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Transgenic mice expressing Na⁺-K⁺-ATPase in smooth muscle decreases blood pressure

¹Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and ²Department of Physiology, University of Arizona Health Sciences Center, Tucson, Arizona

Submitted 6 March 2007 ; accepted in final form 25 April 2007

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The Na⁺-K⁺-ATPase (NKA) is a transmembrane protein that sets and maintains the electrochemical gradient by extruding three Na⁺ in exchange for two K⁺. An important physiological role proposed for vascular smooth muscle NKA is the regulation of blood pressure via modulation of vascular smooth muscle contractility (5). To investigate the relations between the level of NKA in smooth muscle and blood pressure, we developed mice carrying a transgene for either the NKA ₁- or ₂-isoform (_1sm+ or _2sm+ mice) driven by the smooth muscle-specific -actin promoter SMP8. Interestingly, both -isoforms, the one contained in the transgene and the one not contained, were increased to a similar degree at both protein and mRNA levels. The total -isoform protein was increased from 1.5-fold (_1sm+ mice) to 7-fold (_2sm+ mice). The increase in total NKA -isoform protein was accompanied by a 2.5-fold increase in NKA activity in _2sm+ gastric antrum. Immunocytochemistry of the ₁- and ₂-isoforms in _2sm+ aortic smooth muscle cells indicated that -isoform distributions were similar to those shown in wild-type cells. _2sm+ Mice (high expression) were hypotensive (109.9 ± 1.6 vs. 121.3 ± 1.4 mmHg; n = 13 and 11, respectively), whereas _1sm+ mice (low expression) were normotensive (122.7 ± 2.5 vs. 117.4 ± 2.3; n = 11 or 12). _2sm+ Aorta, but not _1sm+ aorta, relaxed faster from a KCl-induced contraction than wild-type aorta. Our results show that smooth muscle displays unique coordinate expression of the -isoforms. Increasing smooth muscle NKA decreases blood pressure and is dependent on the degree of increased -isoform expression.

vascular contractility; hypertension; Na⁺-K⁺-ATPase -isoforms

In the case of salt-sensitive hypertension, NKA is hypothesized (5, 13) to participate, via modulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and contractility, by coupling to the Na⁺/Ca²⁺ exchanger (NCX). A variety of studies have demonstrated that decreases in NKA activity are associated with an elevated blood pressure. These include pharmacological manipulation with cardiac glycosides as well as genetically induced decreases in NKA expression (5, 7, 11). However, to validate causality rather than correlation, gain of function studies are needed to demonstrate whether increases in NKA activity can produce decreases in blood pressure.

NKA is a heterodimer composed of an -subunit, responsible for catalytic function, and a -subunit (2, 4, 7–9, 16). In aorta, two (₁ and ₂) (30) of the four known -isoforms (2, 4, 7) are present; 70% of the total NKA is composed of the ₁-isoform and the remaining 30% is the ₂-isoform (30). To investigate a gain of NKA function on blood pressure, we developed mice that carry transgenes for either the NKA ₁- or ₂-isoform, using the smooth muscle-specific -actin promoter (6) SMP8. The -isoforms in smooth muscle from the _1sm+ mouse line were increased by 1.5-fold and up to 7-fold in the _2sm+ mouse line. An important, but surprising observation was that the -isoform not contained in the transgene was upregulated to a similar degree. We show that an increase in NKA -isoforms leads to a decreased blood pressure, which is dependent on the degree of increased vascular NKA expression. Thus increased expression of vascular NKA can lead to hypotension and perhaps provides a novel pathway for treatment of hypertension.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Generation of transgenic mice. ₁- or ₂-isoform NKA rat cDNA (gift from Dr. Jerry Lingrel, University of Cincinnati) was inserted via HindIII restriction sites into transfer vector pSAHSK + GSSS (Fig. 1, A and B) containing AscI and SnabI restriction sites (generated by Genomatix, Cincinnati, OH). Resultant clones were screened for cDNA orientation using XbaI and NotI restriction endonucleases for vectors containing the ₁-isoform or EcoRI and NotI enzymes for vectors with the ₂-isoform. The ₁- or ₂-isoform NKA cDNA was then cloned via AscI and SnabI sites into target vector pSMP8 + RPNKAA + human growth hormone poly(A) (generated by Genomatix) containing the -actin smooth muscle-specific promoter SMP8 (6) and a human growth hormone polyadenylation sequence (Fig. 1C). Proper construction of the transgenes was verified by sequencing the 5' and 3' ends of the cDNA using primers targeted to the plasmid at the 3' end of the promoter (5'-GTGTTAGTTGAGAACTGTGG-3') and the 5' end of the polyadenylation sequence (5'-GAAGGACACCTAGTCAGACA-3'). Sequencing was performed by the University of Cincinnati DNA Core. The ₁- or ₂-isoform transgenes were linearized (Fig. 1D) via two NotI sites, purified, and microinjected into the pronucleus of fertilized mouse eggs (FVB/n) that were then implanted into pseudopregnant females by the University of Cincinnati Transgenic Mouse Core (Cincinnati, OH). Resultant founder mice produced from this procedure were identified by PCR and Southern blot analyses and then subsequently bred to wild-type (WT) FVB/n mice.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Linear map of the Na+-K+-ATPase (NKA) -isoform transgenes. HindIII-bound NKA 1- or 2-isoform rat cDNAs (A) were cloned into transfer vector pSAHSK + GSSS (Genomatix) (B). The -isoform cDNA was then inserted into target vector pSMP8 + RPNKAA + human growth hormone (hGH) poly(A) (Genomatix) via AscI and SnabI sites (C). The transgene was released at NotI sites for pronuclear microinjection (D).

