Na+ pump {alpha}2-isoform specifically couples to contractility in vascular smooth muscle: evidence from gene-targeted neonatal mice

Daniel A. Shelly,1 Suiwen He,2 Amy Moseley,2 Craig Weber,3 Michelle Stegemeyer,1 Ronald M. Lynch,3 Jerry Lingrel,2 and Richard J. Paul1

Departments of 1Molecular and Cellular Physiology and 2Molecular Genetics, Biochemistry, and Microbiology, College of Medicine, University of Cincinnati, Ohio 45267-0576; and 3Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724-5017

Submitted 9 September 2003 ; accepted in final form 15 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The relative expression of {alpha}1 - and {alpha}2-Na+/K+-ATPase isoforms found in vascular smooth muscle is developmentally regulated and under hormonal and neurogenic control. The physiological roles of these isoforms in vascular function are not known. It has been postulated that the {alpha}1-isoform serves a "housekeeping" role, whereas the {alpha}2-isoform localizes to a subsarcolemmal compartment and modulates contractility. To test this hypothesis, isoform-specific gene-targeted mice in which the mRNA for either the {alpha}1- or the {alpha}2-Na+/K+-ATPase isoform was ablated were utilized. Both of these knockouts, and , are lethal; the latter dies at birth, which allows this neonatal aorta to be studied. Isometric force in -aorta was more sensitive to contractile agonists and less sensitive to the vasodilators forskolin and sodium nitroprusside (SNP) than wild-type (WT) aorta; -aortas had intermediate values. In contrast, neonatal -aorta was similar to WT. Western blot analysis indicated a population of 70% {alpha}1- and 30% {alpha}2-isoforms in the WT. Thus in terms of the total Na+/K+-ATPase protein, the -aorta (at 70%) would be similar to the -aorta (at 65%) but with a dramatically different phenotype. These data suggest that individual {alpha}-isoforms of the Na+/K+-ATPase differ functionally and that the {alpha}2-isoform couples more strongly to activation-relaxation pathways. Three-dimensional image-acquisition and deconvolution analyses suggest that the {alpha}2-isoform is distributed differently than the {alpha}1-isoform. Importantly, these isoforms do not localize to the same regions.

sodium; potassium; ATPase; contraction; transgenic


THE NA+ PUMP HAS LONG BEEN hypothesized to be a major player in {beta}-adrenergic relaxation of vascular smooth muscle (49, 50). The Na+ pump is a transport ATPase of the plasma membrane, which maintains low cytosolic Na+ and high K+ concentrations in all animal cells. The Na+/K+-ATPase is a heteromeric enzyme comprised of a 112-kDa {alpha}-subunit and a 53-kDa glycosylated {beta}-subunit (28). The {alpha}-subunit is the catalytic component of the holoenzyme and contains both the cation and the nucleotide-binding sites (28). The link to modulation of smooth muscle contractility was postulated to be Ca2+ extrusion via the Na+/Ca2+ exchanger that is attributable to a lowered intracellular Na+ concentration with activation of the Na+ pump. Additional evidence was provided by ultrastructural studies that showed colocalization of the Na+ pump and the Na+/Ca2+ exchanger (35). The role of the Na+ pump in Ca2+ homeostasis was extended to include modulation of the filling state of the sarcoplasmic reticulum (SR), which also occurs via linkage to the Na+/Ca2+ exchanger (35, 35) in a subsarcolemmal compartment.

Such a functional compartment involving the Na+ pump has also been proposed based on studies of the coupling of Na+-pump activity and glycolysis. Hoffman and colleagues (42, 47) proposed a membrane compartment with ATP preferentially used by the Na+/K+-ATPase of human red cell ghosts. We showed in vascular smooth muscle that oxidative metabolism is coupled to actin-myosin ATPase activity, but the ATP usage of the Na+ pump is uniquely coupled to aerobic glycolysis (33, 43, 44). This compartmentalization of energy metabolism with function has been demonstrated for a wide variety of cell types (18, 30, 45) including skeletal muscle (19). We postulated that this is due to a glycolytic enzyme cascade associated with the plasma membrane and localized near the Na+ pump (13) or preferential access of glycolytic enzymes to the subsarcolemmal space (32).

