INTRODUCTION

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THE CELLS in the cardiovascular system are constantly exposed to an environment of cyclical mechanical strain exerted by blood pressure. Mechanical strain affects cellular morphology and function (16, 51) and is thought to be involved in a variety of pathophysiological processes, such as the myogenic response, ventricular hypertrophy, and arrhythmias (3, 14, 57). Our recent studies have shown that stretch regulates the expression of the Na⁺-K⁺-adenosinetriphosphatase (Na⁺-K⁺-ATPase) -isoforms in cultured rat aortic smooth muscle cells (ASMC) (43). Because Na⁺-K⁺-ATPase participates in the modulation of cardiac and vascular smooth muscle contractility and excitability (5) and changes in its functional state may contribute to the development of hypertension (6, 27, 40, 42), it now becomes important to clarify the underlying mechanisms of the isoform regulation. For this reason, we focused the current study on the stretch-induced signals that are responsible for the regulation of Na⁺-K⁺-ATPase isoform expression.

Na⁺-K⁺-ATPase is a membrane-associated enzyme responsible for the transport of a range of cellular substrates and electrolytes by actively establishing a transmembrane Na⁺ gradient as the primary driving force (37). Two subunits, and , constitute the active pumping unit of this enzyme. Three isoforms of the -subunit (₁, ₂, and ₃) and two of the -subunit (₁ and ₂) have been reported in mammalian cells (21). We have demonstrated that all three -isoforms are expressed in ASMC (35). Although the -isoforms possess distinct affinities to a group of specific inhibitors, i.e., digitalis glycosides, other functional differences are poorly defined. A number of factors and disease conditions, including glucocorticoids (54), insulin (22), and K⁺ deficiency (2), can differentially regulate the expression of these isoforms in various tissues. These isoforms are also differentially expressed in the cardiovascular system in hypertensive rats (15, 34, 44). To test whether the mechanical strain exerted by blood pressure serves as a signal to influence the expression of these isoforms, we used an in vitro model and stretched cultured ASMC and found an upregulation of both ₁- and ₂-isoforms (43). Moreover, the upregulation of the ₂-, but not that of the ₁-isoform, was suppressed by Gd³⁺, a blocker of stretch-activated channels, suggesting that stretch-induced ion fluxes may participate in the regulation of the expression of at least the ₂-isoform (43). However, the details of how mechanical stimuli are translated into regulatory signals still need to be clarified. Numerous studies have identified putative mechanotransducers that translate mechanical stimuli into intracellular biochemical activities in various types of cells (49). Of these mechanotransducers, intracellular Na⁺ and Ca²⁺ have been suggested to be involved in the regulation of Na⁺-K⁺-ATPase isoform expression (29, 56). In the present study, we examine the roles of Na⁺ and Ca²⁺ influx in the regulation of the -subunit expression of Na⁺-K⁺-ATPase in ASMC by stretch.

	METHODS

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Preparation of cultured ASMC. ASMC were isolated from male Sprague-Dawley rats weighing 150-200 g, as described earlier (35). The aortas were isolated and incubated for 30 min in minimal essential medium supplemented with 2.3 mM Ca²⁺, 0.33 mg/ml soybean trypsin inhibitor, and 200 U/ml collagenase (type I). The tissues were then cleaned of adventitia, minced with scissors, and incubated further for 90-120 min in a fresh aliquot of the same medium plus 15 U/ml elastase (type III). The cells were then filtered and resuspended in culture medium (Medium 199 + 10% fetal bovine serum) and seeded in 100-mm petri dishes. Experiments were performed on confluent ASMC between the 3rd and 7th passages.

Stretch of ASMC. The cells were seeded at 5,000/cm² (24,000 cells/culture well) on type I collagen-coated Flex I and Flex II plates (Flexercell International, McKeesport, PA) and grown to confluence under nonstretch conditions for 8-10 days as described previously (43). Flex I plates containing a flexible silicone elastomer substratum were then mounted in the Flexercell strain unit and subjected to 10% average (22% maximum) surface elongation for cycles of 3 s on/3 s off continuously for durations required by the specific experiments. Control Flex II plates, containing the same collagen-coated silicone elastomer substratum plus a rigid polystyrene bottom, were placed in the same culture conditions but not mounted in the strain unit.