Genotyping of transgenic mice. Founder mice and subsequent transgenic offspring were identified by PCR. DNA from tail biopsies (1 cm) was isolated using ethanol precipitation. Primers targeting the 5' end of the ₁-isoform cDNA (5'-ATGTCCCTTTCCTTCTTCGC-3') and the 3' end of the SMP8 promoter (5'-GGATCATCAAAGGCTTTACAGC-3') or the 5' end of the human growth hormone poly(A) tail (5'-CAGGTTGTCTTCCCAACTTG-3') and the 3' end of the ₂-isoform cDNA (5'-GGGTGGAGAAGGAGACGTAC-3') were generated for PCR analysis of _1sm+ and _2sm+ mice, respectively. PCR conditions included denaturation at 94°C for 3 min, 30 cycles at 94°C for 1 min, 57°C for 1 min, 72°C for 1 min, and elongation at 72°C for 10 min. PCR products were electrophoresed on 1% agarose gels containing ethidium bromide for visualization.

Southern blot analysis. For Southern blot, tail DNA (extracted the same as for genotyping) was digested with HindIII restriction enzyme and separated on a 1% agarose gel. DNA was transferred to a nylon membrane and probed with a random primed ³²P-labeled (random primer DNA labeling kit; Invitrogen, Carlsbad, CA) 1,360-bp fragment of the ₁-isoform cDNA (isolated using BamHI and XbaI restriction enzymes) or 1,965-bp fragment of the ₂-isoform cDNA (isolated using BamHI and EcoRI restriction enzymes). The probes hybridized to a 4-kb product of the HindIII-digested tail DNA.

Western blot analysis. Aorta was dissected free from connective tissue and fat, and the endothelium was removed. Gastric antrum was rinsed free of digestive debris, and the mucosa was removed. Hearts were removed of any fat and connective tissue and rinsed thoroughly of blood. Tissues were frozen in liquid N₂. Aorta was homogenized with a dental amalgamator (Caulk, Milford, DE) and antrum and heart 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 homogenization buffer was supplemented with the following inhibitors used at 1:100: phosphatase inhibitor cocktails I and II and protease inhibitor cocktail (Sigma, St. Louis, MO). Protein homogenates were then assayed to determine concentration, with BSA used as a standard (Bio-Rad protein assay, Hercules, CA).