The nature and functional significance of the compartmentalization of the Na+ pump is further complicated by the existence of multiple Na+/K+-ATPase isoforms of the {alpha}- and {beta}-subunits (9, 10, 22, 54). The {beta}-subunit is glycosylated and may be involved in assembly of the holoenzyme and targeting to the plasma membrane. The {alpha}-subunit is the catalytic subunit and the site for inhibition by cardiac glycosides. Four {alpha}-subunit isoforms have been identified ({alpha}1, {alpha}2, {alpha}3, and {alpha}4), and each has a unique tissue distribution (29, 51, 52, 54). The existence of multiple {alpha}-subunits with different expression patterns (2) that can be developmentally regulated (41) suggests specialized functional roles for these isoforms. In rodents, {alpha}-isoforms have marked differences in affinities for ouabain (21), kinetics for ion transport, and sensitivity to Ca2+ (21, 54). The physiological significance of these isoforms is less well understood (10, 46). The widespread distribution of the {alpha}1-isoform suggests that it subserves generalized cellular Na+ and K+ homeostasis or has what is often called a "housekeeping" role. Owing to their tissue-specific localization and developmental changes, the other {alpha}-isoforms are likely to play more specific roles.

A possible basis for Na+/K+-ATPase isoform-specific function in vascular smooth muscle was put forth by Juhaszova and Blaustein (23) on the basis of immunohistological evidence. They reported that the {alpha}1-isoform was ubiquitously distributed over the surface of the cells. In contrast, the isoform with the high ouabain affinity was confined to a reticular distribution within the plasma membrane that paralleled the underlying sarcoplasmic reticulum (24). In view of this differential localization, these investigators suggested that the {alpha}1-isoform is the one responsible for maintaining a low "bulk" cytosolic Na+ concentration. The high ouabain affinity isoform(s) was postulated to have a major role in modulating Ca2+ in a subsarcolemmal compartment via the Na+/Ca2+ exchanger. This in turn was postulated to modulate the filling of the SR and thereby influence numerous cell processes that depend on mobilization of stored Ca2+ (24). This type of functional compartmentalization of {alpha}-isoforms has recently received strong support from studies by the Lingrel laboratory on cardiac (20) and skeletal (15) muscle from mice in which one copy of the {alpha}1- or {alpha}2-gene was ablated. It was shown that genetic reduction of the {alpha}1-isoform led to hypocontractile muscle, whereas reduction of the {alpha}2-isoform resulted in a hypercontractile state. These results illustrated a potential specific role for the {alpha}2-Na+/K+-ATPase isoform in Ca2+ signaling during striated muscle contraction.

The relative expression of the {alpha}-isoforms of Na+/K+-ATPase can be altered by hormonal and neurogenic mechanisms (1, 9, 17, 25, 40, 53). Importantly, in disease states such as hypertension and diabetes, the distribution of the {alpha}-isoforms is also known to be altered (11, 16, 34). However, the physiological significance of the different Na+/K+-ATPase isoforms in smooth muscle remains uncertain.

In the present study, we utilized {alpha}-isoform-specific knockout mice to study the functional significance of these Na+-pump isoforms to vascular function. The -mice are not born; however, the -mice are born but die within minutes of birth. We developed apparatus to measure contractility in neonatal mouse aorta, thereby making it possible to assess function in the absence of the {alpha}2-isoform. This is in contrast to previous studies on heterozygotes in which the {alpha}2-isoform was only reduced. Importantly, although the reduction in the total Na+/K+-ATPase protein in the -aorta is of similar magnitude to that of the -aorta, there were striking functional differences. These data suggest that the Na+-pump {alpha}2-isoform may be preferentially linked to excitation-contraction coupling in vascular smooth muscle.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and tissue preparation. All work described herein complies with the protocol approved by the University of Cincinnati Institutional Animal Care and Use Committee. The targeted mice used in these studies were previously described (20). For the present study, neonatal wild-type (WT) and heterozygous -mice were derived from five litters, and WT, -, and homozygous -mice were derived from six litters. Neonatal mice were weighed and then killed by decapitation within minutes of birth. Thoracic aorta (5–6 mm) was dissected and rinsed in cold bicarbonate-buffered physiological saline solution (PSS), and loose fat and connective tissue were removed. The PSS contained (in mM) 118 NaCl, 4.73 KCl, 1.2 MgCl2, 0.026 EDTA, 1.2 KH2PO4, 2.5 CaCl2, and 5.5 glucose and was buffered with 25 Na2HCO3; pH, when bubbled with 95% O2-5% CO2, was 7.4 at 37°C.