Intracellular Na⁺ measurement. After being stretched for 1 h, 2 h, 1 day, 2 days, or 4 days, the cells were washed six times with ice-cold 0.15 M LiCl (EM Science Suprapur) and treated with 0.6 ml 50 µM nystatin (Sigma) dissolved in water in each well overnight to release intracellular Na⁺. An Na⁺ standard curve ranging from 0 to 174 µM [0-400 parts/billion (ppb)] was prepared using a standard solution (Atomic Absorption Standard, EM Science), and the results are presented in Fig. 1. For the intracellular Na⁺ measurements, 0.5 ml solution was taken from each well and diluted to 5 ml with water for measurement with an ICP-Emission Spectrophotometer (Perkin-Elmer Optima 3000) at 589-nm wavelength. The water used in the intracellular Na⁺ measurements was obtained from a Milli-Q+ apparatus (Millipore) and had a resistance of 18.2 M/cm. Intracellular volume of cell monolayers was determined by a methylglucose (MG) uptake method (18). After the medium was aspirated and washed with 1 ml of glucose-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline solution [HBSS; containing (in mM) 130 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 2 pyruvic acid, 10 HEPES, pH = 7.4] at room temperature, 0.5 ml of glucose-free HBSS containing 0.2 µCi/ml of 3-O-[¹⁴C]MG and 10 mM cold 3-O-MG was added and incubated for 30 min at room temperature. The intracellular volume for each condition was determined in triplicate. After the incubation period, the wells were rinsed twice with 1 ml ice-cold glucose-free HBSS containing 1 mM phloretin and 1% (vol/vol) ethanol. The cells in each well were digested with 0.5 ml of HBSS containing 0.1% Triton X-100 solution, and samples were taken for protein determination and scintillation counting. The intracellular volume was determined to be 6.64 ± 0.13 µl/10⁶ cells (n = 6). Intracellular Na⁺ concentrations of the samples were derived based on the amount of Na⁺ extracted from the cells, and the total intracellular volume per well as determined by the above methods.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Standard curve for Na+ measurement (r2 = 1.000). Na+ concentrations of the samples were between 108.6 and 173.6 µmol/l [250-400 parts/billion (ppb)].

Treatment of ASMC with Gd³⁺ and isradipine. To examine the effect of Gd³⁺ on stretch-induced intracellular Na⁺ changes, we treated some of the cells with 50 µM Gd³⁺ during the stretch. To investigate the effect of Ca²⁺ entry through L-type channels, isradipine was dissolved in dimethyl sulfoxide (DMSO) and added to the cell culture medium to make the final concentration of isradipine 1 µM. For control experiments, the same amount of DMSO (final concentration 0.01%) was added to the medium.

Preparation of samples for Western blot analysis. After the stretch protocol, ASMC from individual culture wells were washed with cold phosphate-buffered saline. The plates were then scraped in a homogenization buffer [in mM: 250 sucrose, 50 tris(hydroxymethyl)aminomethane (Tris), 1 EDTA, pH 7.4]. Initial centrifugation was at 20,000 g for 1 min at 4°C. The pellet was resuspended in a lysis buffer (in mM: 140 NaCl, 10 Tris, 1.5 MgCl₂ and 0.5% Triton X-100, pH 8.6) and centrifuged at 20,000 g for 3 min at 4°C. The supernatant was used for electrophoresis/Western blot analysis. Protein concentrations were determined by the bicinchoninic acid protein assay (38) using bovine serum albumin as a standard. Final concentrations of the protein in the individual samples were in the range of 2-3 mg/ml.

Gel electrophoresis and immunoblotting. First, standard curves for the ₁-isoform (cell extract protein ranging from 0 to 30 µg) and the ₂-isoform (cell extract protein ranging from 0 to 40 µg) were constructed to determine the linear range and the appropriate protein amount for quantitation of each isoform (Fig. 2). For experimental sample measurements, 10 and 20 µg of the cell extract protein were loaded 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% sodium dodecyl sulfate (20) and then transferred to polyvinylidene fluoride membrane by electroblotting (47). After preincubation in Tris-buffered saline (in mM: 20 Tris · HCl, 137 NaCl, pH 7.5) containing 5% (wt/vol) nonfat dried milk (Carnation) and 1.0% (vol/vol) Tween 20 for 1 h at room temperature, the blots were probed with monoclonal antibodies, McK1 and McB2, directed against the ₁- and ₂-isoforms of Na⁺-K⁺-ATPase, respectively, as described before (34). The specificity of McK1, anti-₁, and McB2, anti-₂, for corresponding subunit proteins is well established and published (13, 48). McK1 does not crossreact with ₂ or ₃, and McB2 does not crossreact with ₁ and ₃. The blots were treated with the secondary antibody, horseradish peroxidase-labeled sheep anti-mouse immunoglobulin (Amersham). Blots were then treated with enhanced chemiluminescence reagent (Amersham) and exposed to X-ray film for visualization of the bands.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Standard curves for quantitation of the 1 (A)- and 2 (B)-isoforms. Aliquots of aortic smooth muscle cell extracts were used for the standard curves. The r2 is 0.992 for the 1 curve and 0.994 for the 2 curve. OD, optical density.