Protein from aorta (10–40 µg) and protein from antrum or heart (2.5–20 µg) were incubated at 37°C for 30 min in Laemmli buffer supplemented with 5% -mercaptoethanol followed by separation by SDS-PAGE (Genemate 4–20% express gels; ISC BioExpress, Kaysville, UT) at 100 V for 3 h and then electrotransferred at 220 mA to polyvinylidene difluoride membranes (Millipore, Bedford, MA) overnight at 4°C. The membranes were incubated in 1% blocking reagent (Roche Diagnostics, Indianapolis, IN) in TBST (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature. Blots were then incubated with monoclonal anti-₁-antibody that binds to the NH₂ terminus of the ₁-isoform (6F; Developmental Studies Hybridoma Bank, University of Iowa), polyclonal anti-₂-antibody that recognizes an intracellular loop region of the ₂-isoform [affinity purified from ₂-antisera using the synthetic HERED peptide (gift from Dr. Thomas Pressley)] (26), or polyclonal KETYY antibody that recognizes the COOH terminus of NKA -isoforms (kindly provided by Dr. Jack Kyte) in TBST for 1.5 h at room temperature. Equal loading was verified with the monoclonal anti-actin C4 antibody (gift from Dr. James Lessard) or Ponceau S staining of the actin band. Peroxidase-conjugated goat anti-mouse (Bio-Rad) or anti-rabbit (Calbiochem, La Jolla, CA) secondary antibodies were used.

Blots were developed with an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ) and exposed with Blue Basic autorad film (ISC BioExpress). Multiple exposures were taken to ensure linearity of the signal. Relative protein expression in transgenic mice compared with paired WT mice was quantitated by densitometry using ImageQuant 5.2 software (Molecular Dynamics, Amersham Biosciences). All summarized results consist of data from at least two Western blots representing two different protein samples. For each genotype, antrum from an average of three mice and aorta from an average of seven mice were pooled for each sample.

Quantitative real-time PCR. Mouse antrum was dissected in ice-cold PSS and rinsed of all debris; the tissue was immediately frozen in liquid N₂. Total RNA was isolated using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to manufacturer's instructions. Total RNA concentration and purity were measured spectrophotometrically. Total RNA (2 µg for each sample) was treated with RQ1 RNase-free DNase I (Promega, Madison, WI) before cDNA synthesis. cDNA synthesis was performed with random hexamer primers using the SuperScript III first-strand synthesis system (Invitrogen). cDNA was used in real-time PCR reactions to quantitate relative mRNA levels of the NKA -isoforms using published primer sequences for the ₁- and ₂-isoforms (22) and GAPDH as an internal control (12). iQ SYBR green Supermix (Bio-Rad) was used for real-time PCR reactions according to manufacturer's instructions. Reactions were run for 50 cycles at 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s using the iCycler real-time PCR machine and iCycler software (Bio-Rad). cDNA reactions in which reverse transcriptase was omitted were used as negative controls. Threshold cycles (automatically calculated by iCycler software) for GAPDH mRNA were compared between transgenic and paired WT samples for normalization. Each antrum sample was assayed in triplicate, and each real-time PCR reaction was performed at least twice per sample. Three paired antrum samples per transgenic line were used in the analysis. Relative -isoform expression (in fold) in transgenic lines compared with paired WT mice was calculated using the delta-delta threshold cycle method (25).

Western blot analysis of the NKA -subunits. Gastric antrum from _1sm+, _2sm+, and paired WT mice were isolated and homogenized as described in Western blot analysis above. Crude protein homogenates remained either untreated or denatured and digested with glycosidase peptide N-glycosidase F (New England Biolabs, Beverly, MA) according to manufacturer's instructions. Protein samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes as described in Western blot analysis above. Blots were probed with the polyclonal anti-₁- or anti-₂-antibodies spETB1 or spETB2 (kindly provided by Dr. Pablo Martin-Vasallo). Three different antrum samples per genotype were used for analysis where each sample contained three pooled antrum. Relative NKA -isoform protein quantitation was performed as described in Western blot analysis above.

Fluorescent, enzyme-linked measurements of NKA activity. Gastric antrum from _1sm+, _2sm+, and paired WT mice were dissected and homogenized as described in Western blot analysis above, with the exception of the homogenization buffer. The homogenization buffer contained 0.25 M sucrose, 30 mM imidazole, and 1 mM Na⁺-free EDTA and was not supplemented with any protease or phosphatase inhibitors.

