Effect of cyclic stretch on alpha -subunit mRNA expression of Na+-K+-ATPase in aortic smooth muscle cells

Nancy Sevieux1, Jawed Alam2, and Emel Songu-Mize1

1 Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, and 2 Department of Molecular Genetics, Ochsner Medical Foundation, New Orleans, Louisiana 70112


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that protein expression of both alpha 1- and alpha 2-catalytic subunits of the Na+-K+-ATPase is elevated after a 2- to 4-day chronic cyclic stretch regimen in cultured aortic smooth muscle cells (ASMC). In the present study, we investigated whether cyclic stretch affects mRNA expression of the alpha -isoforms of the Na+-K+-ATPase. Using a stretch apparatus, rat ASMC were cyclically stretched 10 or 20% of their length for 1, 3, or 6 h. alpha -Isoform mRNA levels were measured using Northern analysis. A 3-h 10% stretch had no significant affect on mRNA expression for either isoform, but a 20% stretch increased mRNA of both isoforms approximately twofold. Whereas a 6-h 20% stretch increased alpha 1 mRNA by 3.3-fold, alpha 2 was not affected any further. Actinomycin D blocked the stretch-induced stimulation of mRNA expression of both alpha -subunits. In conclusion, cyclic stretch stimulates the mRNA expression of both alpha 1- and alpha 2-subunits of Na+-K+-ATPase. The sensitivity of the two genes to the degree and duration of stretch is different. The stretch-induced increase of mRNA may be a result of increased transcription.

sodium pump; transcription


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE SODIUM PUMP or Na+-K+-ATPase is a ubiquitous integral protein of the outer plasma membrane of animal cells (16, 17). 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 the Na+-coupled transporters (9). The functional macromolecule consists of two dimers composed of noncovalently interacting alpha -, beta -, and smaller gamma -subunits (4). Four alpha - and three beta -subunits have been identified (4, 33). Aortic smooth muscles cells (ASMC) express three alpha -isoforms, alpha 1, alpha 2, and alpha 3 (26). The alpha -subunit is responsible for the catalytic activity, whereas the beta -subunit appears to be involved in the insertion and stabilization of the alpha beta complex in the membrane (11).

The functional significance of the molecular isoforms of the Na+-K+-ATPase and the regulation 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 expression are observed in cardiovascular tissues in several animal models of experimental hypertension (14, 19, 23, 28, 29). In vascular tissue, Na+-pump activity appears to be enhanced in hypertension (27, 28). Recently, in an in vitro smooth muscle cell culture model, we reported that mechanical strain upregulates protein expression of two catalytic alpha -subunits (alpha 1 and alpha 2) of Na+-K+-ATPase (18, 32). In that study, we demonstrated, using a cell culture system and the Flexercell Strain Unit, that applied stretch to rat vascular ASMC caused an increase in protein expression of the alpha 1- and alpha 2-isoforms of the Na+-K+-ATPase (32). Gadolinium (Gd3+), a nonselective stretch-activated cation channel blocker, inhibited the upregulation of protein expression of the alpha 2-subunit but had no effect on alpha 1 (32). Therefore, we concluded that the ions entering through stretch-activated channels play a role in alpha 2 protein expression but not alpha 1. We also speculated that the increased vascular wall stretch may contribute to elevated vascular Na+-pump activity observed in hypertensive animals. The purpose of the current study was to explore the effect that stretch stimulus has on mRNA expression of the alpha 1- and alpha 2-isoforms of the Na+-K+-ATPase in ASMC. For this purpose, we used cultured ASMC and applied stretch using the Flexercell Strain Unit to cyclically stretch the cells 10 or 20% (of length) for 1, 3, or 6 h. The effects of Gd3+ and actinomycin D, a transcription inhibitor, on alpha 1 and alpha 2 mRNA expression were also tested.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of cultured ASMC. ASMC were isolated from 4-5 male Sprague-Dawley rats weighing 200-250 g as described earlier (30). 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 of bovine serum albumin, 0.33 mg/ml of soybean trypsin inhibitor, and 25 U/ml of collagenase. The tissues were then cleaned of adventitia, minced with scissors, and further incubated for ~3 h at 37°C in a fresh aliquot of the same medium plus 15 U/ml of elastase. Afterward, the cells were washed with complete medium four times and centrifuged at 1,500 rpm (Beckman TJ-6, Rotor TH-4, 1-89) for 10 min. The final pellet was resuspended in ~7 ml of culture medium, and the cell suspension was seeded in a T-75 flask and grown to confluency. Cells were used between the third and seventh passages.

