Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0576
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ABSTRACT |
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Organ culture specifically inhibits vasorelaxation to acute hypoxia and preferentially decreases specific voltage-dependent K+ channel expression over other K+ and Ca2+ channel subtypes. To isolate further potential oxygen-sensing mechanisms correlated with altered gene expression, we performed differential display analysis on RNA isolated from control and cultured coronary arterial rings. We hypothesize that organ culture results in altered gene expression important for vascular smooth muscle contractility important to the mechanism of hypoxia-induced relaxation. Our results indicate a milieu of changes suggesting both up- and downregulation of several genes. The altered expression pattern of two positive clones was verified by Northern analysis. Subsequent screening of a porcine cDNA library indicated homology to the ryanodine receptor (RyR). RT-PCR using specific primers to the three subtypes of RyR shows an upregulation of RyR2 and RyR3 after organ culture. Additionally, the caffeine- and/or ryanodine-sensitive intracellular Ca2+ store was significantly more responsive to caffeine activation after organ culture. Our data indicate that organ culture increases expression of specific RyR subtypes and inhibits hypoxic vasorelaxation. Importantly, ryanodine blunted hypoxic relaxation in control coronary arteries, suggesting that upregulated RyR might play a novel role in altered intracellular Ca2+ handling during hypoxia.
ryanodine receptor; vascular smooth muscle; hypoxia
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INTRODUCTION |
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A DECREASE IN OXYGEN TENSION (PO2), or hypoxia, modulates coronary, and most systemic, arterial tone, causing a vasodilation to maintain delivery of adequate oxygen to the heart and other organs. The mechanism(s) by which systemic vascular smooth muscle (VSM) senses these changes in PO2 remains unclear. Common theories include the modulation of ion channel function, an acute decrease in available ATP necessary for force maintenance, direct regulation of intracellular Ca2+ concentration ([Ca2+]i), and modulation of Ca2+ sensitivity (6, 8, 9, 19). We have shown that these theories cannot completely account for the hypoxia-induced relaxation of porcine coronary arteries (20). Additionally, considerable attention has been given to small heme-containing proteins as the oxygen sensor where an PO2-dependent change in redox status may transduce the appropriate response (12, 14, 15).
In a recent investigation, we demonstrate the specific inhibition of relaxation to acute hypoxia after organ culture of porcine coronary arteries (25). Organ culture of VSM is reported to cause changes in [Ca2+]i handling (4, 7) and has been associated with altered gene expression (21). Chronic hypoxia leads to the regulation of several hypoxia-inducible genes that modulate long-term responses to decreases in PO2 (1, 13, 22). It is conceivable that the expression of several genes important for relaxation to acute hypoxia changes after organ culture. For example, a change in the expression of those genes responsible for regulating [Ca2+]i could cause altered Ca2+ handling during hypoxia. We have demonstrated that organ culture abolishes the reduction in [Ca2+]i during acute hypoxic relaxation (25). We have also shown that organ culture results in the downregulation of specific voltage-dependent K+ channels that are likely involved in Ca2+-dependent hypoxia-induced relaxation (25). It is also clear that these are not the only pathways that are involved in the mechanism of hypoxic relaxation (20).
In this investigation, we use differential display analysis and subsequent screening of a cDNA library to study organ culture-induced changes in gene expression as novel oxygen sensors modulating hypoxic vasorelaxation. We identified increased expression of the ryanodine receptor (RyR) that was supported by functional studies. Importantly, ryanodine blunted hypoxic relaxation in control coronary arteries, suggesting that upregulated RyR might play a novel role in altered intracellular Ca2+ handling during hypoxia.
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METHODS |
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Organ culture conditions. Organ culture was performed as previously described (25). Briefly, left descending coronary arteries were dissected from adult porcine hearts obtained from the slaughterhouse on the day of death, and two rings per artery were cleaned of adhering connective tissue. One ring was cultured in sterile DMEM plus 1% antibiotic solution at 37°C. A paired ring (control) was stored in the same solution at 4°C. Storing these tissues at 4°C does not significantly alter contractility compared with freshly isolated arteries. After 24 h, rings were prepared for organ bath, molecular, or biochemical studies. All organ culture preparations were performed under sterile conditions in a culture hood.
