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(Hypertension. 1996;27:827-832.)
© 1996 American Heart Association, Inc.

Articles

Regulation of Na⁺,K⁺-ATPase -Subunit Expression by Mechanical Strain in Aortic Smooth Muscle Cells

Emel Songu-Mize; Xiang Liu; Janet E. Stones; Lin J. Hymel

From the Department of Pharmacology and Experimental Therapeutics (E.S.-M., X.L., J.E.S.), Louisiana State University Medical Center, and Department of Physiology (L.J.H.), Tulane University School of Medicine, New Orleans, La.

Correspondence to Dr Emel Songu-Mize, Department of Pharmacology, LSU Medical Center, 1901 Perdido St, New Orleans, LA 70112. E-mail emize@lsumc.edu.

	Abstract

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Abstract We have previously demonstrated that vascular sodium pump activity is stimulated in several rat models of hypertension. In addition, others have reported an upregulation of mRNA for the Na⁺,K⁺-ATPase ₁-subunit in hypertension. To test the effect of sustained, cyclic, stretch-relaxation stimuli on the expression of ₁- and ₂-subunits of Na⁺,K⁺-ATPase in vascular smooth muscle cells, we used the Flexercell strain unit to stretch rat aortic smooth muscle cells for several days on a collagen-coated silicone elastomer substratum. Six-second cycles of stretch-relaxation were applied to obtain 10% average surface elongation (22% maximum) for 4 days. Control cells were not stretched but were grown on a similar surface. The effect of Gd³⁺, a blocker of stretch-activated channels, was also investigated. At the end of 4 days, protein expression of ₁- and ₂-subunits was determined by Western blot analysis. Intensity of the bands for ₁- and ₂-subunits was quantified with the use of a computerized image analyzer. In the stretched cells, both the ₁- and the ₂-subunit protein-band intensities were significantly increased compared with those of the nonstretched cells. Treatment with 50 µmol/L Gd³⁺ during the application of stretch prevented the upregulation of ₂-expression but not that of ₁-expression. Sodium pump activity, the functional counterpart of Na⁺,K⁺-ATPase, was inhibited as a result of stretch; Gd³⁺ had no effect on this variable. Our results suggest that in vascular smooth muscle, stretch may be a signal for the upregulation of both the ₁- and ₂-isoforms. However, a differential response of the two isoforms to the blocker of stretch-activated channels implies involvement of different mechanisms. This alteration in protein expression is not reflected in the function of the enzyme.

Key Words: muscle, smooth, vascular • cells, cultured • Na(+)-K(+)-exchanging ATPase • mechanoreceptors

	Introduction

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Vascular tissue is one of the primary targets of the modulatory influence of high arterial pressure. Several alterations occur as a result of hypertension, at both the gross and molecular levels, that affect the function of the vascular system. Vascular smooth muscle responds to neural stimuli, circulating hormones or factors, and endothelium-derived factors. Mechanical stimuli such as transmural pressure, which results in vascular wall stretch, also affect the responsiveness of vascular smooth muscle cells via stretch-activated (SA) ion channels.^{1 2 3 4} SA channels are frequently nonselective cation channels permeable to Na⁺, K⁺, and Ca²⁺, whose order of selectivity varies with the cell type. In smooth muscle cells from resistance vessels, SA channels are thought to promote increased contractile tone by mediating influx of Na⁺ and Ca²⁺, which depolarizes the cell and activates voltage-gated Ca²⁺ channels.⁵ SA channels are also thought to participate in cell volume regulation in vascular smooth muscle⁶ and in control of cell proliferation.⁷

Na⁺,K⁺-ATPase, a membrane-bound ion transport enzyme, is an important component that determines the resting membrane potential and therefore plays a role in the resting tone and responsiveness of the vasculature to vasoactive substances.^{8 9} The sodium pump appears to be differently regulated in hypertension in cardiovascular tissues. We have previously demonstrated that vascular sodium pump activity is stimulated in several rat models of hypertension.^{10 11} In addition, others have reported an upregulation of the mRNA of the aortic Na⁺,K⁺-ATPase ₁-subunit in two rat models of hypertension.¹² The signal for the increased expression of the catalytic -isoform is not known. We hypothesized that strain resulting from elevated pressure may be a signal that initiates a cascade of events leading to increased expression of the enzyme.