Stretch procedure. The cells were seeded at 1 × 105 cells/well on six-well collagen-coated BioFlex plates and grown to confluency under nonstretch conditions for 8-10 days. BioFlex plates containing a flexible silicone elastomer substratum were then mounted in the Flexercell Strain Unit and subjected to 10 or 20% surface elongation for 3-s stretch/3-s relaxation cycles continuously for durations required by the specific experiments. Control cells were placed in the same experimental conditions but were not stretched.

Northern blot analysis. After experimental treatments, the plates were placed on ice and the cells were washed twice with cold phosphate-buffered saline at pH 7.4. Cells from up to nine wells were pooled, and total RNA was isolated with the guanidinium isothiocyanate-acid-phenol extraction method of Chomczynski and Sacchi (7). Northern blot analyses were carried out as described by Alam et al. (1). Briefly, 10-µg RNA samples were electrophoresed in a 1% agarose/6% formaldehyde denaturing gel, transferred to Zeta-Probe GT membranes, and cross-linked to the membrane for 1 min in an ultraviolet cross-linker (Spectrotronics). Blots were prehybridized for 1-3 h and hybridized for 24-36 h with isoform-specific cDNA probes (gift from Dr. Lingrel, Univ. of Cincinnati) labeled with 32P (dCTP) by random priming (Prime It II kit; Strategene). Hybridization and washing conditions were as described previously (1). The membranes were scanned with the use of a PhosphorImager (Molecular Dynamics, CA), and the density of the bands was analyzed using ImageQuant software (Molecular Dynamics) and normalized against 18S ribosomal RNA as described below.

mRNA stability studies. ASMC were stretched 20% of their length for 3 h. Immediately after stretch, 1 µg/ml of actinomycin D was added to designated wells. Total RNA was extracted at 0, 3, 6, 9, and 12 h after the actinomycin D was added. Northern analysis was carried out for alpha 1- and alpha 2-subunit mRNAs as described earlier.

Signal normalization. After capillary transfer of the RNA to the Zeta-Probe membrane and cross-linking, the membrane was scanned while still wet using a Storm 860 instrument (Molecular Dynamics) set for blue fluorescence. The band densities of the ethidium bromide-stained 18S ribosomal RNA was determined using the PhosphorImager as described by Eykholt et al. (8). After hybridization, membranes were scanned again for determination of alpha 1 and alpha 2 band densities. The results are presented as the ratio of the band densities of the alpha 1- or alpha 2-subunit to the band densities of the ethidium bromide-stained 18S ribosomal RNA (alpha 1 or alpha 2 mRNA/18S rRNA). Photographs of a representative gel (Fig. 1A) and its corresponding membrane (Fig. 1B) are included.


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Fig. 1.   Visualization of 28S and 18S RNAs on agarose gel and membrane. A representative gel (A) and its corresponding membrane (B) show the 28S and 18S bands. Ten-microgram total RNA samples from nonstretch control cells and cells cyclically stretched 10 and 20% for 3 h were applied to each lane as shown. RNA samples from rat hearts were used as controls.

Experimental design and statistical analysis. Three different experiments were performed to determine the following: the effect of 1- and 3-h 10 and 20% stretch; the effect of 6-h 10 and 20% stretch with or without Gd3+; and the effect of 20% stretch with or without pretreatment with actinomycin D on mRNA expression. For each experiment, 12 BioFlex plates were plated (3 or 4 plates/condition depending on the experiment). Six to nine wells were randomly pooled for each condition for extraction of RNA. Every pooled sample constituted n of 1. Experiments were repeated at least once.

Analysis of variance (ANOVA) followed by Tukey-Kramer's or Fisher's protected least-significant differences test was used to determine differences among means of treatment groups. A confidence limit of 95% was considered significant.

