INTRODUCTION

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NA⁺-k⁺-atpase, or the Na pump, is a ubiquitous integral protein of the outer plasma membrane of animal cells (22, 27). The electrochemical gradient produced by this enzyme plays a role in several cellular functions, including maintenance of resting membrane potential of most tissues, osmotic balance of the cell, and generation of the Na⁺ gradient, which supplies the energy that fuels Na-coupled transporters (17). The functional macromolecule consists of two dimers composed of noncovalently interacting - and -subunits and a smaller -subunit. To date, four -subunit and three -subunit isomers have been identified (4, 46). Aortic smooth muscle cells (ASMC) express three -isoforms: ₁, ₂, and ₃ (39). The -subunit is responsible for catalytic activity, whereas the -subunit appears to be involved in the insertion of the - complex into the membrane (18).

The regulation of vascular Na⁺-K⁺-ATPase and the functional significance of the molecular isoforms of the enzyme in disease states such as hypertension are not fully understood. However, alterations in vascular smooth muscle Na⁺-K⁺-ATPase activity and its protein and gene expression are observed in cardiovascular tissues in several animal models of experimental hypertension (19, 29, 34, 41, 42). In the vascular tissue, Na pump activity appears to be enhanced in hypertension (41, 42). To address the contribution of the mechanical strain component of elevated blood pressure to Na pump regulation, we used an in vitro smooth muscle cell culture model and report that chronic mechanical strain contributes to the upregulation of two catalytic -subunits of Na⁺-K⁺-ATPase. This effect of stretch was manifested as an increase in protein (28, 45) and mRNA expression (40) of the ₁- and ₂-isoforms of Na⁺-K⁺-ATPase, suggesting that a chronically applied cyclic mechanical strain can regulate vascular Na⁺-K⁺-ATPase activity.

The purposes of the current study were to determine whether cyclic stretch affects the activity of the Na pump when applied acutely and, if so, what mechanisms may be involved in the short-term effect of stretch on Na pump function in vascular smooth muscle cells.

	METHODS

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Preparation of cultured ASMC. ASMC were isolated from four to five male Sprague-Dawley rats weighing 200-250 g as described earlier (43). Briefly, aortas were excised and placed in culture medium (medium 199) containing 10% fetal bovine serum and 1% penicillin-streptomycin. After three washes in serum- and antibiotic-free medium 199, the aortas were incubated for 30 min at 37°C in medium 199 supplemented with 0.83 mg/ml bovine serum albumin, 0.33 mg/ml soybean trypsin inhibitor, and 25 U/ml collagenase. The tissues were then cleaned of adventitia, minced with forceps, and incubated further for ~3 h at 37°C in a fresh aliquot of the same medium plus 15 U/ml elastase. Afterward, the cells were washed with complete medium four times and centrifuged at 1,500 rpm (model 1-89, Beckman TH-4) for 10 min. The final pellet was resuspended in ~7 ml of culture medium, and the cell suspension was seeded in a T-25 flask and grown to confluency. Cells were used between the third and seventh passages.

Stretch protocol. The cells were seeded at 1 × 10⁵ cell/well on six-well collagen-coated BioFlex plates containing a flexible silicone elastomer substratum and grown to confluency under nonstretch conditions for 8-10 days. BioFlex plates were then mounted in a Flexercell Strain Unit (Flexercell International; McKeesport, PA) and subjected to 20% surface elongation with 3-s stretch/3-s relaxation cycles continuously for the 2-30 min required for the specific experiments. Control cells were placed in the same experimental conditions but were not stretched.