NKA activities were determined with an enzyme-linked assay measuring the rate of ADP production as linked to the rate of NADH fluorescence decrease in the absence or presence of 1 mM ouabain. Crude protein (50 µg) was incubated for 20 min at 37°C in solution with pH 7.4 containing 100 mM NaCl, 20 mM KCl, 8 mM MgCl₂, 40 mM Tris, 1 mM EGTA, 1 mM Na⁺ azide, 25 mM choline chloride, 1 U/ml lactate dehydrogenase, 1 U/ml pyruvate kinase, 1 mM phosphoenolpyruvate, and 80 µM NADH. The reaction was initiated with the addition of 1 mM ATP. NADH fluorescence was continuously followed (excitation = 340 nm, emission = 460 nm) for 20 min with the use of a spectrofluorometer (PTI Delta Scan-1; Photon Technology International, South Brunswick, NJ). Rates were calculated as nanomoles ATP hydrolyzed per minute per milligram of protein. Three _1sm+ or _2sm+ and their paired WT antrum samples were used in the NKA activity measurements, where each protein sample consisted of three pooled antrum. NKA activity was measured for each sample at least twice.

Aorta and cell isolation. Adult mice were euthanized in a precharged CO₂ chamber followed by removal of the entire thoracic aorta (exposed from aortic arch to diaphragm). The aorta was dissected free from all associated fatty tissue and removed by cutting the intercostal arteries. Blood was immediately flushed from the isolated vessel with PBS + 0.1% BSA. To produce cell cultures, aortae were incubated in a PBS solution containing collagenase B (6 mg/ml) and BSA (6 mg/ml) and then incubated at 37°C for 40 min. Cells were dispersed from the digested aorta by trituration, diluted in PBS, and centrifuged at 1,000 g for 3 min. The cell pellet was suspended in 250 µl of DMEM supplemented with 5% FBS and 1% penicillin-streptomycin. Cell suspension from one aorta (250 µl) was plated as 50-µl aliquots onto the center of six glass coverslips housed in individual wells of a six-well plate. The six-well plate was then placed into a 37°C incubator equilibrated with 5% CO₂. After 16 h, 5 ml of fresh DMEM were added to each well. The PBS contained (in mM) 2.7 KCl, 1.5 KH₂PO₄, 138 NaCl, and 8 Na₂HPO₄, at pH 7.2.

Immunocytochemistry. At least 96 h after isolation, cells on coverslips were fixed with 3% paraformaldehyde, rinsed with 25 mM glycine, and permeabilized with 0.1% Triton X-100 (17). The cells were incubated with rabbit polyclonal antibody raised against the NKA ₂-isoform (26) for 2 h at 25°C. For ₁-isoform labeling, cells were incubated with the ₁-isoform-specific antibody NASE (26) for 12 h. After labeling with -isoform-specific antibodies was completed, cells were washed three times (5 min each) in PBS to remove unbound primary antibody and then incubated with a secondary anti-rabbit IgG labeled with Texas red (Jackson ImmunoResearch, West Grove, PA) for 45 min at 25°C. Specificity was determined by incubating the antibody in the presence of its peptide antigen (26) before incubation with the sample. Under this condition, significant labeling was not observed. Coverslips were mounted onto glass slides using a 50% glycerol-saline solution containing the antibleach agent paraphenylendiamine (0.1%).

For standard wide-field imaging, slides were mounted on the stage of an Olympus IX-70 microscope equipped with a x60 1.4 numerical objective. Illumination was provided by a 100-W Hg lamp, and images were acquired by a liquid-cooled charge-coupled device camera (Roper Scientific, Tucson, AZ) equipped with a Kodak charge-coupled device array (KAF1401E). Image analysis was performed on a Silicon Graphics INDY2 workstation using customized software.

