScript III first-strand synthesis system (Invitrogen, Carlsbad, CA). mRNA levels of the following genes were measured using previously published primers: NCX1 (41), SERCA2a (42), PMCA (32), and glyceraldehyde-3-phosphate dehydrogenase [GAPDH, (15)]. Reaction conditions were optimized to ensure that PCR product formation was within the exponential phase and not saturated. The following annealing temperatures and number of cycles were thus determined: NCX1, 58°C and 37 cycles; PMCA, 55°C and 37 cycles; SERCA2a, 55°C and 41 cycles; and GAPDH, 55°C for 35 cycles, using a PTC thermocycler from MJ Research (Watertown, MA). The following thermocycling conditions were used for all reactions: 94°C for 5 min, appropriate number of cycles of denaturation at 94°C for 30 s, annealing for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 7 min. PCR products were gel electrophoresed on a 1% agarose gel and visualized with ethidium bromide. Gel images were analyzed by densitometry using ImageQuant 5.2 software (Molecular Dynamics). mRNA expression for each antrum sample was normalized to its GAPDH expression. All samples were run in triplicate for each PCR reaction.
Thoracic aorta was dissected from male littermate mice as described for Tissue isolation21) after the completion of the experiment.2-5% CO2 for pH 7.4 at 37°C. The resting tension of the aorta was adjusted to 30 mN to set an initial length in the range of maximum force generation. All tissues were weighed, and the thickness was calculated as previously described (
To quantitate the rates of relaxation we measured relaxation half-times. Force development was elicited in aorta by raising the bath medium KCl to 50 mM using a KCl concentrate (3M). This concentration was chosen since it elicits maximum developed force (21, 34). To initiate each experiment, three control KCl-induced contraction-relaxation cycles were performed with a contraction of 15 min followed by PSS for 15 min to ensure reproducible isometric forces. The aortas were then incubated in PSS supplemented with 10 μM cyclopiazonic acid (CPA, SERCA inhibitor, Sigma) for 10 min before contraction with KCl. Sodium orthovanadate (10 μM, PMCA inhibitor, Sigma) was introduced for 5 min before the rinsing out of the KCl. In other experiments, 10 μM KB-R7943 (NCX reverse-mode inhibitor, Tocris, Ellisville, MO) was added for the last 10 min of a KCl-induced contraction. After each experimental condition treated with KCl, the KCl was rinsed from the bath with PSS supplemented with the inhibitors that were present in the medium before rinsing (See Fig. 5A for details). When the aortas were incubated with KB-R7943, KCl was rinsed from the bath with Na+-free PSS and the inhibitor KB-R7943 (necessary to inhibit both the forward and reverse modes of NCX). Relaxation half-times were taken as the time (in s) for aorta to relax to 50% of its peak force using Acqknowledge software (BioPac). Na+-free PSS was made by equimolar replacement of NaCl with N-methyl-d-glucamine and NaHCO3 with choline bicarbonate as previously described (24).
For the CPA modification of developed force, the force developed by aortas in response to raising bath medium KCl to 50 mM was measured using Acqknowledge software. KCl was rinsed from the bath with PSS, and aortas were incubated with 10 μM CPA for 10 min before subsequent KCl stimulation. The KCl-induced developed force after CPA administration was then measured.
Calculations for estimates of contribution to relaxation.
To estimate the relative contribution of the Ca2+ transport proteins to the relaxation of force, we made the following assumption. We assumed that the majority of Ca2+ clearance from the intracellular space would be attributable to SERCA, NCX, and PMCA and that Ca2+ clearance by other mechanisms would be negligible. The percent contribution of SERCA + PMCA to the relaxation half-time was calculated using a linear model as follows: 100·[1 − (relaxation half-time in the presence of CPA and vanadate/relaxation half-time in PSS)] (24).
The significance of Western blot density ratios of α2sm+ to WT mice were determined using Fisher exact, rank sign or Student's t-test, as appropriate. Differences between the expression of the various proteins were tested using a one-way analysis of variance (SigmaStat 11.0). The n value is representative of the number of different protein samples used.
For all mRNA, force development, and relaxation half-time data, significant differences were determined using an unpaired Student's t-test. A P value < 0.05 was accepted for assigning statistical significance.