Measurement of Na pump activity in ASMC. Na pump activity was determined in cultured ASMC using a modified ouabain-sensitive ⁸⁶Rb⁺ uptake technique as previously described (43). When the cells were ready for stretch experiments, the medium was removed, and the cells were washed with Krebs-Henseleit buffer [composed of (in mM) 27.2 NaHCO₃, 119 NaCl, 1 NaH₂PO₄, 1.2 MgSO₄, 1.8 CaCl₂, 11 dextrose, and 5 KCl; pH 7.4, bubbled with 5% CO₂-95% O₂] and supplied with 1 ml of fresh Krebs solution. Some wells contained 2 mM ouabain for determination of ouabain-resistant ⁸⁶Rb⁺ uptake. After a 5-min preincubation period with ouabain, ⁸⁶RbCl [~10⁶ counts per minute (cpm) per well, 1.11-1.35 µCi/ml] was added to start the uptake reaction, at which time the stretch was also initiated. ⁸⁶Rb⁺ uptake reaction was terminated after 2-30 min of stretch, as determined by the individual protocol, by stopping the stretch and removing the incubation medium. The cells were then washed twice with Krebs buffer and lysed in 0.1% SDS in 0.5 N NaOH. An aliquot of the lysed cells was removed for scintillation counting, and another sample was saved for protein determination. Na pump activity was determined by subtracting the ouabain-resistant uptake, i.e., uptake of ⁸⁶Rb⁺ in the presence of 2 mM ouabain, from the total uptake. Na pump activity is expressed as nanomoles of (⁸⁶Rb⁺ + K⁺) taken up per milligram of cell protein per minute.

Measurement of intracellular Na+. Intracellular Na⁺ was measured as described in an earlier study (28). After the stretch protocols, the cells were washed six times with ice-cold 0.15 M LiCl Suprapur and treated with 0.6 ml of 50 µM nystatin in each well for 2 days to release intracellular Na⁺. In our pilot experiments, treatment of ASMC with 50 µM nystatin was very effective in releasing intracellular Na⁺. A Na⁺ standard curve was prepared using a standard solution (Atomic Absorption Standard, EM Science). For the intracellular Na⁺ measurement of the samples, a 0.5-ml aliquot was taken from each well and diluted to 8 ml with water for measurement with an ICP emission spectrophotometer (Perkin-Elmer Optima 3000) at a wavelength of 589 nm. 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. The intracellular volume of cell monolayers was determined by the methyl-D-glucose (MG) uptake method (24). Briefly, the medium was aspirated and washed with 1 ml of glucose-free HEPES-buffered saline solution [composed of (in mM) 130 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 2 pyruvic acid, and 10 HEPES; pH 7.4] at room temperature. HEPES-buffered saline solution (0.5 ml) containing 0.2 µCi/ml of 3-O-[methyl-¹⁴C]MG and 10 mM of cold 3-O-MG was then 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 of ice-cold glucose-free HEPES-buffered saline solution containing 1 mM phloretin and 1% (vol/vol) ethanol. The cells in each well were digested with 0.5 ml of HEPES-buffered saline solution containing 0.1% Triton X-100 solution, and samples were taken for protein determination and scintillation counting. The intracellular volume was determined to be 6.67 µl/10⁶ cells. 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 methods described above.

Statistical analysis. ANOVA followed by Scheffé's test was used. A confidence limit of 95% was considered significant. Experiments were designed with randomization of drug treatments using several six-well Bioflex culture plates per experiments. n represents number of wells for each treatment.

	RESULTS

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To determine the effect of short-term cyclic stretch on the functional counterpart of the Na⁺-K⁺-ATPase, Na pump activity, in cultured ASMC, we measured ouabain-sensitive uptake of ⁸⁶Rb⁺ (+ K⁺) into the cells during the application of 2- to 30-min cyclic stretch. Na pump activity was stimulated by 125, 70, 65, and 35% above baseline nonstretch controls at the end of 2-, 5-, 10-, and 30-min cyclic stretch, respectively (Fig. 1). The baseline nonstretch Na pump activity was 4.47 ± 0.21 nmol · mg protein¹ · min¹ (n = 5) at the 2-min time point, and the magnitude of stimulation by stretch appeared to be reduced during the course of 30 min from 125% at 2 min to 35% at 30 min above baseline activity (Fig. 1). The active K⁺ uptake representing Na pump activity was linear in nonstretch controls for the duration of 30 min. The ouabain-resistant uptake of ⁸⁶Rb⁺ (+ K⁺) was not altered by stretch or any of the drug treatments and constituted 30-40% of the total uptake.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Rate of Na pump during the course of a 30-min stretch period in aortic smooth muscle cells in culture. Bars represent means ± SE of Na pump activity measured as the ouabain-sensitive 86Rb+ (+ K+) uptake rate under control nonstretch and stretch conditions, as described in METHODS. *Significant differences, P < 0.05, between stretch and corresponding nonstretch controls. ANOVA and Scheffé's post hoc test were applied; n = 5 cell culture wells for each group.