Contractility measurements. The aortic rings were threaded between two triangular holders made from 100-µm stainless steel wire. The rings were gently rotated on the wires to remove the endothelium. A lack of response to 10–5 M ACh was used to functionally test for the presence of endothelium. The aortas and holders were then attached between a fixed post and a Harvard apparatus differential capacitor force transducer for isometric force measurements. Resting tension on each aorta was set to 2 mN to approximate the tension calculated for an in vivo aortic pressure of 100 mmHg, and this passive tension was maintained throughout the experiment. Data were acquired using BioPac instrumentation and analyzed using the accompanying AcqKnowledge software. Forces were normalized to the length of the aorta. The aortas were challenged with 0.1 µM U-46619 or 50 mM KCl and at least two contraction-relaxation cycles were performed to insure reproducible forces. Cumulative concentration-isometric relations to U-46619 were undertaken with additions when steady-state forces were achieved, which was generally between 5 and 10 min. Cumulative concentration-relaxation relations to forskolin or sodium nitroprusside (SNP) were similarly undertaken using the concentration of U-46619 required to achieve 80% of the maximum isometric force (ED80).

Western blot analysis. Aortas were dissected from adult and neonatal mice and frozen in liquid N2. Na+/K+-ATPase protein extracts were prepared by homogenizing 4–6 aortas in glass homogenizers suspended in a solution that contained (in mM) 1.5 NaCl, 5 EDTA, 20 sodium metabisulfite, 0.3 PMSF, 60 imidazole, and 0.1% Triton X-100. Protein concentration was measured according to a standard Bradford assay (Bio-Rad). Proteins were separated essentially as previously described (26). Samples were incubated for 30 min at 37°C in 50 mM Tris·HCl (pH 6.9), 5% SDS, 1% {beta}-mercaptoethanol, and 10% glycerol and were then electrophoresed through 10% polyacrylamide gels. The separated proteins were transferred overnight to PVDF membranes (Amersham Life Sciences) and blocked with 5% nonfat dry milk in TBST [5 mM Tris·HCl (pH 7.4), 150 mM NaCl, and 0.01% Tween 20] for 1 h at room temperature. The blots were incubated in TBST containing {alpha}1-isoform-specific monoclonal {alpha}6f (University of Iowa Developmental Hybridoma Bank), {alpha}2-isoform-specific monoclonal McB2 (generously provided by Dr. Kathleen Sweadner), or {alpha}3-isoform-specific monoclonal XVIF9-G10 (Affinity Bioreagents) for 1 h at room temperature. Immunoreactivity was visualized after incubation with peroxidase-conjugated anti-mouse secondary antibody (Calbiochem) using Kodak BioMax MR X-ray film and the enhanced chemiluminescence (ECL) system (Amersham Life Sciences) following the manufacturer's recommendations. For quantitative analysis, the signals were quantified by densitometry using ImageQuant software.

Cell isolation and culture. Neonatal mice (<24 h old) of C57Blk6/129SV background were decapitated, and the aortas were removed. Aortas from two neonates were digested in PBS that contained 6 mg/ml each of collagenase B and BSA for 40 min. The cells from the digested aortas were diluted with PBS and then centrifuged at ~1,000 g for 3 min. The cell pellet was suspended in DMEM supplemented with 5% FBS and 1% penicillin-streptomycin and plated onto six coverslips coated with poly-L-lysine. The cells were then placed into a 37°C incubator equilibrated with 5% CO2 and allowed to attach and spread. Cells were used within 24 h. The PBS contained (in mM) 137 NaCl, 2.7 KCl, 8.0 Na2PO4, and 1.5 KH2PO4, pH 7.3.