Quantitation of the Western blot analysis. The fluorograms were scanned and the intensity of the bands for ₁ and ₂ was quantified as optical density units using a computerized image analyzer (M-2 model, Imaging Research). The highest band density measured, 1.3 optical density units (OD), still allowed a reasonable level of light transmission for quantitation and was within the linear range of the standard curve (Fig. 2). To normalize band density values from different blots, an internal control sample was loaded to each gel and the density was designated as 1.

Statistical analysis. Analysis of variance (ANOVA) followed by a Newman-Keuls test was used where applicable. A confidence limit of 95% was considered significant.

	RESULTS

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To determine whether Na⁺ entry is involved in stretch-induced upregulation of the -isoforms we investigated the effect of stretch on intracellular Na⁺ levels. Intracellular Na⁺ concentrations of the cells were measured after 1 h, 2 h, 1 day, 2 days, and 4 days of cyclical stretch. The Na⁺ standard curve had a good linear relationship in the range from 0 to 174 µM (0-400 ppb) (Fig. 1). A significant increase in intracellular Na⁺ was detected after 1- and 2-h stretch periods (30 and 23%, respectively; Fig. 3). Furthermore, 50 µM Gd³⁺ blocked the stretch-induced intracellular Na⁺ increase. However, no difference was found between stretch and nonstretch groups after 1-, 2-, or 4-day stretch periods, with or without Gd³⁺ (Fig. 3). A slightly higher basal Na⁺ concentration observed at 1 day may be due to the design of the experiments, which include several steps such as washing and extraction of the cells at different time points. However, an ANOVA among the treatment groups at this time point indicated no effect of stretch or Gd³⁺ on intracellular Na⁺. Table 1 summarizes the results of statistical analyses among the individual groups.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of stretch and Gd3+ (Gd) on intracelluar Na+ in aortic smooth muscle cells (ASMC). * Significant difference from the control group at the same time point (P < 0.001). ANOVA and Newman-Keuls test were used for multiple and individual comparisons, respectively (n = 6 for each condition).

To explore the role of Ca²⁺ entry in regulation of the -isoforms, we treated the cells with the calcium channel blocker isradipine (1 µM) while stretching the cells and determined the expression of the -isoforms. The concentration of isradipine was chosen on the basis of a related study (31) in which 1 µM isradipine blocked 90% of ⁴⁵Ca²⁺ entry stimulated by the same cyclical stretch protocol used here. Stretching the cells for 2 days upregulated both the ₁- and ₂-isoforms (Figs. 4 and 5). After a 2-day period, isradipine inhibited the expression of ₁-isoform in cells under nonstretch conditions by 37% but failed to suppress the stretch-induced upregulation of this isoform (Fig. 4). The same treatment did not affect the ₂-isoform expression, either with or without stretch (Fig. 5). Extension of the treatment to a 4-day period exhibited similar results (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of 2-day stretch and isradipine (Isr) treatment on the expression of 1-isoform of Na+-K+-ATPase in cultured ASMC. A: representative immunoblot of the ASMC membrane extracts. Rat kidney homogenate was used as a control. B: quantitation of the 1-isoform protein abundance. Bar graphs represent mean ± SE for band densities (OD) from immunoblots for 1-isoform protein expression. * Significant difference (P < 0.05; large * for the effect of the stretch and small * for the effect of isradipine). ANOVA and Newman-Keuls test were used for multiple and individual comparisons, respectively (n = 6 for each condition).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of 2-day stretch and isradipine treatment on the expression of 2-isoform of Na+-K+-ATPase in cultured ASMC. A: representative immunoblot of the ASMC membrane extracts. Rat brain homogenate was used as a control. B: quantitation of the 2-isoform protein abundance. Bar graphs represent mean ± SE for band densities (OD) from immunoblots for 2-isoform protein expression. * Significant difference (P < 0.05). ANOVA and Newman-Keuls test were used for multiple and individual comparisons, respectively (n = 6 for each condition).

Stretching the confluent cells for up to 4 days did not affect the total protein content per well or the yield of membrane protein extraction. In addition, the isradipine treatment had no effect on these variables (Table 2).