To determine whether the stretch-induced stimulation of the enzyme was reversible, we designed the following experiment: two experimental groups were set up, group 1 and group 2. In group 1, Na pump activity was measured during a 10-min stretch, whereas in group 2, Na pump activity was measured 1 h after 10-min stretch. The corresponding controls for each group were not stretched. Stretch stimulated Na pump activity in group 1 compared with nonstretch controls (Fig. 2). However, in group 2, there was no difference between stretch and nonstretch controls. Baseline Na pump activity appeared to be slightly lower in group 2 compared with group 1 (Fig. 2). This is not suprising because the cells in group 2 were exposed to different experimental conditions, i.e., ⁸⁶Rb⁺ uptake was measured 1 h after stretch.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   The reversibility of stretch-induced stimulation of the Na pump. Bars represent means ± SE of Na pump activity measured as the ouabain-sensitive 86Rb+ (+ K+) uptake rate under control nonstretch and stretch conditions, as described in METHODS. In group 1, Na pump activity was measured during a 10-min stretch period. In group 2, Na pump activity was measured 1 h after a 10-min stretch period. There are corresponding nonstretch controls for each group. *Differences between nonstretch vs. stretch in group 1 and both stretch and nonstretch in group 2, P < 0.05. ANOVA and Scheffé's post hoc test were applied; n = 10 cell culture wells for each experimental condition.

Because stimulation of stretch-activated channels allows entry of Na into the cell, we measured the levels of intracellular Na⁺ at several time points during stretch. We found that intracellular Na⁺ was also elevated significantly 220, 122, 60, and 75% above baseline nonstretch values (12.38 ± 0.38 mM, n = 9) at 2, 5, 10, and 30 min after stretch, respectively (Fig. 3). Gd (50 µM) inhibited the stretch-induced increase in intracellular Na⁺ at 2 min (Fig. 3).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Intracellular Na+ concentrations during the course of 30-min stretch period in aortic smooth muscle cells in culture: the effect of Gd. Bars represent means ± SE of intracellular Na+ concentrations under control nonstretch and stretch without or with (+) Gd. Na measurements were made at the end of each stretch period, as described in METHODS. *Significant difference, P < 0.05, between stretch and corresponding nonstretch controls. +Significant differences between 2-min stretch vs. 2-min (+) Gd and 0-min (+) Gd. ANOVA and Scheffé's post hoc test were applied; n = 9 cell culture wells for each group.

Blocking the stretch-activated channels with 50 µM Gd or the Na⁺/H⁺ exchange pump inhibitor dimethyl amiloride (DMA; 50 µM) (36), alone or together, did not affect the stretch-activated increase in Na pump activity (Fig. 4).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effect of Gd and dimethyl-amiloride (DMA) on Na pump activity under nonstretch and stretch conditions. Bars represent means ± SE of the Na pump activity rate measured during a 10-min nonstretch control or stretch conditions. Cells in designated wells were preincubated for 10 min with 50 µM Gd, 50 µM DMA, a combination of the two drugs, or vehicle before the stretch regimen. Na pump activity was measured as described in METHODS. *Significant difference, P < 0.05, between stretch and corresponding nonstretch controls. ANOVA and Scheffé's post hoc test were applied; n = 5-8 cell culture wells for each group.

Experiments using pharmacological inhibitors, glybenclamide and nifedipine, indicated no involvement of K⁺ channels and voltage-dependent L-type Ca²⁺ channels, respectively (data not shown). In addition, 50 µM PD-98059, which inhibited stretch-induced mitogen-activated protein kinase (MAPK) phosphorylation (44), had no effect on stretch-induced activation of the Na pump. Protein kinase C (PKC), phospholipase C (PLC), and protein phosphatases did not appear to be involved in stretch-induced stimulation of the Na pump (data not shown). However, 10 µM H-89 (20), a protein kinase A (PKA) inhibitor, attenuated but did not reverse the stimulatory effect of stretch on Na pump activity (Fig. 5). H-89 at a higher concentration (20 µM) did not have an additional effect.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Effect of the protein kinase A inhibitor H-89 on Na pump activity. Bars represent means ± SE of the Na pump activity rate without or with H-89. Cells in designated wells were preincubated for 30 min with 10 µM H-89 or vehicle before the stretch regimen. Na pump activity was measured as described in METHODS. *Significant differences between 10-min stretch vs. 0-min control and 0-min H-89. **Differences of 10-min H-89 vs. 10-min vehicle control and 0-min with and without H-89, P < 0.05. ANOVA and Scheffé's post hoc test were applied; n = 8 cell culture wells for each group.