Immunocytochemistry. The cells on coverslips were fixed with 2% paraformaldehyde, rinsed with 50 mM glycine, and permeabilized with 0.1% Triton X-100 (31). The cells were incubated with a rabbit polyclonal antibody against the Na+/K+-ATPase {alpha}2-isoform (a gift of Dr. Tom Pressley, Texas Tech Univ.) and a mouse monoclonal antibody against the Na+/K+-ATPase {alpha}1-isoform (Upstate Biotechnology) for 1 h at 25°C. Cells were washed three times (5 min each) in PBS to remove unbound primary antibody, and then the washed coverslips were incubated with a secondary anti-rabbit IgG labeled with FITC (American Qualex) and anti-mouse IgG labeled with Cy5 (Jackson Immunoresearch Laboratories) for 45 min at 25°C. Coverslips were mounted onto glass slides using a 50% glycerol-saline solution that contained paraphenylenediamine (0.1%) to retard fading.

Imaging. Slides were mounted on the stage of an Olympus IX-70 microscope equipped with a x60 1.4-numerical aperture objective. Illumination was provided by a 100-W Hg lamp, and images were acquired using a liquid-cooled charge-couple device (CCD) camera (Photometrics) equipped with a Kodak CCD array (KAF1401E). Three-dimensional image acquisition and deconvolution were performed using a DeltaVision Restoration microscopy system (Applied Precision, Issauah, WA). Image deconvolution was carried out using an iterative approach based on regularization (7).

Statistics. Concentration-response curves for each type of mouse were fitted to a logistic equation using Origin software; the fitting parameters were EC50, Fo, and Hill parameter. Data are presented as means ± SE; n refers to the number of neonatal mice. Standard ANOVA and the Bonferroni modified t-test were used when multiple comparisons were made. Results were considered significant for a P value < 0.05.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of -, -, and -neonatal mouse aortas. The -, -, and -mice in which one or both copies of the {alpha}1- and {alpha}2-isoforms of the Na+/K+-ATPase was ablated were generated via gene targeting (20). In a variety of tissues from neonatal -mice, no {alpha}2-isoform was detected by Western blot analysis (36). Mice lacking the {alpha}2-isoform gene are born but die within a few minutes of birth. These neonates appear normal with no histological differences compared with WT littermates. Birth weights of the neonates averaged 1.53 ± 0.13 g (n = 55) and no subgroup (WT, , , and ) differed from the mean.

Contractile parameters of neonatal aorta. Based on our hypothesis, the {alpha}2-isoform is specifically coupled to SR Ca2+ filling; therefore, we would predict that receptor-mediated contraction, which is linked by inositol 1,4,5-trisphosphate (IP3) to SR Ca2+ release, would be altered in aortas from the {alpha}2-isoform gene-ablated neonates. Activation by KCl depolarization, on the other hand, is largely associated with Ca2+ influx and is less dependent on the SR. Figure 1 shows the time course of isometric forces developed in response to the receptor-mediated thromboxane A2 analog U-46619 (10–7 M; Refs. 8, 56); contractile parameters from these experiments are summarized in Table 1. ACh (10–5 M), which was used to assess functional endothelium-dependent relaxation in adult aorta, did not elicit significant relaxation in this neonate preparation (Fig. 1). Depolarization with KCl (40 mM) generated approximately one-third less force than U-46619 (Table 2). It is worth noting that phenylephrine, an {alpha}-adrenergic agonist, was ineffective in the neonatal aorta, which is surprising in light of its ability to stimulate adult mouse aorta (27). Maximum forces generated in response to U-46619 (0.1 µM) were not different between any of the classes (, , and ) compared with respective WT aortas. This was true for either the absolute force or force normalized to the length of the aorta. This suggests that the contractile apparatus itself was not altered. Force measured in response to U-46619 developed by the WT aorta from mice used to develop the {alpha}1-knockout mice was larger than that from {alpha}2-WT animals (see Table 1). This was not true for KCl stimulation of any other contractile parameter (see Tables 1, 2, 3) for WT aorta. The basis for the difference in force between WTs for U-46619 contractures is not known. In our studies with genetically altered mice, we have found that aortas from mice from different laboratories but with reportedly identical genetic backgrounds sometimes have slightly different contractile properties. For this reason, our comparisons are between sibling neonatal mice.



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Fig. 1. Superimposed isometric force-time course records of neonatal mouse aortas. Addition of U-46619 (10–7 M) to initiate contraction and ACh (10–5 M) as a test for the presence of endothelium are indicated (arrows); aortas were then rinsed three times. Maximum force was 1.26 mN/mm in the -isoform and 1.66 mN/mm in the -isoform.