Materials and reagents. Medium 199, fetal bovine serum, bovine albumin, soybean trypsin inhibitor, collagenase, elastase, phenol, gadolinium, and actinomycin D were obtained from Sigma (St. Louis, MO). Penicillin and streptomycin were purchased from Mediatech (Herndon, VA). Guanidinium thiocyanate and a random priming kit were acquired from Stratagene (La Jolla, CA). Formaldehyde was obtained from Fisher Scientific (Fair Lawn, NJ). Zeta-Probe GT was purchased from Bio-Rad (Hercules, CA). 32P was acquired from NEN (Boston, MA). The Flexercell Strain Unit and the BioFlex plates were purchased from Flexcell International (McKeesport, PA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine whether cyclic stretch affects expression of the genes encoding the alpha 1- and alpha 2-subunits of the Na+-K+-ATPase in cultured ASMC, we measured the steady-state levels of the corresponding mRNAs by Northern blot analysis (Fig. 2). At the end of a 3-h stretch protocol, 10% stretch had no significant effect on the mRNA level of the alpha 1-subunit, whereas 20% stretch produced an approximate twofold increase compared with the corresponding nonstretch controls (ANOVA, P < 0.05, n = 6; Fig. 2A). alpha 2-Subunit mRNA expression appeared to be more sensitive to the degree of stretch, i.e., a 10% stretch produced a 1.5-fold increase, but this change did not reach significance until a 20% stretch was applied, which produced an ~2-fold increase at the end of a 3-h stretch (ANOVA, P < 0.05, n = 13-18; Fig. 2B). Ten and twenty percent stretch for 1 h had no effect on mRNA expression of both the alpha 1- or alpha 2-subunits (data not shown).


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Fig. 2.   Effect of degree of stretch on Na+-K+-ATPase alpha 1 and alpha 2 mRNA expression. Aortic smooth muscle cells were stretched cyclically at 10 or 20% for 3 h. Bars represent means + SE of the ratio of band densities of the alpha 1 (A)- and alpha 2-subunit (B) mRNA to the 18S ribosomal RNA. The membranes were scanned using a PhosphorImager, and the band densities were calculated using ImageQuant software. star Significant difference compared with nonstretch controls and 10% stretch. ANOVA and Tukey-Kramer's test were used in which P < 0.05 was considered significant; n = 6 for alpha 1; n = 13-18 for alpha 2.

We next determined the effect of a 6-h stretch and investigated the role of Gd3+, a nonselective stretch-activated channel blocker, on mRNA expression of both alpha -isoforms. For these experiments, 10 or 20% cyclic stretch protocol was applied, and the cells were stretched in the presence or absence of 50 µM Gd3+ (Fig. 3). For alpha 1-subunit, a 20% stretch caused an ~3-fold increase in mRNA expression (ANOVA, P < 0.05, n = 14; Fig. 3A). Incubation of the cells with 50 µM Gd3+ did not produce an effect on mRNA expression under stretch or nonstretch conditions. In the case of the alpha 2-subunit, a 20% stretch caused an ~1.5-fold increase in mRNA expression compared with the nonstretch controls, and Gd3+ did not prevent this enhancement. Gd3+ had no effect on nonstretch controls (ANOVA, P < 0.05, n = 8-10; Fig. 3B). There was no change in mRNA expression for either alpha 1- or alpha 2-isoforms at the end of a 6-h 10% stretch regimen (data not shown).


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Fig. 3.   Effect of 20% stretch and gadolinium on Na+-K+-ATPase alpha 1 and alpha 2 mRNA expression. Aortic smooth muscle cells were stretched cyclically at 20% for 6 h with and without 50 µM Gd3+. Northern blot analyses were performed. Bars represent means + SE of the ratio of band densities of the Na+-K+-ATPase alpha 1 (A)- and alpha 2-subunit (B) to the 18S ribosomal RNA without (filled bars) and with (crosshatched bars) Gd3+. The membranes were scanned using a PhosphorImager, and the band densities were calculated using ImageQuant software as described in METHODS. star Significant difference compared with nonstretch controls with or without gadolinium. ANOVA and Tukey-Kramer's test were used in which P < 0.05 was considered significant; n = 8-10 samples for each group.

