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
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ABSTRACT |
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We previously demonstrated that protein expression of both
1- and
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
-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.
-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
1 mRNA by 3.3-fold,
2 was not
affected any further. Actinomycin D blocked the stretch-induced stimulation of mRNA expression of both
-subunits. In conclusion, cyclic stretch stimulates the mRNA expression of both
1-
and
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
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INTRODUCTION |
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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 -,
-, and smaller
-subunits
(4). Four
- and three
-subunits have been identified
(4, 33). Aortic smooth muscles cells (ASMC) express three
-isoforms,
1,
2, and
3
(26). The
-subunit is responsible for the catalytic
activity, whereas the
-subunit appears to be involved in the
insertion and stabilization of the
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 -subunits
(
1 and
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
1- and
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
2-subunit but had no effect on
1 (32). Therefore, we concluded that the
ions entering through stretch-activated channels play a role in
2 protein expression but not
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
1- and
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
1 and
2 mRNA expression were also tested.
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METHODS |
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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 1- and
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 1 and
2 band densities.
The results are presented as the ratio of the band densities of the
1- or
2-subunit to the band densities of
the ethidium bromide-stained 18S ribosomal RNA (
1 or
2 mRNA/18S rRNA). Photographs of a
representative gel (Fig. 1A)
and its corresponding membrane (Fig. 1B) are included.
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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).
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RESULTS |
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To determine whether cyclic stretch affects expression of the
genes encoding the 1- and
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
1-subunit, whereas 20% stretch produced an
approximate twofold increase compared with the corresponding nonstretch
controls (ANOVA, P < 0.05, n = 6; Fig.
2A).
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
1- or
2-subunits (data not shown).
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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 -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
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
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
1- or
2-isoforms at the end of a 6-h 10% stretch regimen
(data not shown).
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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 1 and
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
1 and
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
1 and
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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DISCUSSION |
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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 1- and
2-subunits of the Na+ pump. These findings
are in good agreement with our previous report, in which the
Na+-pump
-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
-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 1-,
2-, and
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 -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
1- or
2-subunits (18). Rayson (24) reported that intracellular Ca2+ increased the
transcription rates of both
1 and
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 1- and
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
-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
1 and
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
unit
(11, 12, 21).
-Subunit expression was also not
addressed in this study. The
-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 1- and
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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jerry B. Lingrel of the University of Cincinnati for
providing the DNA probes for 1- and
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.
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FOOTNOTES |
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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.
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