 

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Table 1. Contractile parameters for receptor-mediated stimulation of mouse aorta

 

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Table 2. Contractile parameters for KCl contracture of mouse aorta

 

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Table 3. EC50 values for concentration-force relations

 



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Fig. 2. Cumulative U-46619 concentration-isometric force relations. A: - and -aortas. B: -, -, and -aortas. Error bars represent SE (omitted for clarity for -aortas). ED50 values were 2.75 x 10–8 ± 1.97 x 10–9 and 2.72 x 10–8 ± 1.50 x 10–9 for - and -aortas, respectively.

 


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Fig. 3. Cumulative forskolin concentration-isometric force relations. A: - and -aortas. B: -, -, and -aortas. ED50 values were 1.41 x 10–7 ± 3.10 x 10–8 and 1.32 x 10–7 ± 1.43 x 10–8 for - and -aortas, respectively.

 


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Fig. 4. Cumulative sodium nitroprusside (SNP) concentration-isometric force relations. A: - and -aortas. B: -, -, and -aortas. ED50 values were 7.35 x 10–9 ± 1.11 x 10–9 and 6.40 x 10–9 ± 1.48 x 10–9 for - and -aortas, respectively.

 
To assess contractile kinetics, we measured the half-time (t1/2) of force development and relaxation after washing out the stimulus. The only statistically significant difference was that the t1/2 for force development in the -aortas was approximately half of that for the WT, with the values for -aortas intermediate. For the -aortas, the t1/2 value was not different from the WT. Measurements of the relaxation dynamics were less precise due to the small forces and the difficulty in rapidly removing U-46619, which required multiple rinses (see Fig. 1). There were no statistically significant differences compared with the respective WTs (see Table 1). For KCl stimulation (see Table 2), there were no differences in maximal force for any class of neonates. The changes in t1/2 for force development and relaxation for the -aortas were less than those of the WT, with the -aortas intermediate. These differences were similar in direction but considerably smaller than those for U-46619 stimulation and were not statistically significant.

Stimulus sensitivity of neonatal aorta. Figure 2 shows the cumulative U-46619 concentration-force relations. The -values were significantly leftward shifted, which indicates a greater sensitivity to receptor-mediated stimulation than WT; the relation for the -aortas was intermediate. There was no difference in sensitivity between the - and WT aortas. The EC50 value and statistical summary for these experiments are given in Table 3.

Relaxation by A or G kinase. Activation of the Na+ pump by A kinase has been proposed to underlie the relaxation to {beta}-adrenergic agonists (50). However, little is known as to whether there is any {alpha}-isoform specificity to this process. We investigated A kinase-mediated relaxation using forskolin, which directly activates adenylate cyclase. Aortas were stimulated to their EC80 levels with U-46619. Forskolin elicited a concentration-dependent relaxation that was less effective in - than in WT aortas (Fig. 3); values for the -aortas were intermediate. There were no significant changes in the EC50 values for relaxation to forskolin. The major difference was a significant suppression of the maximum relaxation observed. A summary and statistical analyses for these experiments are given in Table 3. A similar pattern was observed for relaxation via the G kinase pathway activated by SNP (Fig. 4), with the exception that the -aortas did not differ from WT. No differences in relaxation mediated either by A or G kinase pathways were observed between - and WT aortas (see Figs. 3 and 4; Table 3).

Expression of {alpha}-Na+/K+-ATPase isoforms in WT aorta. Calibration of the absolute levels of {alpha}1- and {alpha}2-isoforms is very difficult due to the limited amount of neonatal aortic tissue. To accomplish the calibration, we first established the neonatal distribution relative to the adult aorta and then calibrated the more abundant adult tissue. The distributions of {alpha}-isoforms of the neonate were similar to those of the adult based on relative antibody binding. Western blots of adult and neonate aortas are shown in Fig. 5. The gel-density ratios of anti-{alpha}1- to anti-{alpha}2-isoforms were 0.88 ± 0.18 in neonatal aortas and 0.88 ± 0.25 in adults. The anti-{alpha}1-to-anti-{alpha}2 density ratio in the neonatal aorta divided by that of the adult in the same gel was 1.12 ± 0.23 (n = 4). Thus although these gel-density ratios cannot be interpreted as protein ratios, they do indicate that the protein ratios are similar in neonates and adults.



