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(Hypertension. 1995;26:177-185.)
© 1995 American Heart Association, Inc.
Articles |
Endothelial Stimulation of Sodium Pump in Cultured Vascular Smooth Muscle
From the Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma, and Unidad de Investigación y Servicio de Geriatría, Hospital Universitario de Getafe (L.R.-M.), Madrid, Spain.
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Abstract |
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Abstract We studied vascular sodium pump activity and its regulation by vasoactive agents and endothelium in cultured aortic vascular smooth muscle cells from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Baseline sodium pump activity (ouabain-inhibitable 86Rb+ uptake) was similar in cells from both rat strains. Angiotensin II and endothelin-1 increased ouabain-inhibitable 86Rb+ uptake more in SHR than WKY cells, whereas no effects were obtained with sodium nitroprusside, 8-bromo-cGMP, or iloprost. We examined the influence of endothelium on vascular sodium pump activity either by coculturing smooth muscle and endothelial cells or by using conditioned medium. Both coculture for 24 hours with endothelial cells and treatment with conditioned medium increased smooth muscle cell sodium pump activity, this effect being higher in SHR cells. These results suggest that the endothelium may modulate sodium pump activity in the underlying smooth muscle by releasing a diffusible compound, which is more active on SHR smooth muscle. The conditioned medium obtained in the presence of inhibitors of angiotensin-converting enzyme, endothelin-1–converting enzyme, cyclooxygenase, lipoxygenase, and nitric oxide synthase had no effect on the ability of conditioned medium to increase sodium pump activity, suggesting that angiotensin II, endothelin-1, eicosanoids, and nitric oxide are not involved in this stimulatory effect. The nature of the possible endothelial factor involved is still unknown, but it possesses a molecular weight between 25 and 50 kD, is heat stable, and is sensitive to trypsin treatment. We propose it could be a growth factor.
Key Words: endothelium • muscle, smooth, vascular • Na+-K+–exchanging ATPase • ouabain • hypertension, sodium-dependent
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Introduction |
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The role of endogenous ouabainlike factors (OLFs), inhibitors of the Na+,K+-ATPase, in the development of high blood pressure is controversial.1 Some recent works, however, reinforce their involvement in hypertension and propose that these OLFs are indistinguishable from ouabain.2 In addition, an elevated ouabainlike activity in response to sodium intake has been reported in the spontaneously hypertensive rat (SHR).3
The effects of ouabain on vascular resistances are well known; it causes vasoconstriction by a direct effect on vascular smooth muscle cells (VSMCs) or by the release of norepinephrine from perivascular nerve endings.4 Indeed, the contractile responses evoked by cardiac glycosides are enhanced in the SHR vasculature.5 6 In addition to vasoconstriction, the inhibition of Na+,K+-ATPase can increase vascular resistances by interfering with vasodilator endothelial factor or factors.7 8 9 Thus, ouabain inhibits endothelium-dependent vasodilation by blocking nitric oxide (NO) release from endothelial cells (ECs),8 10 by reducing the effects of NO in the underlying VSMCs,9 11 or by antagonizing the action of a still unidentified endothelium-dependent hyperpolarizing factor.12
On the other hand, there is growing evidence that ECs may modulate the vasoconstriction induced by sodium pump blockade. Indeed, the contractions evoked by a K+-free solution in rat mesenteric arteries are attenuated in the presence of endothelium and seem to be mediated by NO release.13 Furthermore, the removal of vascular endothelium potentiates the ouabain-induced contractions in human placental arteries and veins14 and guinea pig carotid arteries.15 These effects appear to be due to the loss of a diffusible endothelial factor, not related to NO, prostaglandins, or leukotrienes and not similar to the endothelium-derived hyperpolarizing factor previously mentioned.14 15 It is possible that this endothelial factor can act by stimulating the Na+,K+-ATPase activity in the underlying VSMCs, antagonizing its blockade by ouabain or OLF, or both.14 15 16
The aim of the present work was to further characterize the influence of ECs on vascular sodium pump activity, as well as the possible modification of sodium pump activity in hypertension. For this purpose, we used cultured VSMCs from normotensive Wistar-Kyoto rats (WKY) and SHR to avoid interferences from other vascular components. To analyze the action of endothelium on Na+,K+-ATPase activity, we used bovine aortic ECs.