	DISCUSSION

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In this study, cyclical stretch for 2 days or longer upregulated the ₁- and ₂-isoform expression of Na⁺-K⁺-ATPase in cultured ASMC, confirming our recent findings (43). In the previous study we also found that Gd³⁺ blocks the stretch-induced upregulation of ₂- but not ₁-isoform (43). In the present study, we found that intracellular Na⁺ exhibited a transient elevation after 1 and 2 h of stretch and returned to baseline levels after prolonged stretch for 1, 2, and 4 days. To our knowledge, this is the first direct demonstration of stretch-induced intracellular Na⁺ increase in ASMC. We also demonstrated in this study that Gd³⁺ blocks this transient increase in intracellular Na⁺ by stretch. In addition, the L-type channel blocker isradipine had no apparent effect on stretch-induced upregulation of ₁- and ₂-isoforms. Taken together, these findings strongly suggest a long-term regulatory role for intracellular Na⁺ in the upregulation of ₂-isoform expression. Under our experimental conditions, stretch for 4 days or less does not evoke stretch-induced cell growth as measured by total protein per well or extracted membrane protein per well, as shown here (Table 2) and in the previous study (43). Because neither total protein nor extracted protein showed any significant change, the observed upregulation or downregulation of Na⁺-K⁺-ATPase -isoforms is selective and cannot be ascribed to growth-related protein synthesis.

The major function of Na⁺-K⁺-ATPase is to transport Na⁺ out of and K⁺ into the cell to maintain transmembrane ion gradients, and it is therefore not surprising that numerous studies have demonstrated the importance of intracellular Na⁺ on the regulation of Na⁺-K⁺-ATPase in ASMC as well as in other cell types, as measured by enzyme activity, binding sites, and gene expression. It appears that various routes of Na⁺ entry lead to similar responses. As long as there is an increase in intracellular Na⁺, the cell will have to increase its Na⁺-K⁺-ATPase transport capacity to maintain a physiological intracellular Na⁺ concentration as a compensatory measure. Experimental manipulations, such as chronic treatment with ouabain (7, 10), steroids (24, 25), low-K⁺ medium (28), and monensin (39), in ASMC as well as in other cell types, almost invariably lead to stimulation of Na⁺-K⁺-ATPase, as measured by enzyme activity, binding sites, or gene expression. All of the above approaches converge on the resulting intracellular Na⁺ increase, which is thought to be the initial signal for the upregulation (8). In fact, Yamamoto et al. (56) showed that increasing intracellular Na⁺ by veratridine, an Na⁺ channel activator, stimulates the transcription of Na⁺-K⁺-ATPase ₁- and ₁-isoform genes in ASMC. In further support of this notion, we detected a transient intracellular Na⁺ increase after 1- and 2-h stretch periods. Because in this study Gd³⁺ blocked the stretch-induced intracellular Na⁺ increase, the source of increased Na⁺ is most likely the increased activity of stretch-activated, nonselective cation channels. The role of intracellular Na⁺ as a short-term regulator of this enzyme is well established, that is, an immediate elevation of intracellular Na⁺ initially stimulates the pumping rate of the enzyme within seconds to minutes, thus reestablishing the transmembrane gradient (37). In our experiments, the elevated state of intracellular Na⁺ lasted at least 2 h, suggesting that the increased turnover rate of the enzyme was not able to keep up with the influx of Na⁺. The return of intracellular Na⁺ to baseline levels after a prolonged period of cyclical stretch (1 day) in our study could be due to the increased production of new pump sites, as supported by increased production of the catalytic subunits ₁ and ₂. Similar to our findings, Rayson and Edelman (30) reported that, in the kidney, superfusion of rat outer medullary tubular segment with ouabain doubled intracellular Na⁺ levels in 2-4 h. However, after 18 h of superfusion, intracellular Na⁺ was restored to control levels, and the Na⁺-K⁺-ATPase activity increased by 60% (30, 31), suggesting that the initial intracellular Na⁺ surge was the driving force for the increase in activity. Preliminary results from our laboratory indicate that mRNA for ₁ and ₂ increases after a 6-h stretch, suggesting a regulation at the gene expression level by stretch (unpublished observations).