To test the effect of sustained, cyclic, stretch-relaxation stimuli on the expression of ₁- and ₂-subunits of Na⁺,K⁺-ATPase in vascular smooth muscle cells without the interference of other factors inherent to the in vivo system, we used a cell culture system and a device, the Flexercell Strain Unit,¹³ to apply stretch to rat aortic smooth muscle cells (ASMC) for several days. The effect of Gd³⁺, a blocker of SA, nonselective cation channels, on stretch modulation of Na⁺,K⁺-ATPase was also investigated. At the end of 4 days, protein expression of ₁- and ₂-subunits was determined by Western blot analysis, and the functional counterpart of the enzyme, sodium pump activity, was also determined.

	Methods

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Preparation of Cultured ASMC
ASMC were isolated from male Sprague-Dawley rats weighing 150 to 200 g as described earlier.¹⁴ The aortas were isolated and incubated for 30 minutes in minimal essential medium supplemented with 0.2 mmol/L Ca²⁺, 15 U/mL elastase (type III), 0.4 mg soybean trypsin inhibitor, and 200 U/mL collagenase (type I). The tissues were then cleaned of adventitia, minced with scissors, and incubated further for 90 to 120 minutes in a fresh aliquot of the same medium. The cells were then filtered and resuspended in culture medium (medium 199+10% fetal bovine serum) and seeded in 100-mm culture dishes. Experiments were performed on confluent ASMC between the third and seventh passages.

Stretch Protocol
The cells were seeded at 5000/cm² (24 000 cells per culture well) on type I collagen-coated Flex I and Flex II plates (Flexercell International Corp) and grown under nonstretch conditions for 8 to 10 days before application of stretch. Flex I plates containing a flexible silicone elastomer substratum were then mounted in the Strain Unit and subjected to 10% average (22% maximum) surface elongation at 10 cycles per minute (3 seconds on/3 seconds off) continuously for 4 days. This protocol was selected to provide a dynamic, nearly physiological strain stimulus that mimics the effect of the cardiac cycle on the aortic wall. Although a faster strain frequency would be required to closely mimic the pulse frequency of the rat, we chose 10 cycles per minute because it was the highest frequency that we believed provided a well-regulated strain amplitude by the instrument. Control Flex II plates, containing the same collagen-coated silicone elastomer substratum plus a rigid polystyrene bottom, were grown in parallel but not mounted in the strain unit.

Preparation of Samples for Western Blot Analysis
After the stretch protocol, ASMC from individual culture wells were washed with cold PBS. The plates were then scraped in a homogenization buffer (sucrose 250 mmol/L, Tris 50 mmol/L, EDTA 1 mmol/L, pH 7.4). Initial centrifugation was at 20 000g for 1 minute at 4°C. The pellet was resuspended in a lysis buffer (NaCl 140 mmol/L, Tris 10 mmol/L, MgCl₂ 1.5 mmol/L, and Triton X-100 0.5%, pH 8.6) and centrifuged at 20 000g for 3 minutes at 4°C. The supernatant was used for electrophoresis/Western blot analysis. Protein was estimated by the method of Lowry et al¹⁵ with bovine serum albumin as a standard. The final concentration of protein in the preparation was 2 to 3 mg/mL. The protein amount per culture well was 67.4±1.6 µg (n=44) and was not affected by the treatments (Table).

View this table: [in this window] [in a new window]   Table 1. Total Cell Protein Amount and Relative Cell Homogenate Protein Concentrations of Cultured Smooth Muscle Cells: Effect of Stretch and Gadolinium

Gel Electrophoresis and Immunoblotting
We loaded 5 and 15 µg of the cell extract protein for ₁- and ₂-isoforms, respectively, onto the gel for electrophoresis. The cell extracts and prestained molecular weight standards (Bio-Rad) were subjected to polyacrylamide gel electrophoresis on 10% polyacrylamide gels in the presence of 0.1% SDS¹⁶ and then transferred to polyvinylidene fluoride membrane by electroblotting.¹⁷ After a preincubation in Tris-buffered saline (mmol/L: Tris HCl 20, NaCl 137, pH 7.5) containing 5% (wt/vol) nonfat dried milk (Carnation) and 1.0% (vol/vol) Tween 20 for 1 hour at room temperature, the blots were probed with monoclonal antibodies McK1, McB2, and McBX3 directed against the ₁-, ₂-, and ₃-subunits of Na⁺,K⁺-ATPase, respectively, as described previously.¹⁸ The blots were treated with the secondary antibody, horseradish peroxidase-labeled sheep anti-mouse immunoglobulin (Amersham). Blots were then treated with enhanced chemiluminescence reagent (ECL, Amersham) and exposed to x-ray film for the visualization of the bands.

Quantitation
The fluorograms were scanned, and the intensity of the bands for ₁- and ₂-subunits was quantified as optical density units with the use of a computerized image analyzer (model M-2, Imaging Research Co).