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Fig. 5. Western blots. Top: wild-type (WT) adult and neonatal aortas probed with {alpha}1- (4 lanes, left) and {alpha}2- (4 lanes, right) isoform-specific antibodies. Numbers above lanes represent the micrograms of protein added to the gel. The {alpha}1- and {alpha}2-isoforms are similarly expressed in neonate and WT aortas. Bottom: blot used for calibration of {alpha}-isoforms, using increasing concentrations of extracts of NIH/3T3 cells (3 lanes, left) and adult WT aortas (3 lanes, right). Probes were anti-{alpha}1- and pan-anti-{alpha}-Na/K+-ATPase ({alpha}-NKA). These data yielded a calculated {alpha}1-isoform concentration of 63.6% of the total {alpha}-Na/K+-ATPase.

 

Our measurement of the {alpha}-isoform protein distribution in the adult aorta utilized a method in which the relative amount of {alpha}1-isoform to total Na+/K+-ATPase is calculated by calibrating the gel density of the antibody specific for the {alpha}1-isoform relative to that of an antibody that recognizes all Na+/K+-ATPase isoforms. This was done using NIH/3T3 fibroblasts or kidney preparations as a standard as they contain a known percentage of the {alpha}1-isoform. In adult aortas, the {alpha}1-isoform constituted 68.8 ± 3.8% (n = 4 pooled batches of 5–6 arteries each) of the total Na+/K+-ATPase. A similar analysis, but based on the {alpha}2-isoform using brain as a standard, indicated that the {alpha}2-isoform was 38.1 ± 9.7% of the total (n = 4, pooled batches of 7 and 9 aortas each). These data indicate that {alpha}1- and {alpha}2-isoforms are the predominant {alpha}-isoforms present in mouse aorta. These {alpha}1- and {alpha}2-isoforms are also reported to be the sole {alpha}-isoforms in rat aortas (48). This relative expression in WT adults is similar to that of adult hearts (P. James and J. Lingrel, unpublished data) with ~70% {alpha}1- and 30% {alpha}2-isoforms.

We used an immunohistological approach to visualize the spatial distribution of the {alpha}1- and {alpha}2-isoforms. Figure 6 shows micrographs of WT neonatal aorta cells stained with fluorescence-labeled anti-{alpha}1- and {alpha}2-antibodies. Three-dimensional image-acquisition and deconvolution analyses demonstrate that the {alpha}2-isoform has a distinctly different distribution than the {alpha}1-isoform. Although the {alpha}1-isoform appears largely to be uniformly distributed across these cells, the {alpha}2-isoform is clearly more localized. The absence of {alpha}2-antibody labeling in cells isolated from -mice (not shown) demonstrates specificity of this antibody for the {alpha}2-protein. This is consistent with previous studies that show specificity in astrocytes isolated from embryonic mice from -relative to WT mice (12).



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Fig. 6. Fluorescence micrograph showing {alpha}1- and {alpha}2-Na+/K+-ATPase distributions in smooth muscle cells isolated from WT neonatal mouse aortas after 24 h in culture. Secondary fluorophore-labeled antibodies were used to independently detect primary anti-{alpha}1-monoclonal and anti-{alpha}2-polyclonal antibodies. Scale bar, 10 µm.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
These Na+/K+-ATPase gene-targeted mice provide a unique animal model to test the hypothesis that the different amounts and spatial distributions of the {alpha}-isoforms are indicative of differential functions. A comparison of the - to the -isoform cannot be made, because the -isoform is embryonic lethal. One can make a reasonable comparison in terms of the extent of {alpha}-isoform reduction relative to the total Na+/K+-ATPase. Based on Western blot analyses, we estimated a 70:30 {alpha}1-to-{alpha}2-isomer ratio in WT neonatal aortas. A reduction of one copy of the {alpha}1-isoform gene would be anticipated to lead to one-half of the {alpha}1-Na+/K+-ATPase as occurs in both heart (20) and skeletal muscle (14). Thus the total Na+/K+-ATPase in -aortas would be reduced by ~35%. The magnitude of this reduction is similar to that expected (~30%) due to ablation of both copies of the {alpha}2-gene in the -aorta. Thus despite a similar reduction in the total Na+/K+-ATPase, striking functional differences were observed that are dependent on whether the reduction was due to the {alpha}2- or the {alpha}1-isoform.