Smooth muscle contractility measurements. Thoracic aorta was dissected from male littermate mice. After removal of fatty tissue, the aorta was cut into two pieces (one piece with the endothelium intact and the other half with the endothelium removed by gently rubbing the tissue between the thumb and index finger). Weight and calculated thickness (15) of each aortic segment were not different between _1sm+ or _2sm+ mice and their respective WT pairs, averaging 0.81 ± 0.02 mg and 114 ± 3 µm (n = 36), respectively. The aortic strips were threaded between two steel triangles 100 µm in diameter. The triangles were mounted in an organ bath system between a fixed post and a Harvard Apparatus differential capacitor force transducer (South Natick, MA). Isometric force was recorded continuously (Biopac Instruments and Acqknowledge software, Goleta, CA). Tissues were maintained in PSS and bubbled with 95% O₂-5% CO₂ for pH 7.4 at 37°C. Resting tension of the aorta was adjusted to 30 mN to set an initial length in the range of maximum isometric force generation. The tissues were then challenged with at least two contraction-relaxation cycles with 50 mM KCl (added from concentrate) to ensure reproducible forces. The tissues were then exposed to increasing cumulative concentrations of KCl or phenylephrine (PE). ACh (10^–5 M) was used to verify the presence of endothelium in aorta. All forces were analyzed and normalized to cross-sectional area as previously described (15).

Smooth muscle kinetics measurements. Contraction was elicited via depolarization by raising medium KCl to 50 mM, and then relaxation was initiated by rinsing with PSS. Relaxation half-times were calculated as the time (in s) for aorta to relax to 50% of its peak force. Force development half-times were calculated by measuring the elapsed time required for each vessel to reach 50% of its peak force from baseline in response to 50 mM KCl. Similar measurements were performed for challenges with 1 µM PE.

Blood pressure measurements. Systolic blood pressure (SBP) was measured in male littermate mice using a computerized tail-cuff system (BP-2000 blood pressure analysis system; Visitech Systems, Apex, NC). The mice were allowed to acclimate to the apparatus for 5 min before the start of measurement. Ten preliminary and 10 experimental pressures were acquired for 5 consecutive days/wk. Pressures were taken on the same days each week and at the same time each day. Data were accepted if the mouse had valid readings for at least 5 of the 10 experimental pressures taken. Blood pressures were obtained during a 2-wk period. The daily blood pressure averages per mouse were averaged to determine the mean SBP for that mouse.

Statistical analysis. The data are presented as means ± SE, where n is equal to the number of samples. Significance was determined with an unpaired Student's t-test, with a P 0.05 taken as significant where only two variables were compared. For force-concentration relations, multivariate analysis using ANOVA and Holm-Sidak post hoc analysis was applied using SigmaStat 3.1.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Generation of NKA -isoform transgenic mice. Transgenic mice were generated by inserting ₁- or ₂-isoform cDNA (31) between the smooth muscle-specific -actin promoter, SMP8 (6) and a polyadenylation sequence in the SMP8 + RPNKAA + human growth hormone poly(A) plasmid (Fig. 1). After pronuclear microinjection with the linearized transgene, one NKA transgenic founder for the ₁-isoform (line 09) screened from 14 mice by PCR and Southern blot analyses was identified (Fig. 2). Two NKA transgenic founders for the ₂-isoform (line 79 and line 84) were identified from 35 resultant mice (Fig. 2). Transmission of the transgenes from founder mice to offspring was verified by Southern blot (Fig. 2B). Both _1sm+ line 09 mice and _2sm+ line 84 mice have normal litter sizes of eight and seven pups, respectively, although the frequency of litters born is less in _1sm+ line 09 than in WT mice. As calculated from PCR analysis, the transgene was passed onto 44% of offspring in _1sm+ line 09 mice and to 51% of _2sm+ line 84 mice. The _2sm+ line 79 mice have small litter sizes, averaging only 3.6 mice, and a low frequency of births; transmittance of the transgene appeared normal with 53% of the offspring carrying the transgene. In all transgenic lines, Mendelian ratios of male to female mice were observed. Because of the small litter sizes in line 79 _2sm+ mice, studies in _2sm+ mice were confined to line 84. There were no gross anatomic abnormalities in NKA transgenic mice as measured by body and wet tissue weights of both smooth and nonsmooth muscle tissues (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Identification of NKA -isoform transgenic mice. A: PCR analysis. Primers targeting the 1-isoform transgene yielded a 354-bp product, and primers targeting the 2-isoform transgene yielded a 345-bp product. Linearized constructs of the -isoform transgenes served as positive controls. H2O and wild-type (WT) mouse DNA served as negative controls (data not shown). B: Southern blot. Probes containing 1- or 2-isoform cDNA hybridized to a 4-kb fragment of tail DNA that was digested with HindIII. The linearized -isoform transgene constructs served as positive controls (data not shown), and WT mouse DNA served as negative controls.