We previously reported that mice expressing a transgene for the NKA α2-isoform in smooth muscle had elevated levels of the α2-isoform mRNA and protein (34). Importantly and surprisingly, there was also a coordinate increase of similar magnitude of the α1-isoform, which was not contained in the transgene. α1- and α2-Isoform proteins were increased six- to sevenfold in gastric antrum and approximately twofold in aorta from α2sm+ relative to WT mice. This differential tissue expression reflects the hierarchy of the α-actin SMP8 promoter that has a greater expression in the stomach than in the aorta (43).
Expression of Ca2+ transport proteins.
Our initial characterization of aorta from the α2sm+ mice indicated that a major phenotypic effect was an increase in the rate of relaxation from contraction. Since NKA is linked to the regulation of intracellular Ca2+ via a functional coupling to the Na+-Ca2+ exchanger (NCX), this was anticipated. However, since the NKA α1-isoform was also increased despite the transgene containing only α2-isoform cDNA, we investigated whether other Ca2+ transport proteins may also have been upregulated. We used Western blot analysis to measure the protein levels of NCX, PMCA, and SERCA in the aorta and antrum from NKA α2sm+ mice relative to paired WT mice. Figure 1A shows that protein levels of NCX, PMCA, and SERCA each increased in the gastric antrum and in aorta from α2sm+ mice relative to WT mice. Summary data indicating a three- to fivefold increase in each Ca2+ transport protein from a minimum of three different α2sm+ samples, where each sample represents either seven to eight pooled aorta or three pooled antrum, are presented in Table 1. This level of increases in these Ca2+ clearance proteins is similar in magnitude to the increases observed for the α-isoforms in α2sm+ mice previously reported (34). In 26 of the 27 total measurements, the density of α2sm+ mice was greater than that of the WT mice. Table 2 shows a significant fourfold increase for both antrum and aorta in the α2sm+-to-WT ratio for the Ca2+ clearance proteins (NCX, PMCA, and SERCA). For contractile-associated proteins (actin, calponin, and myosin light chain-20), changes were small and not statistically significant.
To determine whether there was a general increase in protein expression profiles in smooth muscles in α2sm+ mice relative to paired WT mice, we compared the relative gel densities for the protein levels of aorta and antrum using Coomassie blue-stained SDS-PAGE gels. A typical gel is shown in Fig. 2. With the validation of the visual impression of little differences, the density ratio of α2sm+ to WT protein was 0.97 ± 0.09 and 0.78 ± 0.10 for antrum and aorta (n = 4, each; not significant), respectively.
For a direct comparison to the Western blot data for the Ca2+ clearance proteins, we also probed for contractile-associated proteins actin, calponin, and myosin light chain-20 in the gastric antrum and aorta from Fig. 1B; there were no statistically significant differences in expression (Table 1). As an alternative analysis, we used the Fisher exact test to compare the number of gels with a twofold or greater increase for Ca2+ clearance proteins (14 of 26) to those for contractile-associated proteins (3 of 26); the null hypothesis, i.e., that the data from NKA α2sm+ and paired WT mice were not different, was rejected at the P = 0.003 level. These data further support that an overall upregulation of smooth muscle proteins was unlikely.
Ca2+ transport protein distribution.
A prerequisite for the Ca2+ clearance function is that the upregulated proteins must be similarly distributed as the endogenous protein. We compared the distributions of Ca2+ transport proteins using immunocytochemistry. In Fig. 3, wide-field micrographs show the distribution of NCX, PMCA, and SERCA2 in WT and α2sm+ aortic smooth muscle cells in primary culture. PMCA is distributed throughout the cell and does not appear to concentrate in subcellular structures other than near the nucleus in what appears to be Golgi-like structures. SERCA2 is distributed throughout the cell with areas of significant concentration near the nucleus and at the plasma membrane. Unlike PMCA, NCX and SERCA are clearly distributed with subcellular compartments throughout the cell. Importantly, the distributions of NCX, PMCA, and SERCA are similar in WT and α2sm+ cells. This is comparable with that previously reported for the distributions of α1 NKA- and α2 NKA-subunits in these mice (34).
mRNA levels of Ca2+ transport proteins.