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Methods |
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Experimental Animals
Commercially available 20-week-old male WKY and SHR weighing 300 to 350 g were used (Iffa-Credo). The rats were fed from 4 weeks of age at the facilities of the Facultad de Medicina de la Universidad Autónoma (Madrid). For determination of mean arterial pressure, rats were anesthetized with pentobarbital (70 mg/kg IP), carotid arteries were cannulated and connected to a transducer (Letica), and pressure was registered on a polygraph (2006 Letica). Mean arterial pressure (mean±SEM) was 98±5 mm Hg in WKY (n=16) and 172±9 mm Hg in SHR (n=16, P<.05). Rats were then exsanguinated and aortas carefully dissected and removed. The procedures followed were in accordance with European institutional guidelines.
Cell Culture
Primary cultures of rat VSMCs were obtained by enzymatic
dissociation of WKY and SHR arteries. Thoracic aortas were cut into
small pieces, placed in Dulbecco's modified Eagle's medium (DMEM,
Gibco) containing 0.1% bovine serum albumin (BSA, Sigma Chemical Co)
and 4 mg/mL collagenase (type II, Sigma), and incubated for
90 minutes at 37°C in a humidified atmosphere of 5% CO2
and 95% air. The resulting cell suspension was washed three times by
centrifugation with fresh DMEM containing 0.1% BSA. Cells were then
resuspended in culture medium consisting of DMEM supplemented with 10%
fetal calf serum (Gibco), 100 U/mL penicillin, 100 µg/mL
streptomycin, and 2.5 µg/mL amphotericin B (Sigma) and seeded into
25-cm2 culture flasks (Nunc) coated with 1% gelatin
(Sigma). Cells were characterized as smooth muscle by
immunocytochemical staining with smooth muscle–specific monoclonal
antibody to 
-actin; 98% of these cells were positively stained.
Experiments were performed in VSMCs pooled from at least four rats of
each strain. Cultures from identically treated lines between passages 6
and 16 were used.
To obtain bovine aortic EC cultures, bovine aorta was opened and cleaned carefully with phosphate-buffered saline (PBS). The endothelial source then was incubated with PBS containing 2 mg/mL collagenase (type II, Sigma) for 10 minutes at room temperature. The resulting cell suspension was collected and washed twice by centrifugation with fresh DMEM containing 0.1% BSA. Cells were resuspended in culture medium and treated as previously described for VSMCs. ECs were characterized by immunocytochemical staining with a monoclonal antibody QBEnd/10 raised against a CD34 endothelial marker17 ; approximately 95% of these cells were positively stained. ECs were subcultured and passaged in 80-cm2 flasks (Nunc) with 0.05% trypsin/0.02% EDTA (Gibco). Experiments were performed with ECs pooled from at least three bovine aortas. Cultures from identically treated cell lines between passages 4 and 16 were used.
86Rb+ Uptake
86Rb+ uptake was studied according to
the method described by Magargal and Overbeck18 with minor
modifications. After trypsinization, VSMCs were resuspended in culture
medium and plated into 24-well culture plates at a density of
approximately 5x104 cells per well. VSMCs were cultured
until they reached confluence (about 4 days after plating) and then
were growth arrested by replacement of culture medium with serum-free
DMEM containing 0.1% BSA and antibiotics (vehicle medium) for 24
hours. The medium was then removed and replaced with vehicle medium
with or without 1 mmol/L ouabain, which has been shown to completely
inhibit the Na+,K+-ATPase
activity.19 The cells were preincubated at 37°C for 15
minutes; then 3.8 µmol/L 86Rb+ (1 µCi/mL;
specific activity, 4.68 mCi/mg; New England Nuclear) was added to every
well, and the cells were incubated at 37°C for different time periods
(1 to 120 minutes). The K+ concentration of the incubation
medium was 5.4 mmol/L; therefore, the ratio of [Rb+] to
[K+] was 7x10-4. The medium was aspirated,
and cell layers were rapidly rinsed six times with 100 mmol/L ice-cold
MgCl2.
Intracellular 86Rb+ was extracted with 500 µL of 5% trichloroacetic acid at 4°C for 1 hour. Aliquots of 400 µL from every well were used for determination of 86Rb+ by counting in an LS2800 liquid scintillation counter (Beckman Instruments, Inc). After removal of trichloroacetic acid, cell protein was solubilized by incubation with 200 mmol/L NaOH at 4°C overnight. Protein determination was performed according to the method described by Bradford20 with the use of BSA as standard. 86Rb+ uptake was expressed as nanomoles per milligram protein. Ouabain-sensitive 86Rb+ uptake (Na+,K+-ATPase activity) was estimated as the difference between total and ouabain-insensitive uptakes. Sodium pump activity in the presence of different compounds or effectors was measured by adding them in wells either with or without ouabain 15 minutes before and during the incubation period with 86Rb+. This activity was related to the baseline sodium pump calculated for each experiment.