To determine whether stretch-induced increase in mRNA may be a result of an increase in transcription, we pretreated the cells with 1 µg/ml of actinomycin D, an RNA transcription inhibitor, for 30 min and measured the mRNA levels at the end of a 3-h stretch (Fig. 4). Twenty percent stretch significantly increased alpha 1 and alpha 2 mRNA levels by 2- and 1.5-fold, respectively, as determined in the previous experiments, compared with the levels in control nonstretch cells. Actinomycin D blocked the stimulation of alpha 1 and alpha 2 mRNA induced by a 20% stretch (Fig. 4, A and B; ANOVA, P < 0.05, n = 4). To rule out the possibility that the stretch-induced increase in mRNA may be due to a decrease in degradation of the message, mRNA stability experiments were performed. Half-lives for alpha 1 and alpha 2 mRNA in control cells and 20% stretch cells were >12 h. In addition, the rate of decay of the mRNA for both control and stretch cells was similar (data not shown).


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Fig. 4.   Effect of 20% stretch and actinomycin D on Na+-K+-ATPase alpha 1 and alpha 2 mRNA expression. Aortic smooth muscle cells were preincubated for 30 min with 1 µg/ml of actinomycin D or vehicle and were then stretched cyclically at 20% for 3 h. Bars represent means + SE of the ratio of band densities of the alpha 1 (A)- and alpha 2-subunit (B) mRNA to the 18S ribosomal RNA without (filled bars) and with (crosshatched bars) actinomycin D. The membranes were scanned using a PhosphorImager, and the band densities were calculated using ImageQuant software. star Significant difference compared with nonstretch controls and stretch with actinomycin D. ANOVA and Fisher's test were used in which P < 0.05 was considered significant; n = 4 samples for each group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have demonstrated that application of 20% cyclic stretch to cultured ASMC increased the steady-state mRNA levels within 3 h for both alpha 1- and alpha 2-subunits of the Na+ pump. These findings are in good agreement with our previous report, in which the Na+-pump alpha -subunit protein expression was also upregulated in response to cyclic stretch. It appears that the effect of a long-term stretch that resulted in an increased protein expression of the two catalytic subunits of the Na+ pump may involve the induction of gene expression. A transcriptional regulation may be likely, since the stretch-induced mRNA induction was blocked by actinomycin D. Furthermore, stability experiments yielded half-lives of the mRNA of greater than 12 h and showed no difference in decay rates of mRNA from stretch and control cells. Therefore, the stretch-induced increase observed after 3 and 6 h is likely to be due to an increase in transcription rate and not a change in message stability. Gd3+ did not block mRNA induction for either isoform, although it did block protein expression in the previous study (32). The effect of Gd3+ may be posttranscriptional and suggests involvement of Na+ and/or other monovalent ions on protein expression of the alpha -isoforms.

It is well established that Na+-K+-ATPase, or its functional counterpart the Na+ pump, participates in the modulation of vascular smooth muscle contractility and tone (5). Activation of the vascular smooth muscle Na+ pump causes hyperpolarization and relaxation, and inhibition causes depolarization and contraction (13). Our collective studies to date suggest a role for increased mechanical strain in regulation of the vascular Na+-K+-ATPase. Changes in the functional state of the vascular Na+ pump or its regulation may either contribute to the development of hypertension (5, 23) or be a compensatory response to the elevated pressure. If this response to cyclic stretch occurs in vivo, this may constitute a compensatory relaxation in the vascular smooth muscle in the face of increased intravascular strain. However, it is not clear to what extent this mechanism is effective in reducing the intravascular tension in essential hypertension. Our experiments are performed using cells from normotensive rats. Whether this mechanism is fully operative in cells from hypertensive animals has not been determined. It is possible that in genetically hypertensive animals, this compensatory mechanism is compromised, thus contributing to the rise in blood pressure. Although Na+-K+-ATPase gene has been implicated in hypertension, it is believed that >50 genes are important for the regulation of blood pressure (10). It is entirely possible that a genetic alteration in either the Na+-pump protein or its regulation (transcriptional/posttranscriptional?) by stretch may contribute to the etiology of high blood pressure.