Coordinate expression of NKA -isoforms.

View this table: [in this window] [in a new window]   Table 2. Summary of Western blot data of NKA -isoforms

View larger version (14K): [in this window] [in a new window]   Fig. 3. NKA -isoform protein in mouse antrum and aorta. Representative Western blots for the relative NKA -isoform contents in 1sm+ (A) and 2sm+ (B) mouse smooth muscle tissues compared with paired WT tissue samples are shown. Actin was used as a loading control. Western blot data are summarized in Table 2.

Because upregulation of the -isoform not contained in the transgene was observed at the protein level in NKA transgenic mice, we wanted to verify whether the coordinate upregulation occurred at the transcriptional level as well. We measured mRNA levels of the -isoforms in gastric antrum from _1sm+ or _2sm+ mice relative to respective paired WT antrum using real-time PCR analysis. Coordinate upregulation of the -isoforms was also evident at the mRNA level in NKA transgenic mice (Fig. 4). The mRNA for ₁- and ₂-isoforms was increased 4.3- and 2.5-fold, respectively, in _1sm+ mice and 10.7- and 8.6-fold in _2sm+ mice.

View larger version (16K): [in this window] [in a new window]   Fig. 4. NKA -isoform mRNA levels in gastric antrum. mRNA levels in 1sm+ and 2sm+ mouse antrum, measured by real-time PCR, are presented as a percentage of the paired WT mRNA. 1- and 2-isoforms were elevated 4.3 ± 2.2- and 2.5 ± 0.9-fold, respectively, in 1sm+ mice and 10.7 ± 2.9- and 8.6 ± 3.2-fold in 2sm+ mice. Values are means ± SE (n = 3 samples). *P < 0.05.

Relative -isoform levels.

View larger version (26K): [in this window] [in a new window]   Fig. 5. -Subunit protein in NKA transgenic (TG) mice. A: representative Western blots of NKA 1- or 2-isoforms in gastric antrum from 1sm+ and 2sm+ mice. Antrum protein was deglycosylated for -subunit detection, resulting in an approximate molecular mass of 30 kDa. B: quantitation of Western blots for 1- and 2-isoform expression in 1sm+ and 2sm+ mouse antrum represented as a percentage of paired WT antrum. Values are means ± SE; n = 3 samples, where each sample consisted of 3 pooled antrum per genotype.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Immunocytochemical micrographs showing NKA -isoforms in primary cultured aortic smooth muscle cells. Wide-field microscopic images were taken of cultured aortic smooth muscle cells incubated with fluorescently labeled anti-1- or anti-2-antibodies. Left: cells from WT mice. Right: cells from 2sm+ mice. The 2-isoform has a reticular distribution in cells from 2sm+ similar to those from WT aorta, whereas the 1-isoform maintained a wide-spread distribution in 2sm+ cells similar to WT. Scale bars = 20 µm.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Systolic blood pressure (SBP) in NKA transgenic mice. SBP was measured using tail cuff sphygmomanometry (see METHODS). 1sm+ and 2sm+ mice were compared with their WT littermates (labeled WT 1 and WT 2, respectively). A: histogram of the daily SBP measurements in WT 1 and 1sm+ mice. B: WT 2 and 2sm+ mice fitted to Gaussian distributions. Black bars and lines represent transgenic data, and gray bars and lines represent WT data. SBP results in 1sm+ mice was similar to those in WT 1 mice (122.7 ± 2.5 mmHg, n = 11, vs. 117.4 ± 2.3 mmHg, n = 12). The 2sm+ mice had a significantly lower SBP than the WT 2 mice (109.9 ± 1.6 mmHg, n = 13, vs. 121.3 ± 1.4 mmHg, n = 11). *P < 0.05.