Conditioned Medium
Confluent cultures of ECs in 80-cm2 flasks (Nunc)
were washed extensively with vehicle medium and then maintained in the
same serum-free medium for 48 hours. The medium then was collected,
centrifuged once for 10 minutes to eliminate possible cell residues,
and stored at -20°C (conditioned medium, CM). Simultaneously, ECs
were detached with trypsin-EDTA, and cell number was determined with a
hemocytometer. Thus, the volume of CM was related to the number of ECs
used for conditioning (CM concentration was then expressed as ECs per
milliliter). 86Rb+ uptake experiments were
performed as previously described except that vehicle medium was
replaced by CM.
In another set of experiments, the medium was conditioned in the presence of different inhibitors such as captopril (1 µmol/L), phosphoramidon (100 µmol/L), NG-nitro-L-arginine methyl ester (L-NAME, 1 and 10 µmol/L), indomethacin (1 and 10 µmol/L), and 5,8,11,14-eicosatetraynoic acid (ETYA, 1 and 10 µmol/L). In these cases, each inhibitor was added to the medium for 48 hours; after the first 24 hours of incubation, the same concentrations of the respective drugs were added again to avoid possible degradation of the drug. The concentrations of these agents in the text and figures reflect the amount present before the second addition. In other experiments, CM was incubated at 37°C with 200 µg/mL trypsin (type IX, Sigma) for 150 minutes; the reaction was stopped by addition of 800 µg/mL trypsin inhibitor (type I-S, Sigma). CM then was incubated again at 37°C for 30 minutes and finally stored at -20°C. The same treatment was applied to the control medium. In other experiments, CM and control medium were placed in boiling water at 100°C for 3 or 10 minutes and stored at 4°C before assay.
The fractions of CM with different molecular weight were obtained by ultrafiltration assays. In these experiments, CM was filtered through membranes with a pore size that retains substances with molecular weights higher than 25 kD (Centriflo, Amicon) or 50 kD (Macrosep, Filtron). The CM (15 mL) was added to the filter tube and centrifuged at 3000g for 150 minutes. Supernatant and pellet were collected and brought to 15 mL (initial volume) with vehicle medium.
Coculture
Coculture of VSMCs and ECs was performed with
"transwell" 24-multiwell plates (Costar). Confluent
monolayers of ECs were grown in the transwells and then introduced into
confluent cultures of VSMCs. The coculture was performed for 24 hours
in vehicle medium. Then, transwells containing ECs were removed, and
86Rb+ uptake was measured in VSMCs as
previously described. In the same conditions, transwells without ECs
were used as controls.
[3H]Ouabain Binding Studies
These experiments were performed in VSMCs according to the
method described by Khalil et al21 with minor
modifications. Briefly, cells (6x105 cells per well) were
inoculated into six-well culture plates and cultured until they reached
confluence (about 4 days after plating). Cells were growth arrested by
replacement of culture medium by vehicle medium for 24 hours. The
medium was then removed, and the cells were washed with 5 mL
K+-free DMEM to remove any trace of vehicle medium that
contained potassium. VSMCs were incubated at 37°C in 95% air/5%
CO2 for 150 minutes in K+-free DMEM containing
a fixed concentration of 100 nmol/L [3H]ouabain (specific
activity, 29 Ci/mmol; Amersham) and different concentrations of
unlabeled ouabain (from 1 to 10 µmol/L). Nonspecific
[3H]ouabain binding was determined by incubation of the
cells in the presence of the labeled and 1 mmol/L unlabeled ouabain.
After incubation, the medium was suctioned, and the cells in each well
were washed seven times with 2 mL ice-cold PBS. After washing, cells
were disrupted with 2 mL of 200 mmol/L NaOH at 4°C overnight.
Aliquots of 1 mL were counted in a Beckman LS2800 liquid scintillation
counter, and protein determination was performed according to the
method described by Bradford.20 We have used the
modification of Dixon22 described by Khalil et
al21 to calculate Kd and
Bmax for ouabain.