Mechanisms underlying this effect of stretch stimulus on the induction of the gene expression and the protein expression are not known. We considered intracellular Na+ a likely candidate. Na+ ion enters the cell during cyclic stretch and increases the intracellular Na+ concentrations. We demonstrated that intracellular Na+ is elevated for at least 2 h during stretch, and this rise in Na+ is blocked by Gd3+ (20), but Gd3+ did not have any effect on stretch-induced mRNA induction. Therefore, it appears that the effect of Gd3+ on protein expression observed earlier may be posttranscriptional (32). Na+ may be entering the cell through other channels, such as store-operated channels. Recently, Arnon et al. (2) and Boulay et al. (6) suggested that in arterial myocytes, one of the major routes of Na+ entry is through store-operated channels. Therefore, Na+ may still be a likely candidate in producing stretch-induced mRNA induction. Furthermore, others have suggested that intracellular Na+ may be important in alpha 1-, alpha 2-, and beta 1-subunit gene expression of the Na+ pump in vascular smooth muscle cells (25, 34).

It is also possible that Ca2+ ions entering the cell through several channels during stretch may play a role in upregulation of the alpha -isoforms. One possible route is the Na+/Ca2+ exchanger that can be activated by increased intracellular Na+ as a result of stretch. Another possibility is the release of Ca2+ from intracellular stores via D-myo-inositol 1,4,5-trisphosphate production as a result of activation of the phospholipase C signaling pathway by stretch (3). A third route of entry is the L-type voltage-gated channels. At the protein expression level, blocking the L-type Ca2+ channels did not appear to affect the stretch-induced protein expression of either alpha 1- or alpha 2-subunits (18). Rayson (24) reported that intracellular Ca2+ increased the transcription rates of both alpha 1 and beta 1 mRNAs in kidney outer medullary tubular segments. We also have to consider indirect but plausible explanations to stretch-induced Na+ pump upregulation. For example, it is possible that any of the ion channels discussed above may be affected by stretch and change the permeability of the membrane, thereby promoting enhanced Na+-pump synthesis to keep up with the ionic leaks such as Na+.

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, which might directly modulate the enzymatic activity of integral membrane proteins. It has been recently reported that in vascular smooth muscle cells, extracellular hyperosmolality induced mRNA and protein expression for alpha 1- and beta 1-subunits (22). The authors demonstrated that this induction was transcriptional and independent of increased intracellular Na+. It is likely, then, that stress produced by hypertonicity may be responsible for the transcriptional regulation. This type of mechanical strain has also been shown to activate mitogen-activated protein (MAP) kinases (15). However, it is not likely that this pathway is involved in stretch-induced upregulation of Na+-pump alpha -subunits in our system. We demonstrated that 42- and 44-kDa MAP kinases were stimulated by stretch but found no involvement of these MAP kinases on stretch-induced Na+-pump activation (31).

It is interesting that degree of stretch, 10% vs. 20% surface elongation, appears to be important in mRNA induction, and alpha 1 and alpha 2 have different thresholds for induction. These findings suggest that the two isoforms may indeed have different functions and may respond to physiological (a temporary increase in cardiac output as seen during exercise) or pathophysiological (essential hypertension) stretch stimuli in a different manner. Although we have not addressed the functional aspect of the Na+ pump in this study, it is well known that the Na+ pump functions only as an assembled alpha beta unit (11, 12, 21). beta -Subunit expression was also not addressed in this study. The beta -subunit may be limiting under certain conditions, such as degree of stretch. Future functional studies are planned.

In conclusion, we demonstrated that cyclic mechanical strain of 10-20% surface elongation of cultured ASMC induces both the alpha 1- and alpha 2-subunit mRNA expression, and stretch affects this change at the transcriptional level. We have also demonstrated that the sensitivity of the two genes to the degree and duration of stretch is different. Differential regulation of the catalytic isoforms of the vascular smooth muscle cell Na+-K+-ATPase by stretch may have important implications in response of the blood vessels to pathophysiological changes in disease states such as essential hypertension.