The contractility values for the -aortas, which were measured in terms of concentration-force and concentration-relaxation relations and the kinetics of force development and relaxation, were identical to those of WT mice. This suggests that there is substantial reserve for the "housekeeping" function suggested for the {alpha}1-isoform. The -aorta, in contrast, was more sensitive to receptor-mediated stimulation for which it had a faster rate of force development than WT. It also was less sensitive to relaxation by either A- or G-kinase pathway activation. The contractility of the -aortas was generally intermediate between - and WT aortas, which suggests dependence on the amount of {alpha}2-isoform. This was not the case for SNP relaxation, where the - and WT aorta behaviors were identical. The basis for this is not known. Our immunocytochemistry data indicate that the {alpha}1- and {alpha}2-isoforms of the Na+/K+-ATPase are located in different regions of mouse aortic smooth muscle cells. This is in agreement with the studies of Juhaszova and Blaustein (23, 24); using astrocytes isolated from embryonic mice, these workers suggested that the {alpha}2-isoform is localized near the SR. All of these aspects are consistent with the hypothesis that the {alpha}2-isoform modulates SR function via colocalization with the Na+/Ca2+ exchanger (35) and the SR Ca2+-ATPase (SERCA). If the {alpha}2-isoform and Na+/Ca2+ exchanger underlie the Ca2+ homeostasis of this subsarcolemmal compartment, one could make the following predictions. In the absence of the Na+/K+-ATPase in this compartment in -aorta, Ca2+ content would be high due to the absence or reversal of Na+/Ca2+ exchange. Coupled to the SR via SERCA, one would anticipate greater Ca2+ loading (38). An increased Ca2+ load of the SR would be expected to lead to larger Ca2+ release per any given level of IP3 produced in response to receptor-mediated stimulation. This is consistent with the greater rate of force increase as well as the greater sensitivity to U-46619 observed in -aorta. For KCl contractures, increases in intracellular Ca2+ are largely due to influx through plasmalemmal Ca2+ channels. Thus the lack of any differences in the contractile responses to KCl is also consistent with our hypothesis, since Ca2+ release from the SR is likely to be less important when stimulated by depolarization. The decreased sensitivity to agonist relaxation may also be explained similarly, in that a "hyperloaded" SR may not be as effective at removal of Ca2+ from the cytosol when activated by these pathways. It is well known that SERCA can be inhibited by elevated SR Ca2+.

There are of course other possibilities. For example, the Na+ pump is composed of {alpha}- and {beta}-subunits for which 3 isoforms have been described for mammalian tissues. It is possible that a decrease in one {alpha}-isoform could affect the {beta}-subunit distribution and thereby affect the function of the Na+ pump (37). Additional experimentation is needed to confirm the mechanism(s) underlying these isoform-specific functional changes.

In rodents, the {alpha}2-isoform has a higher ouabain affinity (Kd ~10–7 M) than the {alpha}1-isoform (Kd ~10–5 M; Ref. 39), whereas in humans, all {alpha}-isoforms have similar, high affinities (Kd ~10–8 M; Ref. 57). Thus a role for a ouabainlike factor in cardiac glycoside toxicity (57) or hypertension (6) would appear more obvious for rodents. However, our results suggest that the {alpha}2-isoform may play a differential role despite a similar affinity for ouabain. Although the ultimate mechanism(s) is not yet known with certainty, our data clearly demonstrate that ablation of the gene for the -Na+/K+-ATPase elicits major changes in vascular contractility. Reduction of Na+/K+-ATPase to a similar degree except by decreasing the {alpha}1-isoform was not associated with any significant changes in contractility in -aorta. This observation correlates with structural evidence that suggests the importance of distinct {alpha}-isoform locations in regulation of contractility. Moreover, the differences in function suggest new therapeutic strategies whereby smooth muscle contractility can be modulated by targeting specific isoforms of the Na+/K+-ATPase.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-66044 (to R. J. Paul) and HL-28573 (to J. Lingrel).


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. Paul, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576 (E-mail: Richard.Paul{at}uc.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.


    REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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