Organ bath studies.
Organ bath experiments were performed as previously described
(25). Briefly, all arterial rings were mechanically
deendothelialized. One cultured ring and its paired control were placed
in a bath containing physiological saline solution (PSS) of the
following composition (mmol/l): 118.3 NaCl, 25.0 NaHCO3,
11.1 dextrose, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 0.026 EDTA, and 2.5 CaCl2.
Bath pH was 7.4 when aerated with 95% O2-5%
CO2 at 37°C. Tissues were allowed to equilibrate for
1 h. Tension was adjusted to 40 mN, which sets the tissue length
in the range for optimal force generation. At least two
contraction/relaxation cycles to 80 mM KCl were performed until maximum
reproducible muscle forces were observed. The absence of the
endothelium was confirmed by lack of a response to substance P
(108 M). A reference contraction using 0.1 µM U-46619
or 40 mM KCl was generated, and, after steady force, hypoxia was
obtained by bubbling 95% N2-5% CO2 through
the baths for ~20 min. The final PO2 of the
bath solution, measured polarographically, was ~1-2%. We define
these conditions as hypoxia. In some experiments, rings were switched
to Ca2+-free PSS (nominally Ca2+ free plus 0.5 mM EGTA) and allowed to stabilize for 10 min. Next, the response to 20 mM caffeine was measured. In separate experiments, rings were
pretreated with 20 µM ryanodine for 15 min and then activated with 40 mM KCl and subjected to hypoxia.
Total RNA isolation. Total RNA was isolated from control and organ cultured coronary arterial rings used in the organ bath experiments. To isolate a maximum quantity of total RNA, additional control and cocultured rings (those not used in organ bath experiments) were also endothelium-denuded and utilized in the protocol. The TriReagent (Molecular Research, Cincinnati, OH) RNA isolation protocol was followed. Briefly, this procedure requires tissue homogenization in the phenol-based monophase solution for complete separation of the nucleoprotein complexes. This was followed by an aqueous phase separation with pure chloroform. Total RNA was precipitated with isopropyl alcohol. RNA sample light absorbencies were measured with a spectrofluorimeter to approximate the sample concentrations. RNA gels were performed using an identical amount of DNA-free RNA and then stained with ethidium bromide to confirm loading.
Differential display. Total RNA samples were DNase treated for 1 h to remove any residual DNA. The samples were used in a reverse transcriptase reaction with one of four oligo(dT) primers (dT, dC, dG, or dA) for first-strand synthesis according to the RNAimage protocol from Gen Hunter (Nashville, TN). The same oligo(dT) primers were end-labeled with 32P and used with one of eight arbitrary primers (provided by Gen Hunter) in the PCR reaction for second-strand synthesis. The combination the four oligo(dT) primers with the arbitrary upstream primers ensures representation of most of the mRNA population. PCR products were then loaded on a 6% denaturing acrylamide gel and run at constant voltage for 30 min to 1 h. The gel was dried at 80°C and placed on film for autoradiography. Changes in mRNA expression were determined by comparing the relative intensity of the bands in control vs. organ-cultured samples. A total of 10 gels was performed using all arbitrary primers separately with the oligo(dT) primer to assure representation of approximately all the RNA population.
PCR-Trap cloning.
We used a standard cloning protocol from Gen Hunter to clone candidate
gene fragments into a readily accessible vector. Briefly, bands of
interest were excised from the gel and reamplified using the primers
from differential display. The amplified PCR product was ligated to the
PCR-Trap vector and transformed into DH5 cells for cloning. The
vector is constructed so only those cells containing the cDNA insert
will retain tetracycline resistance. The cDNA inserts were verified by
PCR amplification using left and right primers (Lgh and Rgh,
respectively, provided by Gen Hunter) constructed to flank the
blunt-ended ligation points. The fragment was isolated from the vector
by HindIII restriction digest and sequenced with left and
right sequencing primers, which recognize vector sites upstream and
downstream of Lgh and Rgh, respectively.