    ACKNOWLEDGEMENTS

We thank Dr. Jerry B. Lingrel of the University of Cincinnati for providing the DNA probes for alpha 1- and alpha 2-subunits of Na+-K+-ATPase and Dr. Kenneth Johnston of the Department of Microbiology, Immunology, and Parasitology, Louisiana State Univ. Health Sciences Center, for assistance with preparation of the probes. We thank Shawn Wiltse for technical assistance and Monica Byrnes (of Learning Resources) for preparation of the figures.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-32270 (awarded to E. Songu-Mize).

Address for reprint requests and other correspondence: E. Songu-Mize, Dept. of Pharmacology and Experimental Therapeutics, Louisiana State Univ. Health Sciences Center, 1901 Perdido, New Orleans, LA 70112 (E-mail: emize{at}lsuhsc.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.

Received 12 January 2000; accepted in final form 21 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alam, J, Shibahara S, and Smith A. Transcriptional activation of the heme oxygenase gene by heme and cadmium in mouse hepatoma cells. J Biol Chem 264: 6371-6375, 1989[Abstract/Free Full Text].

2.   Arnon, A, Hamlyn JM, and Blaustein MP. Na+ entry via store-operated channels modulates Ca2+ signaling in arterial myocytes. Am J Physiol Cell Physiol 278: C163-C173, 2000[Abstract/Free Full Text].

3.   Berridge, MJ. Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993[ISI][Medline].

4.   Blanco, G, and Mercer RW. Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol Renal Physiol 275: F633-F650, 1998[Abstract/Free Full Text].

5.   Blaustein, MP. Sodium ions, calcium ions, blood pressure regulation, and hypertension: a reassessment and a hypothesis. Am J Physiol Cell Physiol 232: C165-C173, 1977[Abstract].

6.   Boulay, G, Brown DM, Qin N, Jiang M, Dietrich A, Zhu MX, Chen Z, Birnbaumer M, Mikoshiba K, and Birnbaumer L. Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry. Proc Natl Acad Sci USA 96: 14955-14960, 1999[Abstract/Free Full Text].

7.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

8.  Eykholt RL, Mitchell MD, and Marvin KW. Direct imaging of Northern blots on an optical scanner using ethidium bromide. Biotechniques 28: 864-866, 868, 870, 2000.

9.   Fleming, WW. The electrogenic Na+, K+-pump in smooth muscle: physiologic and pharmacologic significance. Annu Rev Pharmacol Toxicol 20: 129-149, 1980[ISI][Medline].

10.   Garbers, DL, and Dubois SK. The molecular basis of hypertension. Annu Rev Biochem 68: 127-155, 1999[ISI][Medline].

11.   Geering, K, Theulaz I, Verrey F, Hauptle MT, and Rossier BC. A role for the beta -subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. Am J Physiol Cell Physiol 257: C851-C858, 1989[Abstract/Free Full Text].

12.   Hasler, U, Wang X, Crambert G, Beguin P, Jaisser F, Horisberger JD, and Geering K. Role of beta-subunit domains in the assembly, stable expression, intracellular routing, and functional properties of Na,K-ATPase. J Biol Chem 273: 30826-30835, 1998[Abstract/Free Full Text].

13.   Hendrickx, H, and Casteels R. Electrogenic sodium pump in arterial smooth muscle cells. Pflügers Arch 346: 299-306, 1974[ISI][Medline].

14.   Herrera, VL, Chobanian AV, and Ruiz-Opazo N. Isoform-specific modulation of Na+, K+-ATPase alpha-subunit gene expression in hypertension. Science 241: 221-223, 1988[ISI][Medline].

15.   Itoh, T, Yamauchi A, Miyai A, Yokoyama K, Kamada T, Ueda N, and Fujiwara Y. Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells. J Clin Invest 93: 2387-2392, 1994[ISI][Medline].

16.   Jorgensen, PL. Mechanism of the Na+, K+ pump. Protein structure and conformations of the pure (Na+ + K+)-ATPase. Biochim Biophys Acta 694: 27-68, 1982[ISI][Medline].

17.   Lingrel, JB. Na,K-ATPase: isoform structure, function, and expression. J Bioenerg Biomembr 24: 263-270, 1992[ISI][Medline].