View larger version (19K): [in this window] [in a new window]   Fig. 8. Developed force-concentration relations in endothelium-intact (+E) and endothelium-denuded (–E) aorta from 1sm+ and 2sm+ mice. Developed isometric force was measured in aorta from 1sm+ and paired WT mice (left) and 2sm+ and respective WT aorta (right) in response to KCl (A and C) and phenylephrine (PE; B and D). Symbols are mean values, with bars representing ±SE. For 1sm+ (A and B, circles), there were no differences from either +E aorta (solid symbols and lines) or –E aorta (open symbols and dashed lines) and their respective WT (squares). For 2sm+ (C and D), the +E aorta also did not differ; however, the –E aorta were statistically different from the WT.

View this table: [in this window] [in a new window]   Table 4. Summary of sensitivities (EC50) of 1sm+ and 2sm+ aorta to KCl, PE, or ACh

View larger version (12K): [in this window] [in a new window]   Fig. 9. Force-concentration relations for aorta for the endothelium-dependent vasodilator ACh from 1sm+ (A) or 2sm+ (B) mice. +E aortas (solid symbols) from 1sm+ or 2sm+ mice did not display any differences in the relaxation-concentration relations. –E aortas (open symbols) only had minimal vasorelaxation with ACh. Symbols are mean values, with bars representing ±SE.

View this table: [in this window] [in a new window]   Table 5. Relaxation half-times for 1sm+ and 2sm+ aorta in response to 50 mM KCl or 1 µM PE

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

The first novel result was coordinate expression of the ₁- and ₂-isoforms in smooth muscle; that is, the -isoform contained in the transgene and the -isoform not contained in the transgene were coordinately increased by approximately the same percentages. There is little available evidence on how the levels of NKA are regulated. A coordinate increase in -isoform expression could be due to a decrease in protein degradation, transcriptional upregulation, or stabilization of the transcript. Our real-time PCR data indicate that the coordinate -isoform increases seen at the protein level are due in part to an increase in the RNA message for both isoforms.

There is substantial evidence indicating that the SMP8 promoter is expressed only in smooth muscle (6, 33). This is supported by our results in hearts from NKA transgenic mice, which showed no change in -isoform protein levels. Thus it is unlikely that the coordinate increase in -isoforms is due to general changes in endocrine status or compensation, as this would have affected the -isoform distribution of the heart.

NKA is reported to participate in several signaling pathways via its modulation of [Ca²⁺]_i (28). Alteration of NKA activity by ouabain can translate to activation of signaling proteins, such as NF-B, which may lead to activation or deactivation of expression of various genes (28). This could underlie the coordinate upregulation of the opposite -isoform.

Importantly, the increases in NKA -isoform levels in smooth muscle from NKA transgenic mice were accompanied by increases in total NKA activity. Thus the increase in protein translated to increased NKA function. The measured increase in total NKA activity was less than the observed increase in protein expression in _2sm+ mice. One explanation is that Western blot and NKA activity measurements are two very different parameters that require different sample preparations and different measurement techniques, each with varying degrees of sensitivity of detection, extent of nonspecific signals, and amplification, thus making absolute measurements not necessarily directly comparable. The low NKA density in smooth muscle, 100-fold less than cardiac or skeletal muscle (3, 14), and limited tissue mass decrease the precision of NKA measurements in smooth muscle. Another possibility is the availability of the -subunit, which is required for NKA function (9). In smooth muscle from antrum, we measured little change in -subunit levels from _1sm+ mice, but _2sm+ mice had a 25–40% increase in -subunit protein levels. There is no available evidence on the absolute ratio of to in smooth muscle to our knowledge. There are some indications that the -subunit is synthesized in excess of the -subunit (19, 21). The -subunit could be a limiting factor to increasing the NKA activity. However, the -subunit is also reported to be required for targeting of the -isoform to the plasma membrane (9). Our immunomicrographs showed that the distributions of ₁-and ₂-isoforms in the _2sm+ mice were similar to those in WT mice, which would argue against a -subunit-limited NKA activity.

Increased expression of NKA had a major impact on SBP. The _2sm+ mice were hypotensive compared with WT littermates, whereas _1sm+ mice were normotensive. Because total NKA activity is substantially higher in the _2sm+ mice than in the _1sm+ mice, the differences in SBP are likely a result of the degree of NKA expression. A significant decrease in SBP is relatively unusual in light of the generally small SBP changes in the literature associated with a single gene alteration (29). Our data support the hypothesis that increased NKA activity results in a decreased SBP and moreover that this can be solely a vascular phenomenon. Thus our gain-of-function experiments complement studies of decreasing NKA activity (7, 11, 36), which suggest that the vascular NKA alone could be an important factor in hypertension.