Messenger RNA levels were measured to assess whether the increases in proteins involved in Ca2+ clearance in smooth muscle were due to increased mRNA. Messenger RNA levels of NCX, PMCA, and SERCA normalized to GAPDH expression were not significantly different in NKA α2sm+ antrum relative to WT antrum (Fig. 4). GAPDH levels were also unchanged in NKA α2sm+ antrum.
Contractile function in aorta from α2sm+ and WT mice.
As an initial estimate of the contractile consequences of the increased Ca2+ clearance proteins, we measured the isometric force developed in response to KCl or phenylephrine stimulation, the rate of force development, and the rate of relaxation from an isometric contraction. There were little differences in the former parameters; hence, we concentrated on relaxation, which may be more sensitive to altered Ca2+ clearance. An example of the protocol used for measuring the contributions of the primary players in Ca2+ clearance is shown in Fig. 5A. As previously reported, endothelium-denuded aorta from NKA α2sm+ mice have a half-time of relaxation from a KCl-induced contraction that is significantly decreased (faster relaxation) than a paired WT aorta (Ref. 34, and Fig. 5B).
Since NKA isoforms were increased in α2sm+ smooth muscle, it was of interest to determine whether the increased relaxation was ascribable to an enhanced Ca2+ clearance by NKA-NCX coupling and/or by that attributable to the other major Ca2+ clearance proteins, PMCA and SERCA. When SERCA is inhibited using CPA, and PMCA with sodium orthovanadate (VO4), NCX remains as the sole mechanism of Ca2+ extrusion. Under these conditions, relaxation was significantly slower, the half-times increasing two and a half- to threefold (Fig. 5). After the force declined to ∼50% of the initial value, the relaxation rate dramatically decreased. This may reflect that NCX is a low Ca2+ affinity but a high turnover extrusion pathway versus the high affinity but lower turnover extrusion of Ca2+ exhibited by SERCA and PMCA. There were no significant differences between aorta from α2sm+ and paired WT mice.
We also investigated the effects on relaxation of NCX inhibition, which leaves Ca2+ extrusion by PMCA and sequestration by SERCA as the main mechanisms of Ca2+ clearance (26). An addition of KB-R7943, which blocks the reverse NCX exchange to an aorta contracted with KCl, elicited a 23–27% reduction in the force developed (Fig. 5A). Subsequently, the remaining contraction was relaxed with Na+-free PSS plus KB-R7943, conditions that inhibit both forward and reverse NCX exchange. The half-times of relaxation with NCX inhibited were not different between α2sm+ aorta and paired WT aorta. Moreover, these relaxations were similar to that of the control (Fig. 5B), suggesting that either NCX is not a major player in these aortic relaxations or that SERCA and PMCA activities can compensate for the inhibition of NCX. The ratios of the relaxation half-time from a KCl contracture to that in the presence of CPA and vanadate were similar for WT and α2sm+ aortas, 0.38 and 0.32, respectively. Thus, when SERCA and PMCA are inhibited, NCX and any other Ca2+ clearance activity, for example, mitochondrial, would account for 30–40% of the relaxation rate, using a linear model (24).
Effects of CPA on developed force.
Since SERCA protein levels were elevated in aorta from α2sm+ mice, intracellular Ca2+ store levels could be altered, thereby affecting contractile function. The inhibition of SERCA by CPA elicits a transient increase in force because of the leakage of Ca2+ from the SR but blunted by Ca2+ clearance. This transient increase was ∼5% of the KCl maximum force, and no statistical difference between α2sm+ and WT mice was observed (Table 3). Following the pretreatment with 10 μM CPA, aortas were stimulated by the addition of 50 mM KCl to assess the effects of the loss of SR Ca2+ uptake and enhanced capacitive Ca2+ influx (CCI) on force development. After an incubation with CPA, the KCl-induced force development was enhanced compared with KCl alone, averaging ∼17% (n = 8) for both α2sm+ and WT mice. Neither the force developed in response to KCl alone nor the increase with KCl after CPA was different between α2sm+ and WT mice.
To assess the effects of CCI, aortas were pretreated for 15 min in Ca2+-free PSS plus CPA to deplete SR Ca2+ stores and to activate store-operated Ca2+ channels. We measured the force response to the readdition of [Ca2+]i. As shown in Table 3, there were no significant differences in these CCI-associated increases in force.