18.   Liu, X, Hymel LJ, and Songu-Mize E. Role of Na+ and Ca2+ in stretch-induced Na+-K+-ATPase alpha -subunit regulation in aortic smooth muscle cells. Am J Physiol Heart Circ Physiol 274: H83-H89, 1998[Abstract/Free Full Text].

19.   Liu, X, and Songu-Mize E. Alterations in alpha -subunit expression of cardiac Na+,K+-ATPase in spontaneously hypertensive rats: effect of antihypertensive therapy. Eur J Pharmacol 327: 151-156, 1997[ISI][Medline].

20.   Liu, X, and Songu-Mize E. Effect of Na+ on Na+,K+-ATPase alpha -subunit expression and Na+-pump activity in aortic smooth muscle cells. Eur J Pharmacol 351: 113-119, 1998[ISI][Medline].

21.   McDonough, AA, Geering K, and Farley RA. The sodium pump needs its beta subunit. FASEB J 4: 1598-1605, 1990[Abstract/Free Full Text].

22.   Muto, S, Ohtaka A, Nemoto J, Kawakami K, and Asano Y. Effects of hyperosmolality on Na,K-ATPase gene expression in vascular smooth muscle cells. J Membr Biol 162: 233-245, 1998[ISI][Medline].

23.   Pamnani, M, Clough D, Hout S, and Haddy F. Sodium-Potassium Pump Activity in Experimental Hypertension in Vasodilation. New York: Raven, 1981.

24.   Rayson, BM. [Ca2+]i regulates transcription rate of the Na+/K+-ATPase alpha 1 subunit. J Biol Chem 266: 21335-21338, 1991[Abstract/Free Full Text].

25.   Ruiz-Opazo, N, Cloix JF, Melis MG, Xiang XH, and Herrera VL. Characterization of a sodium-response transcriptional mechanism. Hypertension 30: 191-198, 1997[Abstract/Free Full Text].

26.   Sahin-Erdemli, I, Rashed SM, and Songu-Mize E. Rat vascular tissues express all three alpha -isoforms of Na+-K+-ATPase. Am J Physiol Heart Circ Physiol 266: H350-H353, 1994[Abstract/Free Full Text].

27.   Songu-Mize, E. Vascular sodium pump activity kinetics in early and advanced stages of deoxycorticosterone-salt hypertension in rats. Life Sci 49: 2045-2052, 1991[ISI][Medline].

28.   Songu-Mize, E, Bealer SL, and Caldwell RW. Effect of anteroventral third ventricle lesions on vascular sodium-pump activity in two-kidney Goldblatt hypertension. Hypertension 5: I89-I93, 1983[ISI][Medline].

29.   Songu-Mize, E, Bealer SL, and Caldwell RW. Phasic vascular sodium pump changes in deoxycorticosterone-hypertensive rats. Circ Res 55: 304-308, 1984[Abstract].

30.   Songu-Mize, E, Bealer SL, and Hassid AI. Centrally administered ANF promotes appearance of a circulating sodium pump inhibitor. Am J Physiol Heart Circ Physiol 258: H1655-H1659, 1990[Abstract/Free Full Text].

31.   Songu-Mize, E, and Jacobs M. Effect of cyclic in vitro stretch on aortic smooth muscle cells p42 and p44 mitogen activated kinases (Abstract). FASEB J 12: A403, 1998[ISI].

32.   Songu-Mize, E, Liu X, Stones JE, and Hymel LJ. Regulation of Na+,K+-ATPase alpha -subunit expression by mechanical strain in aortic smooth muscle cells. Hypertension 27: 827-832, 1996[Abstract/Free Full Text].

33.   Sweadner, KJ. Isozymes of the Na+/K+-ATPase. Biochim Biophys Acta 988: 185-220, 1989[ISI][Medline].

34.   Yamamoto, K, Ikeda U, Okada K, Saito T, Kawakami K, and Shimada K. Sodium ion mediated regulation of Na/K-ATPase gene expression in vascular smooth muscle cells. Cardiovasc Res 28: 957-962, 1994[ISI][Medline].


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