To evaluate the potential changes in vascular smooth muscle that may underlie the decreased blood pressure, we measured contractility and relaxation parameters in aorta. Consistent with the absence of change in blood pressure, there were no differences in contractility to KCl or PE stimulation in aortas from the _1sm+ mice. The _2sm+ aortas had moderately increased force in response to KCl (15–20%) or PE stimulation (12–15%). Higher forces are seemingly the opposite of the predicted increased Ca²⁺ extrusion by NKA-NCX coupling.

One might have anticipated that the increase in NKA and fall in blood pressure would both be accompanied by a decrease in aortic smooth muscle contractility. There are several possibilities behind the increase in force seen in endothelium-denuded _2sm+ aorta. 1) The increased Na⁺ gradient could stimulate Na⁺/H⁺ exchange, leading to cytosolic alkalinization, which can lead to higher levels of contractility (20). 2) The relative functional roles of ₁ to ₂ may be important. A modification of the hypothesis of coupled NKA-NCX Ca²⁺ extrusion has been proposed based on the differential distribution (Fig. 6) of -isoforms. Blaustein and colleagues (1, 10) proposed that ₂-NKA and NCX are localized in plasma membrane microdomains with preferential access to Ca²⁺ loading of the sarcoplasmic reticulum (SR). Increases in the ₂-isoform in this compartment would lower SR loading, which could outweigh the effects of ₁ on global [Ca²⁺]_i. For example, a hypoloaded SR could lead to lower spontaneous SR Ca²⁺ release and consequently lower K_Ca channel activity, which would lead to depolarization and greater contractility (35). 3) Another possibility to consider is the role of NKA in signaling. NKA is postulated to lead to possible activation of signal transduction factors like NF-B or Src (28). This may lead to the upregulation and downregulation of genes related to and/or unrelated to ion transport and/or contractility, thereby resulting in changes in aortic contractility, which remains to be elucidated.

One can also envision that either decreases or increases in contractile force, depending on which circulation (renal, central nervous system, etc.), could lead to reduced blood pressure. In some ways, it would be rather remarkable if changes in aortic smooth muscle reflected every microcirculation. However, this was the simplest hypothesis and important to test. The increases in contractile forces in _2sm+ mice were only measured in endothelium-denuded aorta. Endothelium-intact aorta, a perhaps more physiological representative of the in vivo vessel, did not display any differences in contractility, suggesting some basal endothelial compensation. It is possible that the compensation is more effective in other microcirculations or at the resistance vessel level or that other microvasculatures simply differ from aorta. Understanding the mechanism(s) underlying the decrease in blood pressure will require future studies.

Importantly, mechanical factors other than the developed force may be in play in the observed hypotension. Of particular interest, there is a correlation between speed of relaxation and blood pressure. Studies of spontaneously hypertensive rats report slower relaxation rates in caudal and mesenteric arteries (23, 24). We measured relaxation half-times in _1sm+ and _2sm+ mice. The _2sm+ aortas had faster rates of relaxation, indicating possible increased Ca²⁺ extrusion. A decreased blood pressure associated with faster aortic relaxation was recently reported for diabetic rats (27). We would suggest that the observed faster rates of aortic relaxation may be a larger factor leading to the observed hypotension rather than the effects of the moderately increased maximum force.

In summary, our results indicate that a genetically engineered increase in NKA activity is associated with a decreased SBP, which is dependent on the degree of vascular NKA expression. Thus focusing on vascular NKA per se may provide novel insights for treatment of hypertension.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-66044 (R. J. Paul and R. M. Lynch).

	ACKNOWLEDGMENTS

 
We thank Dr. Jerry Lingrel for kindly providing us with the rat Na⁺-K⁺-ATPase ₁- and ₂-isoform cDNAs and Dr. Arthur Strauch for the SMP8 promoter.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. J. Paul, 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.

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