Northern blot hybridization.
Approximately 10 µg of total RNA from each isolation procedure were
loaded on a formaldehyde gel and run at constant voltage for 1 h.
RNA in the gel was transferred to nitrocellulose for 16-18 h.
Nitrocellulose filters were baked at 80°C for permanent fixation of
the RNA followed by incubation in prehybridization solution [50%
formamide, 50 mM NaPO4, pH 6.5, 5× SSC (1 M sodium chloride, 0.3 M sodium citrate), 5× Denhardt's solution, 250 µg/ml salmon sperm DNA, 0.5% SDS, and 1% glycine] at 42°C for 2 h.
Hybridization using 1 × 106
counts · min1
(cpm) · ml
1 radiolabeled cDNA
cloned from PCR-Trap was performed at 42°C in hybridization solution
(30% formamide, 20 mM NaPO4, pH 6.5, 5× SSC, 1×
Denhardt's, 100 µM/ml salmon sperm DNA, 0.5% SDS, and 10% dextran
sulfate) overnight. After one wash in 2× SSC-0.1% SDS and two
washes in 1× SSC-0.1% SDS (each for 15 min at 42°C), filters were
wrapped in plastic and placed in a PhosphorImager cassette for development.
cDNA library screening.
The porcine cDNA library (a generous gift from Dr. Frank Simmen,
University of Florida) was directly cloned into Bluescript phagemid and
stored at a titer of 1.12 × 1010 plaque-forming units
(pfu)/ml. Aliquots of the cDNA library were titered with suspension
media (SM) of the following composition: 5.8 g/l NaCl, 2 g/l
MgSO4, 1 M Tris · HCl, 50 ml/l KCl,
and 0.01% gelatin. The phage were plated at 50,000 pfu/ml on 150-mm
Luria Broth/Agar plates. Ten plates were prepared by infecting XL-1blue cells with the appropriate titer of phage. Duplicate plaque lifts were
made for each plate with 3 M nitrocellulose filter paper. Filters were
allowed to dry, and the plaques were denatured on the filters by washes
with 0.2 M NaOH/1.5 M NaCl, 0.4 M Tris · HCl/2×
SSC, pH 7.6, and 2× SSC. Filters were baked at 80°C and preincubated
at 42°C in hybridization solution (formamide, 20× SSC, 2 M
Tris · HCl, pH 7.6, 100× Denhardt's, 50%
dextran sulfate, and 10% SDS) for 1 h. Radiolabeled cDNA clone
from PCR-Trap (1 × 106 cpm/ml) was added to the
solution to initiate hybridization at 42°C. Filters were washed one
time with a low-stringency wash solution (2× SSC-0.1% SDS) at room
temperature followed by two high-stringency washes (0.2× SSC-0.1%
SDS) at 42°C. The wet filters were wrapped in plastic and placed in a
film cassette for autoradiography. Positive plaques were resuspended by
incubation in SM and a drop of chloroform for 2 h. Coinfection of
XL-1Blue cells with Bluescript phagemid and helper phage resulted in
plasmid excision and packaging into the helper phage. The excision
reaction was used to infect SOLR cells that suppress phage
replication. Therefore, only those cells that were infected by the
ampicillin resistance helper phage were able to expand. Plasmid DNA was
then isolated and sequenced using primers constructed for recognition
of vector sequences flanking the insert.
RT-PCR of RyR subtypes. Total RNA was used for RT-PCR according to the protocol of PE Life Sciences (Boston, MA). In short, an oligo(dT) primer was used with the murine leukemia virus RT for first-strand synthesis. Total RNA (1 µg) was used in this mixture to a final volume of 20 µl, and incubated for 15 min at 42°C. The entire RT product was used for PCR amplification of the desired RyR gene in combination with pig-specific primers (11). PCR parameters were as described by PE Life Sciences for 35 cycles. Annealing temperatures for the three RyR isoforms were as follows: 52°C for RyR1 and -2 and 56°C for RyR3. PCR products were analyzed on 1.2% DNA agarose gels containing ethidium bromide (3 µg/µl) for visualization. For further visual confirmation of identical loading, RT-PCR of the ribosomal subunit, S16, was always also performed with each reaction.