Another possibility for stretch-induced stimulation is that Na pump function is modified by a direct effect of mechanical stimulus on this integral membrane protein. This may be mediated by conformational changes occurring perhaps with the participation of cytoskeletal proteins. Therefore, to determine the involvement of the actin cytoskeleton, we treated the cells with 0.4 µM cytochalasin D (38) for 1 h, an agent that depolymerizes actin microfilament bundles (9). This treatment fully prevented the effect of stretch on the Na pump (Fig. 6A). Pretreatment of the cells for 1 h with 15 µM phalloidin (30), an agent that stabilizes actin in its polymerized form (9), prevented the effect of cytochalasin D on stretch-induced activation of the Na pump. Cytochalasin D and phalloidin had no effect on the Na pump activity of nonstretch control cells (Fig. 6A). Treatment with cytochalasin D did not affect the levels of intracellular Na⁺ (data not shown). To determine the involvement of microtubules, we treated the cell for 5 h with 3 µM colchicine (38), an agent that disrupts microtubule assembly (11), and measured the Na pump activity during a 10-min stretch. Colchicine had no effect on the stretch-induced stimulation of the Na pump (Fig. 6B). A higher concentration (5 µM) of colchicine also had no effect. Cell viability measured as trypan blue exclusion was not affected by these drugs under any of the conditions described in this protocol. There was a 94-97% viability (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Effect of cytochalasin D (Cyto), phalloidin (Phall), and colchicine on Na pump activity under nonstretch and stretch conditions. A: bars represent means ± SE of the Na pump activity rate measured during 10-min nonstretch control or stretch conditions. Cells in designated wells were preincubated for 60 min with 0.4 µM cytochalasin D, 15 µM phalloidin, 60 min with phalloidin, followed by 60 min with cytochalasin D, or vehicle. After preincubation, the cells were washed, supplied with fresh incubation medium, and then stretched for 10 min. B: bars represent means ± SE of the Na pump activity rate without or with colchicine. Cells in designated wells were preincubated for 5 h with 3 µM colchicine or vehicle before the 10-min stretch regimen. Na pump activity was measured as described in METHODS. *Significant difference, P < 0.05, between stretch and corresponding nonstretch controls. ANOVA and Scheffé's post hoc test were applied; n = 9 cell culture wells for each group.

To investigate whether vesicular transport was occurring, we used wortmannin, an agent that inhibits phosphatidylinositol 3-kinase (PI3K) activity. Activation of PI3K is associated with the initiation of vesicular trafficking and targeting of proteins to specific cell compartments (12). Pretreatment of cells with 100 nM wortmannin (32) for 40 min completely blocked the stretch-induced increase in Na pump activity (Fig. 7).

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Effect of wortmannin on Na pump activity under nonstretch and stretch conditions. Bars represent means ± SE of the Na pump activity rate without or with wortmannin. Cells in designated wells were preincubated for 40 min with 100 nM wortmannin or vehicle before the 10-min stretch regimen. Na pump activity was measured as described in METHODS. *Significant differences between 10-min stretch with and without wortmannin and 0-min stretch with and without wortmannin, P < 0.05. ANOVA and Scheffé's post hoc test were applied; n = 9 cell culture wells for each group.

	DISCUSSION

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We demonstrated that cyclic stretch increases Na pump activity acutely, within 2 min, which was not entirely due to an elevation of intracellular Na⁺. The stretch-induced activation of the Na pump was not prevented by the inhibition of Na entry through stretch-activated channels by Gd or through the Na⁺/H⁺ exchange pump into the cell. Likewise, this effect of stretch on Na pump activity was not affected by the inhibition of several ion channels and signal transduction pathway enzymes. Depolymerization of actin filaments and inhibition of vesicular transport, however, successfully prevented this stretch-induced activation. In addition, inhibition of PKA produced a small but significant degree of reversal of stimulation of Na pump activity.