In other experiments, commercial canine kidney cortex Na+,K+-ATPase (specific activity, 1 to 2 U; Sigma) was used. [3H]Ouabain binding was carried out in tubes with the purified enzyme (0.02 U per tube) in 1 mL buffer medium plus freshly added ATP (100 µmol/L) and different concentrations of cold ouabain (from 100 pmol/L to 100 µmol/L). The composition of this buffer medium was (mmol/L) Tris-HCl 50, NaCl 150, MgCl2 2.5, and Na2EDTA 1, pH 7.4. The tubes then were placed in a shaking water bath at 37°C; 15 minutes later, 10 nmol/L [3H]ouabain was added and the tubes were incubated for 60 minutes. Binding was stopped by quickly cooling the tubes by immersion into a bath at 4°C. Samples were diluted with 2 mL ice-cold buffer and rapidly filtered through GF/C (Whatman Ltd) glass fiber filters. The filters were then washed with 10 mL ice-cold buffer, and their radioactivity was measured as previously described. Specific binding was expressed as femtomoles [3H]ouabain per milligram purified enzyme.
32P Release
The activity of purified
Na+,K+-ATPase was measured by
32P release according to the method described by Kelly et
al23 with minor modifications. In these experiments,
different concentrations of ouabain (from 100 pmol/L to 1 mmol/L) and
CM aliquots were used. These compounds were preincubated for 2 hours at
37°C with 700 µL incubation buffer consisting of (mmol/L) NaCl 150,
Tris-HCl 50, Na2EDTA 0.25, MgCl2 5, and ATP 5,
pH 7.4, plus 100 µL of a 0.6 mg/mL (0.05 U/mL) preparation of canine
kidney Na+,K+-ATPase. Subsequently, KCl
(final concentration, 5 mmol/L) and 0.3 µCi of
[
-32P]ATP (Amersham) were added to yield a final
volume of 1 mL. This mixture was incubated for 30 minutes at 37°C,
and the reaction was terminated by addition of 3 mL ice-cold 4%
(wt/vol) charcoal (Norit A, Sigma) in 100 mmol/L HCl, 1 mmol/L
NaH2PO4, and 1 mmol/L
Na4P2O7. After 5 minutes at room
temperature, the samples were centrifuged at 2000g for 10
minutes, and the 32P released was determined in a Beckman
LS2800 liquid scintillation counter.
Drugs and Statistical Evaluation
Angiotensin II (Ang II), endothelin-1, phosphoramidon, ouabain
octahydrate, indomethacin, ETYA, sodium nitroprusside,
8-bromo-cGMP, L-NAME, and saralasin were purchased from Sigma;
captopril from Squibb; and iloprost from Schering.
Data are given as mean±SEM. In all experiments, n indicates the number of independent experiments. Each experiment was performed in three to four replicates (wells) for the different studied cases with the use of VSMCs passaged from the same primary culture derived from at least four rats. Student's t test for unpaired observations and ANOVA were used for statistical analyses. A probability value less than .05 was considered significant. EC50 was calculated according to the procedure of Fleming et al.24
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Results |
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86Rb+ Uptake Studies
Preliminary 86Rb+ uptake experiments were performed for determination of the optimal incubation period with this radioisotope and evaluation of sodium pump activity in WKY and SHR VSMCs. In basal conditions 86Rb+ uptake increased linearly with time up to 40 to 60 minutes; however, the rate of 86Rb+ uptake decreased significantly in the presence of the Na+,K+-pump inhibitor ouabain (1 mmol/L) after 20 minutes of incubation compared with untreated cells (Fig 1). The ouabain concentration used was chosen in agreement with previous results from our laboratory and others.19 As described in "Methods," Na+,K+-ATPase activity was defined in every experiment as the difference between total and ouabain-insensitive 86Rb+ uptakes. The values for total and ouabain-insensitive 86Rb+ uptakes were similar in WKY and SHR cells. Therefore, sodium pump activity (determined by ouabain-sensitive 86Rb+ uptake) was also similar in VSMCs from both rat strains (Fig 1). In the rest of the experiments, a 40-minute incubation period with the radionuclide was selected.