We previously reported that our α2sm+ transgenic mice display a coordinate increase in the NKA α1-isoform, which was not contained in the transgene (total NKA increase 2–7-fold) (34). Cyclic stretch of cultured arterial smooth muscle cells led to an increase in mRNA for both α1- and α2-NKA isoforms, also suggesting some form of coordinated expression (38). We show in this investigation that these mice in addition to increased NKA have corresponding increased levels of the Ca2+ clearance proteins NCX, PMCA, and SERCA in smooth muscle but not contractile proteins actin, myosin light chains, and calponin. Moreover, immunohistochemical data indicate that the Ca2+ clearance proteins expressed in the α2sm+ aorta are distributed similarly to those in the WT.
The NKA is a component of intracellular Ca2+ homeostasis via coupling with NCX in the plasma membrane. NKA and NCX are known to be colocalized (18, 26, 29) and coupled functionally (29). Therefore, an alteration in NKA activity or a change in the number of pumps at the plasma membrane can influence the Na+-Ca2+ exchange rate and/or the direction of NCX function (i.e., whether Ca2+ enters the cell or is extruded) by altering the resting membrane potential and the Na+ gradient. In the case of elevated NKA protein expression, the driving force favors Na+ entry, thus promoting Ca2+ extrusion via NCX and the lowering of intracellular Ca2+ and consequently SR Ca2+ stores. Based on published studies of changes in expression of both cardiac NKA and NCX in animal (27) and human (11, 27, 30) models of disease, we expected that NCX expression would be changed in our α2sm+ transgenic mice.
One prediction is that to compensate for an altered driving force of Na+ and a consequent change in Ca2+ clearance, NCX turnover or the number of NCX transporters may be inversely related to that of NKA. Such a reciprocal relationship has been observed in cardiac muscle in which a reduction of the Na+ pumps lead to an increased expression of NCX during disease states (27). In contrast, studies in diaphragm from α2-isoform gene-ablated mice showed a parallel decrease in the NKA α2-isoform and NCX (35). This would be consistent with our model in which NKA and NCX are rigorously coupled. Perhaps a parallel versus a reciprocal regulation of NKA and NCX is dependent on the tissue function and type.
Specific roles for each α-isoform in the regulation of [Ca2+]i and SR Ca2+ loading have also been proposed (8, 10, 16, 39). Thus changes in total NKA activity might induce alterations in the activity of Ca2+ transport proteins such as NCX, PMCA, or SERCA or their protein expression levels as a long-term compensation because of altered [Ca2+]i in aorta from α2sm+ mice. The apparent coordinate increase in NKA isoforms and Ca2+ clearance proteins in α2sm+ mice may be indicative of some type of functional unit for Ca2+ homeostasis.
At first, it may appear somewhat nonintuitive that increased NKA and likely increased Ca2+ clearance via NCX would lead to the upregulation of other Ca2+ clearance proteins. Our hypothesis is that an increased α2-isoform would lead to an increased Ca2+ clearance in its proposed subsarcolemmal location, which in turn would result in a lower SR Ca2+ load. Decreasing the SR Ca2+ load would likely increase the Ca2+ influx via CCI channels (5, 26). Thus upregulation of Ca2+ clearance proteins could be a compensatory response to this increased CCI.
There are data in disease states for the coordinate expression of the Ca2+ transport proteins in cardiac muscle (11, 13, 27, 33) and, though limited, in smooth muscle (11, 13, 28, 45). Our mice, in which the NKA α-isoform protein is elevated specifically in smooth muscle, allow us to directly examine how an increase in the amount of NKA protein affects the levels of Ca2+ clearance proteins in smooth muscle. In a perinatal diaphragm from α2+/− and α2−/− mice (35), the expression of SERCA or PMCA proteins were altered and accompanied by an upregulation of the α1-isoform of 147–194% in α2+/− and α2−/− mice, respectively (35). Thus data from α2-isoform gene-ablated mice as well as our NKA α2sm+ mice support the hypothesis that a change in the levels of NKA protein can influence the levels of NCX, SERCA, and PMCA proteins, all of which are important in Ca2+ clearance and [Ca2+]i. Altered [Ca2+]i may be part of such a coordinate signaling pathway, since intracellular Ca2+ can be a mitogenic signal (19, 37, 40). Since we found no changes in actin or myosin light chain protein levels, the changes appear to be relatively specific to Ca2+ homeostasis as there was no generalized smooth muscle remodeling.