Statistical analysis. Data were analyzed using the t-test for paired two-sample means or two-way repeated ANOVA with one-factor balance design. Statistical significance was accepted for P < 0.05. Values are expressed as means ± SE. N values represent the number of hearts from which arteries were isolated.
Chemicals. All organ bath chemicals were purchased from Sigma-Aldrich Chemical (St. Louis, MO). RT-PCR reagents were from Applied Biosystems (Atlanta, GA). Differential display and cloning reagents were from Gen Hunter.
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RESULTS |
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Gene expression in control and cultured porcine coronary arteries.
Differential display was used to visualize the relative expression of
mRNA between control and cultured coronary arteries. Figure
1 is a representation of typical results
from these experiments. It is evident that there is a change in
expression of many genes after organ culture exhibiting both up- and
downregulation. Of all the primer sets used, only two sets resulted in
any visible changes in expression totaling about fifteen separate
fragments in all.
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cDNA library screening and identification of candidate genes.
Figure 3A shows the results of
the third passage screen of the porcine cDNA library showing all the
plaques positively labeled with the radiolabeled probe constructed from
the G1 gene fragment. Sequence analysis of the cDNA insert
from these plaques revealed a 400-bp region of a pig-specific gene
(Fig. 3B). Comparison of this fragment with the GenBank
database revealed homology with the skeletal isoform of the RyR. The
G2 probe also positively labeled plaques from the porcine
cDNA library; however, we were unable to get significant sequence
homology from the GenBank database (data not shown). These results
suggest the possible upregulation of the skeletal RyR isoform in
porcine coronary artery after organ culture.
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RT-PCR of RyR isoforms in control and organ-cultured arteries.
There are three known RyR isoforms, RyR1, -2, and -3 (18).
The predominant isoform found in skeletal muscle is RyR1. The relative
expression of all three RyR isoforms after organ culture of porcine
coronary artery was investigated. Figure
4 shows RT-PCR analysis using specific
primers to amplify distinct segments of each RyR subtype. The results
indicate an upregulation of RyR2 and RyR3. There is no change in RyR1
expression according to RT-PCR.
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Activation of caffeine- and/or ryanodine-sensitive intracellular
Ca2+ stores after organ culture.
VSM is known to exhibit a transient increase in isometric force after
treatment with caffeine. This is caused by a temporary increase in
[Ca2+]i as intracellular Ca2+
stores release Ca2+ into the cytosol. We investigated the
function of these stores in control and organ-cultured coronary
arteries. There is a significantly higher increase in isometric force
to caffeine in cultured vessels (Fig.
5C). However, there is no
change in the maximum isometric force generation to KCl (Fig. 5,
A and B), suggesting that activation via
extracellular Ca2+ influx through surface membrane
Ca2+ channels appears normal. These results suggest an
increased reactivity to ryanodine- and/or caffeine-sensitive
intracellular Ca2+ activation. This correlates with an
increased expression of two RyR isoforms and may contribute to altered
[Ca2+]i handling after organ culture during
acute hypoxia, as previously observed (25).
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DISCUSSION |
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In this investigation, we correlate the loss of hypoxic vasodilation after 24 h in organ culture with altered gene expression in the porcine coronary artery to isolate potential oxygen-sensing pathways. Considerable attention has been given to the study of smooth muscle organ culture in an attempt to better understand smooth muscle gene regulation, response to growth factors, and regulation of proliferation (7, 26). Cell and organ culture of VSM has been shown to change expression of both smooth muscle-specific and more ubiquitous genes potentially involved in modulation of these functions (21). In this model, gene expression changes are used as a tool to identify possible candidates contributing to the mechanism of hypoxia-induced vasodilation. Our data indicate that the expression of RyR2 and RyR3 is increased after 24 h of organ culture.