Measurement of Sodium Pump Activity in ASMC
The sodium pump activity was determined in ASMC in culture with the use of a modification of the ouabain-sensitive ⁸⁶Rb⁺ uptake technique as previously described.¹⁴ The medium was removed and the cells were washed with Krebs-Henseleit buffer (pH 7.4, bubbled with CO₂ 5%/O₂ 95%; composition in mmol/L: NaHCO₃ 27.2, NaCl 119, NaH₂PO₄ 1, MgSO₄ 1.2, CaCl₂ 1.8, dextrose 11, KCl 5) and incubated in Krebs-Henseleit buffer without KCl for 30 minutes at 37°C. After 30 minutes, the cells were washed with Krebs-Henseleit buffer and supplied with 0.5 mL of fresh Krebs' solution. Some wells contained 2 mmol/L ouabain for determination of ouabain-resistant ⁸⁶Rb⁺ uptake. After a 2-minute incubation period, ⁸⁶RbCl (approximately 10⁶ cpm per well, 10 to 50 nmol/L) was added to start the uptake reaction, which was terminated after 30 minutes by removing the incubation medium and washing twice with Krebs buffer. Sodium pump activity was determined by subtracting the uptake of ⁸⁶Rb⁺ (plus K⁺) in the presence of 2 mmol/L ouabain (ie, ouabain-resistant uptake) from the total uptake. Sodium pump activity is expressed as nanomoles (⁸⁶Rb⁺ plus K⁺) per milligram protein per 30 minutes. The uptake reaction is linear for at least 30 minutes under the conditions described above.¹⁴ Total cell protein per culture well was 250±5 µg (n=46) and was not affected by stretch or Gd³⁺ treatment (Table).

Data Analysis
ANOVA followed by Scheffé's or Fisher's post hoc tests or Student's t test for paired data was used where applicable. A confidence limit of 95% was considered significant.

	Results

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We assessed the effect of chronic exposure to cyclic mechanical stretch on the expression of the ₁- and ₂-subunit proteins of Na⁺,K⁺-ATPase in cultured ASMC grown to confluence. The ₃-subunit was undetectable in the small samples available from the culture plates. A significant upregulation occurred in both the ₁- and ₂-subunits of the enzyme (Fig 1) when the cells were stretched for 4 days. The expression was approximately 50% above nonstretch controls (P=.00128 and P=.01321 for ₁ and ₂, respectively; Student's t test for paired data) for both isoforms.

View larger version (25K): [in this window] [in a new window]   Figure 1. Effect of stretch on the expression of -subunits of Na+,K+-ATPase in aortic smooth muscle cells. Bars represent the mean and SE for band densities from immunoblots for 1- and 2-subunit protein expression under stretch (hatched bars) and nonstretch conditions (stippled bars). *Significant difference between stretch and nonstretch conditions for 1- (P=.00128) and 2-subunits (P=.01321, Student's t test for paired data, n=25 cell culture wells for each condition). O.D. indicates optical density units.

To determine the possible contribution of the activation of nonselective SA, cation channels on the upregulation of the Na⁺,K⁺-ATPase -subunits, we studied the effect of 50 µmol/L Gd³⁺ in a series of experiments. When included in the incubation medium during the stretch cycle, Gd³⁺ significantly inhibited the upregulation of ₂ expression (Figs 2 and 3). Gd³⁺ had no effect on expression of the ₂-subunit in cells not exposed to stretch, ie, nonstretch controls (Fig 3). Although a similar trend was apparent for the effect of Gd³⁺ on the ₁-subunit (Fig 4), this inhibition did not reach significance (Fig 5).

View larger version (69K): [in this window] [in a new window]   Figure 2. A representative immunoblot of the aortic smooth muscle cell membrane extracts. A 15-µg sample of cell extract protein was applied to each lane. The monoclonal McB2 antibody was used to target the 2-subunit protein. The brain tissue homogenate was used as a control.

View larger version (15K): [in this window] [in a new window]   Figure 3. Effect of stretch and Gd3+ on the expression of 2-subunit of Na+,K+-ATPase in aortic smooth muscle cells. Bars represent the mean and SE for band densities from immunoblots for 2-protein expression under stretch and nonstretch conditions and with (hatched bars) and without 50 µmol/L Gd3+. *P<.05, significant differences between stretch without Gd3+, stretch with Gd3+, and nonstretch with and without Gd3+ (P=.0001 for overall ANOVA; Scheffé's post hoc test was applied for individual comparisons; n=14 cell culture wells for each group). O.D. indicates optical density units.