At this point, we have no information on the mechanism(s) underlying the increased NCX, PMCA, and SERCA protein without major changes in mRNA, though this is not unusual. There are a number of examples of coordinated Ca2+ handling mechanisms (22), particularly those with developmentally imposed changes (1). In addition there are a number of proposed systems for which [Ca2+]i is postulated to play a major role in smooth muscle signaling (17). These can either stabilize proteins by reducing degradation (44) or enhancing production, often by modulating initiation (20). Further experimentation is needed to minimally prove this apparent unit for a coordinate expression of Ca2+ clearance proteins, and more to uncover mechanisms.
Given the increase in Ca2+ clearance proteins in α2sm+ aorta, it was of interest to assess the effects on contractility. Our hypothesis was that the lower blood pressure would be reflected in a decreased vascular smooth muscle contractility. To this end we measured the force developed and the rates, based on half-times, of force development and relaxation, mechanical processes dependent on [Ca2+]i.. We previously published that relaxation half-times from a KCl-induced contraction were faster in aorta from α2sm+ mice compared with WT mice, suggesting that increased NKA could possibly result in enhanced Ca2+ clearance (34). To identify the Ca2+ clearance pathways that may be responsible for this faster relaxation in the NKA transgenic mice, we also measured the relaxation half-times under inhibitory conditions for proteins important in Ca2+ clearance.
When the low-capacity, high-affinity transporters, SERCA and PMCA, were inhibited, aortas relaxed to ∼50% of the initial force and about one third the control rate. The rate then dramatically decreases and, in some cases, the aortas were unable to relax completely to the original baseline. This relaxation is primarily determined by the activity of NCX, a protein with a low affinity for Ca2+ (Kd ≈ 1 μM) but high turnover (5–7, 14). Inhibition of NCX alone did not significantly affect relaxation. Based on relaxation half-times measured in the presence of inhibitors of SERCA and PMCA, we calculated that SERCA and PMCA contributed ∼60–70% of the rate of Ca2+ clearance. Surprisingly, there were little differences in the relative contributions of these transporters to the relaxation rate between aortas from α2sm+ and WT mice.
With the exception of the relaxation rates, contractility was only very moderately altered in α2sm+ aorta compared with the increases in Ca2+ clearance protein expression. There could be several explanations why there were only moderate differences in relaxation half-time. First, all of the expressed protein is not properly targeted and therefore not functional. Our imaging data indicate that no substantial changes in the distributions of Ca2+ clearance proteins occur, supporting our contention that they are functional. Moreover, the percent contributions of SERCA and PMCA for Ca2+ clearance in smooth muscle from α2sm+ and WT mice were similar, suggesting that the proportions of the Ca2+ extrusion pathways were maintained in mice with increased NKA. This is consistent with our Western blot data which show that NKA α-isoforms as well as NCX, SERCA, and PMCA are similarly increased in NKA α2sm+ mice. We would anticipate that this would not be the case if a large fraction of the expressed Ca2+ clearance proteins was not functional. Second, myosin light chain phosphorylation/dephosphorylation or mechanical dissociation of myosin and actin may be the rate-dependent step for relaxation rather than Ca2+ (2, 31), thereby diluting any relation between [Ca2+]i and muscle contractility. Third, [Ca2+]i is dependent not only on the rate of Ca2+ clearance but also on Ca2+ entry pathways, which may be elevated (e.g., L-type Ca2+ channels, store-operated Ca2+ channels) with changes in NKA (26, 36).
The moderate changes in contractility may, in fact, be a consequence and/or the design principle behind the coordinate upregulation of expression of Ca2+ clearance proteins. If Ca2+ entry mechanisms were increased, the coordinate expression would lead to the maintenance of a similar regulation of [Ca2+]i and contractility. Our data suggest the existence of a functional Ca2+ clearance system containing at least NKA, NCX, SERCA, and PMCA. The bases for this coordinate expression remain to be discovered.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-66044 (to R. J. Paul and R. M. Lynch).
No conflicts of interest, financial or otherwise, are declared by the author(s).
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