The cells of the blood vessels are constantly exposed to cyclical mechanical strain exerted by blood pressure. Mechanical strain affects cellular morphology and function (21, 31, 47) and is thought to be involved in a variety of physiological and pathophysiological processes, such as the myogenic response, hypertrophy, and proliferation (10, 47). The sensing and transduction of a mechanical stimuli may involve activation of several cellular structures or mechanisms. These include the putative mechanosensors and mechanotransducers such as surface glycoproteins, integrins, the cytoskeleton, stretch-activated channels and other ion channels, and second and third messengers that act on various aspects of cell function (10). Mechanical stimuli, which result in vascular wall stretch, affect the responsiveness of vascular smooth muscle cells via stretch-activated ion channels (3, 14, 23, 37). Stretch-activated channels are permeable to Na⁺, K⁺, and Ca²⁺. An increase in intracellular Na⁺ stimulates Na pump activity by increasing the turnover rate of the enzyme. Therefore, our first attempt was to verify that intracellular Na⁺ increases as a result of short-term cyclic stretch, which may result in activation of the Na pump. Indeed, intracellular Na⁺ significantly increased during the first 30 min of the stretch, as shown in Fig. 3. Whereas Gd prevented the increase in intracellular Na⁺ concentration (Fig. 3), it did not prevent the stretch-stimulated increase in Na pump activity (Fig. 4). Blocking the Na⁺/H⁺ exchange pump was also without effect (Fig. 4). Clearly, other factors were also involved in activation of the Na pump when stretched acutely. Although Na entering through stretch-activated channels or the Na⁺/H⁺ exchanger was not involved, we cannot eliminate the possibility that intracellular Na⁺ may play a role in stretch-induced stimulation. Waters et al. (48) recently demonstrated that a stimulation of Na⁺-K⁺-ATPase via membrane translocation of the ₁-subunit occurs with stretch in rat alveolar epithelial cells. In that cell system, Na appeared to be an important component of the translocation, in contrast with what we observed in our studies with ASMC. It is entirely possible that details of the mechanisms of the translocation are differently regulated in these two functionally different cells.

To rule out the possible contribution of the activation of K⁺ channels and voltage-gated Ca²⁺ channels, which may be indirectly affected by stretch-activated channels, we used glybenclamide and nifedipine to block K⁺ and Ca²⁺ channels, respectively, before application of stretch. Again, these agents had no effect (data not shown).

Apart from the well-recognized contributions of Na⁺ and K⁺, the components of certain intracellular signaling pathways participate in regulation of Na pump activity (1, 15, 16). Therefore, it was reasonable to hypothesize that a signal transduction cascade initiated by a mechanical signal somehow results in the regulation of Na⁺-K⁺-ATPase. Among the signal transduction pathways, the MAPK pathway is a likely candidate, because it is activated by mechanical strain (25, 26, 33). We reported that p42 and p44 are phosphorylated by the stretch regimen applied in this experiment and that the duration of this effect was similar to that of activation by epidermal growth factor. Moreover, inhibition of MAPK kinase by PD-98059 prevented the effect of stretch on MAPK activation (44). However, inhibition of the pathway with PD-98059 did not block the action of stretch on Na pump activity, suggesting that MAPK pathway involvement is unlikely (data not shown).

The components of the PLC and adenylyl cyclase signaling pathways also participate in regulation of Na pump activity (1). Feschenko and Sweadner (15) demonstrated a conformation dependence of phosphorylation of rat kidney Na⁺-K⁺-ATPase by PKC and PKA. Fisone et al. (16) also reported a synergistic regulation of choroidal plexus Na⁺-K⁺-ATPase by PKC and PKA pathways. Furthermore, receptor-mediated short-term regulation of the Na pump by various agonists, such as stimulation by -adrenergic agonists (2) and inhibition by dopamine (7, 8), has been shown to involve PKA and PKC enzymes. Therefore, we used several agents to inhibit or stimulate the activities of PKC, PLC, and PKA in ASMC before the stretch protocol. Inhibitors of PKC, calphostin and staurosporin, and an inhibitor of PLC, U-73122, had no effect on stretch-induced Na pump activity (data not shown), whereas H-89, an inhibitor of PKA, had a small but significant effect in preventing the effect of stretch, suggesting some involvement of this enzyme.