It has been demonstrated that stretch-induced ion fluxes are mediated by stretch-activated channels (4, 12, 18, 33). In ASMC, the stretch-activated channels fall into two classes: one class includes nonselective cation channels that are responsive to mechanical stimuli and, once activated, are permeable to Na⁺, Ca²⁺, and K⁺; the other class consists of Ca²⁺-activated K⁺ channels (11, 18, 23). A considerable fraction of L-type channels is also activated by stretch, most likely due to membrane depolarization by Na⁺ entry (32). Gd³⁺ is a potent blocker of stretch-activated channels, regardless of the cell type (18, 26, 33, 58).

Although studies have shown that L-type Ca²⁺ channels play an important part in stretch-induced Ca²⁺ influx in isolated ASMC and arteries (23, 45) and ~90% of stretch-activated ⁴⁵Ca²⁺ entry is blocked by isradipine in ASMC (A7r5 cell line) subjected to a similar stretch protocol to the one used in this study (32), our data would preclude entry of Ca²⁺ through L-type channels as a mechanism for stretch-dependent regulation of ₁ and ₂ expression. Our data also suggest that, if there is a role for intracellular Ca²⁺ in stretch-induced regulation of Na⁺-K⁺-ATPase -isoforms, the source of the Ca²⁺ would more likely be Ca²⁺ release from intracellular stores. Our experimental design and apparatus preclude the measurement of intracellular Ca²⁺ because of continuous movement of the cells. However, increased intracellular Ca²⁺ after stretch has been observed in various types of cells and is thought to be responsible for many stretch-associated cellular responses (1, 36, 55). Recently, Tanaka et al. (45) demonstrated that the intracellular Ca²⁺ released from ryanodine-sensitive and -insensitive storage sites accounts for stretch-induced contractions in canine cerebral arteries. In addition, Borin et al. (9) reported that, in ASMC (A7r5 line), increasing intracellular Na⁺ with ouabain treatment augmented the release of intracellular Ca²⁺ evoked by thapsigargin, serotonin, and vasopressin. The intracellular Ca²⁺ increase did not occur in an Na⁺-free medium and was not prevented by the Ca²⁺ channel blocker verapamil. It has also been shown by Rayson (29) in outer medullary kidney tubular segments that intracellular Ca²⁺ elevations increase the transcription rate of the ₁- and ₁-subunit mRNAs. Therefore, the ineffectiveness of isradipine in inhibiting stretch-induced upregulation of the Na⁺-K⁺-ATPase -subunit observed in our study does not exclude a role for intracellular Ca²⁺ in the regulatory process.

The membrane Na⁺/Ca²⁺ exchange mechanism makes it difficult to differentiate between the roles of intracellular Na⁺ and Ca²⁺ in the regulation of Na⁺-K⁺-ATPase -subunit expression. However, because intracellular Na⁺ concentration is on the order of millimolar and intracellular Ca²⁺ on the order of nanomolar (6), even a slight intracellular Na⁺ elevation can cause a considerable intracellular Ca²⁺ increase. Thus it is conceivable that even a slight rise in intracellular Na⁺ caused by stretch-activated channels could be amplified into a stronger intracellular Ca²⁺ signal via Na⁺/Ca²⁺ exchange.

Stretch-induced intracellular Na⁺ and Ca²⁺ changes and the resultant Na⁺-K⁺-ATPase -isoform upregulation may be relevant to the pathophysiology of hypertension. Abnormalities of both intracellular Na⁺ and Ca²⁺ have been implicated in hypertension. Many studies have indicated changes in membrane Na⁺ flux and Na⁺-K⁺-ATPase activity in hypertension (17, 52, 53). Intracellular Ca²⁺ has also been found to be increased in hypertension (46, 50). If the stretch-induced upregulation of Na⁺-K⁺-ATPase -subunit expression in ASMC applies to the vasculature in vivo, this would result in elevated transport capacity in response to high blood pressure. This adaptation can be seen as an effort to reduce blood vessel tone by promoting Na⁺ efflux and therefore reducing intracellular Ca²⁺ through Na⁺/Ca²⁺ exchange. Earlier studies from our laboratory have shown enhanced vascular Na⁺ pump activity in other experimental models of hypertension (40, 41).

In conclusion, we have demonstrated that intracellular Na⁺ is elevated after a cyclical stretch is applied to aortic smooth muscle cells in culture. This elevation of Na⁺ lasts at least 2 h and can be blocked by Gd³⁺. Isradipine did not affect stretch-induced upregulation of either -isoform, suggesting that Ca²⁺ influx through L-type channels does not play a role in the regulatory process. We thus propose that transient intracellular Na⁺ increase mediated by stretch-activated channels may be the initial signal for stretch-induced, long-term upregulation of the ₁- and ₂-isoforms of Na⁺-K⁺-ATPase.