Initial database search results suggest that the G1 cDNA fragment was homologous to a skeletal RyR entry. RT-PCR using primers specific for each RyR isoform indicate that only RyR2 and -3 are upregulated after organ culture. However, since differential display produces 3'-terminal cDNA fragments, use of them as the sole means of specific gene isoform identification within a family is not completely accurate. The smear demonstrated by Northern blot analysis supports the idea that the G1 fragment recognized several RNA species and not just one single band. Second, the library used was an endometrial library that may not have contained the RyR2 and -3 isoforms.
Previous studies indicate not only the altered gene expression after organ culture but also functional changes important in maintaining normal VSM contractility. There is an altered Ca2+ handling associated with the amount of serum used in culture of rat-tail artery (7). Moreover, there is evidence suggesting an altered intracellular store activity after organ culture (3). Our data indicate that there is an increased force response to caffeine in porcine coronary arteries after organ culture. This would suggest that there is an increase in the Ca2+ loading of the ryanodine/caffeine Ca2+ store or that there is an upregulation of the RyR. Overall results from our organ culture model support these interpretations and correlate nicely with an increase in RyR2 and RyR3 expression. Interestingly, maximum force generation to KCl stimulation is not significantly different after organ culture, suggesting that activation through extracellular Ca2+ influx is unaffected.
We have previously described the specific inhibition of relaxation to hypoxia after organ culture of VSM. This inhibition is associated with an inability to reduce [Ca2+]i during acute hypoxia. RyR has been implicated in hypoxic vasoconstriction of pulmonary arteries (2, 10). In fact, inhibition of Ca2+ release from these intracellular stores significantly reduces the force increase to hypoxia in these vessels (10). Additionally, decreased RyR function can modulate Ca2+ sparks potentially associated with some oxygen-sensing mechanisms (5). Our results show an increase in two types of RyR. Both RyR2 [predominantly found in heart and some expression in brain (17)] and RyR3 [ubiquitous expression (17)] are associated with lower activation thresholds compared with RyR1 (18). It is conceivable that an increase in both of these RyR isoforms in systemic VSM could translate into the observed altered intracellular Ca2+ handling during hypoxia and may contribute to the loss of hypoxic vasorelaxation.
In VSM, mitochondrial inhibition may induce Ca2+ spark activity that may lead to hyperpolarization and ultimate vasodilation (23). Additionally, the redox status of RyR, which is possibly influenced by decreased PO2, can alter Ca2+ release (16). One hypothesis from this investigation is that an increase in RyR expression could lead to altered hypoxia-induced Ca2+ sparks and saturation of any oxygen-sensitive hyperpolarization response. We have shown that KCl concentration-response curves are right-shifted in organ-cultured arteries, indicating a more hyperpolarized tissue (24). Ryanodine treatment in control vessels may mimic organ culture results by acutely altering Ca2+ spark activity and reducing that available for hypoxic relaxation (Fig. 6). Therefore, it is possible that the resulting change in intracellular Ca2+ handling coincident with increased RyR expression after organ culture may contribute to the inability to relax to hypoxia. An alternate interpretation based on recent evidence (12) is that mitochondria-dependent Ca2+ uptake altered by hypoxia may contribute to the reduced Ca2+ handling capability after organ culture. Further investigation is necessary to determine any direct relationship between RyR expression and altered oxygen sensitivity in porcine coronary artery.
Here we demonstrate how organ culture causes the increased expression of specific RyR isoforms, RyR2 and RyR3. This increased expression is supported by functional data indicating a larger increase in isometric force in response to caffeine-activated intracellular Ca2+ release. Importantly, ryanodine was found to blunt the hypoxic relaxation in control arteries. These changes in VSM gene expression with organ culture coupled with new sensitive methods for assessing changes in RNA and protein levels provide us with new tools to approach the elusive mechanisms of vascular oxygen sensing.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. J. Paul, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576 (E-mail: george.thorne{at}amedd.army.mil).
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.
First published December 11, 2002;10.1152/ajpcell.00158.2002
Received 9 April 2002; accepted in final form 5 December 2002.
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