View larger version (64K): [in this window] [in a new window]   Figure 4. A representative immunoblot of the aortic smooth muscle cell membrane extracts. A 5-µg sample of cell extract protein was applied to each lane. The monoclonal antibody McK1 was used to target the 1-subunit protein. The kidney tissue homogenate was used as a control.

View larger version (18K): [in this window] [in a new window]   Figure 5. Effect of stretch and Gd3+ on the expression of 1-subunit of Na+,K+-ATPase in aortic smooth muscle cells. Bars represent the mean and SE for band densities from immunoblots for 1-protein expression under stretch and nonstretch conditions and with (hatched bars) and without 50 µmol/L Gd3+. *P<.05, significant differences between stretch without Gd3+ and nonstretch with and without Gd3+. **Significant difference between stretch with Gd3+ and nonstretch with Gd3+ (P=.002 for overall ANOVA; Scheffé's post hoc test was applied for individual comparisons; n=14 culture wells for each group). O.D. indicates optical density units.

To determine whether the upregulation of the protein expression of the two catalytic -subunits of Na⁺,K⁺-ATPase results in an alteration in the functional counterpart of the enzyme, the activity of the sodium pump, we measured the ouabain-sensitive ⁸⁶Rb⁺ uptake under the conditions of applied stretch and nonstretch and in the presence and absence of Gd³⁺. The sodium pump activity was significantly inhibited (22.7%) as a result of stretch (Fig 6). Gd³⁺ did not have an effect on this inhibition. Gd³⁺ also did not have any effect on the sodium pump activity under nonstretch conditions (Fig 6). The ouabain-insensitive ⁸⁶Rb⁺ uptake was not affected by stretch (244±14 versus 265±19 nmol/mg protein per 30 minutes, nonstretch versus stretch, respectively) or Gd³⁺ (245±4 versus 283±6 nmol/mg protein per 30 minutes, nonstretch plus Gd³⁺ versus stretch plus Gd³⁺, respectively).

View larger version (22K): [in this window] [in a new window]   Figure 6. Effect of stretch and Gd3+ on sodium pump activity in aortic smooth muscle cells in culture. Bars represent the mean and SE for sodium pump activities under stretch and nonstretch conditions and with (hatched bars) and without 50 µmol/L Gd3+. *P<.05, significant differences between stretch without Gd3+ and nonstretch with and without Gd3+ (P=.022 for overall ANOVA; Fisher's post hoc test was applied for individual comparisons; n=8 culture wells for each group). prot indicates protein.

Stretching the confluent cells for 4 days did not alter the total protein content per well or the yield of protein extraction (Table). Moreover, 50 µmol/L Gd³⁺ also had no effect on these variables. We interpret the lack of enhancement of total protein content to indicate that there is no general, nonspecific stimulation of protein expression in rat ASMC caused by the applied mechanical strain.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have demonstrated that sustained, cyclic, mechanical stretch applied to rat ASMC in culture induces a significant inhibition of the transport rate of Na⁺,K⁺-ATPase together with significant upregulation of both the ₁- and ₂-subunits. In addition, this upregulation is differentially affected by Gd³⁺, a blocker of SA, nonselective cation channels; it blocked the upregulation of ₂ but not of ₁. Despite the upregulation of -subunit expression, sodium pump activity (the functional expression of the enzyme Na⁺,K⁺-ATPase) was not upregulated. On the contrary, it showed a small but significant decrease.

Although specific blockers of SA channels are presently unknown,¹⁹ on the basis of current knowledge, 50 µmol/L Gd³⁺ can be considered an effective blocker of SA, nonselective cation channels if not a highly selective one. Previous studies have shown that 10 µmol/L Gd³⁺ blocks SA, nonselective cation channels in Xenopus oocytes,²⁰ whereas 20 µmol/L blocks similar channels in cultured chick heart cells.¹⁹ In our studies with the Flexercell Strain Unit, we previously found that 100 µmol/L Gd³⁺ blocked SA Ca²⁺ influx in A7r5 cells.²¹ On the basis of the assumption that 50 µmol/L Gd³⁺ effectively blocks SA nonselective cation channels in ASMC, we believe that blocking of ₂ upregulation by Gd³⁺ is consistent with the role of stretch activation of ionic fluxes in regulation of ₂-protein expression. One possible link between stretch and the sodium pump would be stretch-enhanced Na⁺ entry, which would increase the cells' need for Na⁺ extrusion capability. Yamamoto et al²² found that intracellular Na⁺ stimulates the transcription of ₁- and ₂-subunits of the sodium pump in rat ASMC. Since potassium channels can also be activated by stretch in vascular smooth muscle cells,^{1 2 23} another possible involvement of the sodium pump in stretch response might be to compensate for lost intracellular K⁺. Furthermore, because stretch activates Ca influx through both SA channels and L-type Ca²⁺ channels in vascular smooth muscle cells,^{5 24} including those derived from rat aorta,²¹ elevation of cytosolic Ca²⁺ must be considered as a possible mediator of stretch effects on the sodium pump. Several studies have indicated a link between intracellular Ca²⁺ and modulation of the sodium pump.²⁵ More recently, cytosolic Ca²⁺ has been shown to increase the transcription rate of the ₁- and ₂-subunit mRNAs of the sodium pump in outer medullary kidney tubular segments.²⁶