In addition to the activation of second messenger systems, direct mechanical effects could also explain the stretch-induced regulation of Na⁺-K⁺-ATPase. Such effects may include the direct alteration of the membrane surface tension by mechanical strain as well as conformational strain transmitted via cytoskeletal proteins such as actin (33), which might modulate the enzymatic activity of integral membrane proteins. Coupling of Na⁺-K⁺-ATPase to the actin-binding proteins ankyrin-spectrin/fodrin system has been reported (13). In addition to the structural relationship of the cytoskeleton and Na⁺-K⁺-ATPase, the actin-based cytoskeleton may be functionally associated with Na⁺-K⁺-ATPase. For example, it has been shown that actin filaments stimulate Na⁺-K⁺-ATPase (1, 6). Berterollo et al. (2) have shown that receptor-mediated short-term regulation of the Na pump by the -adrenergic receptor agonist isoproterenol involves the endosomal transport and thus translocation of the Na pump molecules from the cytosol to the plasma membrane (2). Conversely, during inhibition by dopamine, the pump molecules are translocated from the plasma membrane into the cytosol (7, 8). These authors (7) also presented evidence that the actin cytoskeleton is involved in both cases but not in the microtubular system. In addition, PKA and PKC enzymes were involved in the actions of isoproterenol and dopamine on the Na pump, respectively (2, 7, 8).

Our experiments clearly demonstrated that the integrity of the actin cytoskeleton is essential for the stretch-induced activation of the Na pump. Inhibition of actin polymerization with cytochalasin D fully prevented the action of stretch on the Na pump. Phalloidin, which stabilizes actin in the polymerized form, when applied before cytochalasin D, prevented the action of cytochalasin D (Fig. 6A). Cantiello (6), in a cell-free preparation, showed that on phosphorylation by PKA, polymeric actin stimulates Na⁺-K⁺-ATPase activity. These studies are consistent with our findings that actin is necessary for Na pump activation and PKA is also involved. Although microtubular assembly is also regulated by mechanical strain applied to cultured smooth muscle cells (35), the microtubular system did not appear to be involved in stretch-induced activation of the Na pump.

We also have evidence that endosomal translocation may also be involved in our system. Pretreatment with wortmannin, an inhibitor of PI3K, fully prevented the action of stretch on Na pump activity. Activation of PI3K is associated with the initiation of vesicular trafficking and targeting of proteins to specific cell compartments (12). Therefore, we hypothesize that vesicular transport may be involved in our system. Preliminary evidence in our laboratory for protein abundance of Na⁺-K⁺-ATPase supports our hypothesis that translocation of Na pump molecules from the endosomal compartment to the plasma membrane in response to stretch may be involved (unpublished data). As was shown with isoproterenol-induced stimulation of the Na pump (2), the actin cytoskeleton, PKA, and possibly endosomal transport constitute the components of stretch-induced activation of the Na pump in ASMC. The role of PKA may be more complex than just phosphorylating certain proteins, such as the Na pump or actin (6), because the effect of PKA inhibitor was minor in our system. This aspect needs to be investigated further.

It is well established that Na⁺-K⁺-ATPase, or the Na pump, participates in the modulation of vascular smooth muscle contractility and tone (5), and changes in its functional state may either contribute to the development of hypertension (5, 34) or may be an adaptive compensatory response to the elevated pressure. The acute adaptation of the Na pump can be seen as a means to accommodate elevated intravascular pressure by reducing the blood vessel tone in the face of increased intramural pressure. This compensatory reaction may be overridden by numerous other chronic events such as hypertrophy of the vascular wall and exposure to humoral and endothelial factors, as well as the genetic disposition, which ultimately results in progression of hypertension in genetically predisposed individuals.

In conclusion, acute cyclic mechanical strain, or stretch, increases the activity of the Na pump in vascular smooth muscle cells. The mechanism of this effect of stretch involves the participation of the actin cytoskeleton and possible involvement of endosomal transport and PKA. Therefore, we hypothesize that stretch stimulus may induce a translocation of the enzyme. This effect of stretch may be part of a compensatory mechanism during episodes of increased intramural pressure.