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P<.05 vs total 86Rb+ uptake.To study the regulation of sodium pump activity by different endothelial vasoactive factors, we performed similar 86Rb+ uptake experiments in the presence of such compounds added 15 minutes before and during the incubation period. Ang II (from 100 pmol/L to 1 µmol/L) induced concentration-dependent increases in Na+,K+-pump activity in both WKY and SHR VSMCs (Fig 2A); this activity was greater in SHR cells. The angiotensin receptor antagonist saralasin (1 µmol/L) abolished this Ang II–induced effect (data not shown), demonstrating that this effect was mediated by specific receptors, in agreement with previous results from other researchers.25 VSMC sodium pump activity was also stimulated by endothelin-1 (from 100 pmol/L to 100 nmol/L) in both cell types, this stimulation being higher in SHR versus WKY cultures (Fig 2B). On the other hand, the stable prostacyclin analogue iloprost (1 to 100 nmol/L), the NO donor sodium nitroprusside (10 and 100 µmol/L), and the permeable cGMP analogue 8-bromo-cGMP (10 and 100 µmol/L) had no significant effect on ouabain-sensitive 86Rb+ uptake in VSMCs from either SHR or WKY (data not shown). The concentration levels of these agonists were considered appropriate on the basis of previous work from our laboratory.9 26 None of the studied drugs modified the ouabain-insensitive 86Rb+ uptake in VSMCs from SHR and WKY (data not shown).
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To investigate endothelial modulation of vascular sodium pump activity, we performed cocultures with VSMCs and ECs for 24 hours. Coculture increased ouabain-sensitive 86Rb+ uptake in WKY and SHR cells (Fig 3A). Moreover, this effect on sodium pump activity was greater in VSMCs from SHR compared with those from WKY (Fig 3A). The ouabain-insensitive 86Rb+ uptake was also increased by coculture in WKY cells (from 306.9±10.8 to 448.3±8.1 nmol 86Rb+/mg protein; P<.05 versus control value) but not in SHR cells (data not shown).
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In addition, we studied the effect of CM produced by the growth of different numbers of ECs. In VSMCs from WKY and SHR, CM induced an increase in sodium pump activity, this effect being dependent on the number of ECs used for conditioning (Fig 3B). This stimulatory effect was again greater in SHR cells (Fig 3B). In this type of experiment, however, ouabain-insensitive 86Rb+ uptake was not modified by CM in VSMCs from either WKY or SHR (data not shown). In the following experiments, only CM yielding the maximal stimulatory effect was used.
To analyze a possible antagonism between CM and ouabain, we measured sodium pump activity in the presence of increasing concentrations of ouabain (from 10 µmol/L to 1 mmol/L) with either CM or vehicle control medium. In these experiments, we assumed that sodium pump activity was abolished in the presence of 1 mmol/L ouabain; therefore, we considered that the ouabain-sensitive 86Rb+ uptake had a value of zero in these conditions (Fig 4). In both cell types, ouabain induced concentration-dependent decreases on sodium pump activity in the presence of either CM or control medium. The increment in ouabain-sensitive 86Rb+ uptake by CM was abolished by 500 µmol/L ouabain in WKY VSMCs, whereas in SHR cultures a concentration of 1 mmol/L was necessary to produce the same effect (Fig 4).
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To determine some possible vasoactive endothelial factors involved in the stimulatory effect produced by ECs, we tested the effect of different agents. Saralasin (1 µmol/L) did not modify the CM-induced increase of vascular sodium pump activity in VSMCs from WKY and SHR (data not shown). In another set of experiments, the endothelial cultures were treated for 48 hours with the angiotensin-converting enzyme blocker captopril (1 µmol/L), the endothelin-converting enzyme inhibitor phosphoramidon (100 µmol/L), the NO synthase antagonist L-NAME (1 and 10 µmol/L), the cyclooxygenase blocker indomethacin (1 and 10 µmol/L), and the cyclooxygenase and lipoxygenase inhibitor ETYA (1 and 10 µmol/L). None of these drugs modified the ouabain-sensitive 86Rb+ uptake elicited by CM in either WKY or SHR cells (data not shown). The concentration levels of these blockers were chosen in agreement with reported results from our laboratory14 26 27 28 and by others.25 29
We used three different approaches to analyze the characteristics of the endothelial factor or factors that stimulated the sodium pump: heat stability, protease digestion, and stimulation of molecular size with the use of ultrafiltration. Heating of CM at 100°C for 3 minutes did not change its ability to activate the sodium pump in these cell cultures (data not shown). However, the same treatment for 10 minutes dramatically reduced the effects of CM but had no effect in the control case (Fig 5A). In addition, CM incubation for 150 minutes with the nonspecific protease trypsin (200 µg/mL) abolished the ability of CM to stimulate Na+,K+-ATPase (Fig 5B). On the other hand, CM aliquots were ultrafiltered through membranes that had a 25- or 50-kD molecular weight exclusion limit. In both cell types, only CM fractions that contained substances larger than 25 kD or compounds smaller than 50 kD reproduced the CM-induced stimulation of vascular sodium pump activity (Fig 6). Finally, to determine whether stimulation of the sodium pump by CM might result from the greater permeability to Na+ of cultures treated with CM, we loaded WKY and SHR VSMCs with Na+ by incubation in a K+-free buffer during 150 minutes and measured 86Rb+ uptake for 40 minutes in loaded and control cells. Na+ loading increased sodium pump activity in both cell types (WKY, from 171.0±5.4 nmol 86Rb+/mg protein in control to 300.9±19.3 in Na+-loaded cells; SHR, from 175.7±5.5 nmol 86Rb+/mg protein in control to 396.8±13.1 in Na+-loaded cells). In these experimental conditions, the CM produced a similar increment on ouabain-sensitive 86Rb+ uptake in control cells (227±20.8 and 260±9.3 nmol 86Rb+/mg protein in WKY and SHR VSMCs, respectively) and in Na+-loaded cells (189.0±24.1 and 275.6±14.9 nmol 86Rb+/mg protein).