In addition to ionic fluxes, direct mechanical effects may be responsible for the effects we observed, particularly the upregulation of ₁, which was not significantly inhibited by Gd³⁺. Such effects include the direct alteration of membrane surface tension by mechanical strain as well as conformational strain transmitted via cytoskeletal proteins, which might directly modulate the enzymatic activity of integral membrane proteins. Coupling of Na⁺,K⁺-ATPase to the ankyrin-spectrin/fodrin system has been reported.²⁷ Our observed inhibition of pumping activity could be explained through such a mechanism. Although we observed only a 23% reduction of maximally stimulated ouabain-sensitive Rb⁺ influx, if we take into account that both the ₁- and ₂-subunits are upregulated by approximately 50% on a protein basis, the expected maximal turnover rate of the pump would actually decrease more considerably, by approximately 35%. This significant reduction of the efficacy of the pump would have profound effects on cell physiology, and thus upregulating the expression of the catalytic subunits of the pump to compensate would be a fitting response. Since this effect was not Gd³⁺ sensitive, we conclude that it is most likely caused by a direct effect of membrane strain on the pump, which in turn causes the observed upregulation of the ₁-subunit to compensate for the reduction in turnover number. If this is the case, it constitutes an important distinction between the mechanisms controlling the expression of the subunit isoforms.

Although ours is the first report of the effects of mechanical strain on Na⁺,K⁺-ATPase in a cardiovascular cell, other studies have reported upregulation of a variety of proteins associated with cellular responses to mechanical loading, including smooth muscle myosin, elastin, integrin, platelet-derived growth factor, inositol 1,4,5-trisphosphate receptor, and cyclin D.^{28 29 30 31 32 33} Although these responses represent a considerable variety of gene products, all of the affected proteins are intimately involved in cellular responses to mechanical force, its generation, or its tissue distribution. In the present study, we found no significant increase in total protein levels by stretch, and therefore there is no basis to suspect a generalized increase in gene or protein expression.

Na⁺,K⁺-ATPase or the sodium pump is responsible for the maintenance of the cellular membrane potential and can contribute to vascular smooth muscle reactivity and tone.^{34 35 36} Inhibition of the vascular sodium pump and the resultant depolarization of the cellular membrane shift the tone in favor of contraction in the presence of vasoactive substances,³⁷ whereas a stimulation of the pump would favor hyperpolarization and relaxation.^{35 37} Numerous reports in the literature strongly suggest the involvement of Na⁺,K⁺-ATPase in mechanisms underlying hypertension.^{38 39 40} However, the mechanisms responsible for altered sodium pump activity associated with hypertension have not been fully explained. In particular, the role of individual isoforms and the mode of their regulation in hypertension are not known. The expression of the isoforms has been shown to be altered by hormones,^{41 42} potassium deficiency,⁴³ and hypertension.^{12 44} The distribution and the regulation of the isoforms in different tissues vary, suggesting distinct physiological roles for particular isozymes. We have demonstrated that the vascular sodium pump is altered (both stimulated and inhibited) during different stages of deoxycorticosterone acetate hypertension⁴⁵ as well as in other experimental rat models.¹⁰ In addition, others reported an upregulation of the mRNA of the aortic Na⁺,K⁺-ATPase ₁-subunit in two rat models of hypertension.¹² The present study introduces stretch and its resultant signal transduction events as another possible mechanism of regulation of the sodium pump. This mechanism may play an important role in the pathophysiology of hypertension.

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (grant HL-32270 to Dr Songu-Mize); Louisiana State University Medical Center Neuroscience Center of Excellence (Dr Songu-Mize); American Heart Association, Louisiana Affiliate, Inc (Dr Hymel); and Louisiana Stimulus for Excellence in Research (Dr Hymel). The monoclonal antibodies McK1, McB2, and McBX3 were a gift from Dr Kathleen Sweadner (Harvard Medical School, Boston, Mass).

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