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[3H]Ouabain Binding Studies
We performed binding experiments in WKY and SHR VSMCs to calculate
Kd and Bmax values for
ouabain in the same experimental conditions that we used to determine
sodium pump activity for each VSMC preparation. The
Bmax values for WKY and SHR VSMCs were
1.19±0.03 and 0.97±0.05 pmol/mg cell protein, and
Kd values were 2.90±0.07 and 2.88±0.11
µmol/L, respectively (n=10-12). These experiments demonstrate no
significant differences in these parameters between WKY and SHR.
To determine a possible displacement of [3H]ouabain by CM, we designed binding experiments using purified canine kidney Na+,K+-ATPase. We performed initial binding studies with cold ouabain (from 100 pmol/L to 100 µmol/L) in the presence of a fixed [3H]ouabain concentration (10 nmol/L) to validate this method. Ouabain produced a concentration-dependent displacement in [3H]ouabain specific binding that was abolished at 1 µmol/L (data not shown). The EC50 for the ouabain binding assay was 2.96±0.09 nmol/L. As culture medium contains K+, which can displace ouabain binding, parallel experiments with CM or control medium were conducted. Different volumes of CM and control medium (from 25 to 250 µL) produced identical volume-dependent [3H]ouabain displacement (Fig 7A).
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32P Release Studies
To examine whether the CM stimulation was mediated by a direct
effect on Na+,K+-ATPase, we used CM
aliquots in an enzymatic activity assay that measured activity by
32P release. First, we performed a dose-response curve with
ouabain (from 100 pmol/L to 1 mmol/L) to validate the method. Ouabain
induced concentration-dependent decreases in 32P release up
to 50% of control at 1 mmol/L (data not shown). In the same
experimental conditions, CM and control medium did not modify the
Na+,K+-ATPase activity measured by
32P release (Fig 7B).
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Discussion |
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The main objective of this work was to investigate the possible endothelial modulation of sodium pump function in cultured VSMCs. For this purpose, we measured basal sodium pump activity (ouabain-sensitive 86Rb+ uptake) in WKY and SHR VSMCs and found it to be similar in both cell types. This finding agrees with previous results reported in cell cultures by Orlov et al30 but disagrees with the increased sodium pump activity reported in SHR VSMCs reported by other authors.31 32 The reason for such differences is not clear, but the discrepancies could reflect different growing states of VSMC cultures. Some authors used subconfluent proliferating cultures,32 whereas we and others used confluent nonproliferating VSMC cultures.30 An enhancement of sodium pump activity has been proposed during proliferation, caused by the increase in the number of sodium pump sites.33 34 In our experiments, the final cell density was higher in the SHR cultures, consistent with a previously described enhanced growth rate35 ; however, the number of Na+,K+-ATPase units per milligram of cellular protein (Bmax) and the affinity of Na+,K+-ATPase for ouabain were similar in VSMC cultures from both WKY and SHR. Therefore, the number of sodium pump sites and the activity in confluent nonproliferating cultures from WKY and SHR cells did not differ.
We also tested the effects of different endothelium-related vasoactive factors that can modify sodium pump activity. The vasoconstrictor peptides endothelin-1 and Ang II enhanced ouabain-sensitive 86Rb+ uptake in VSMCs from both rat strains, in agreement with previous results from other researchers.25 36 Thus, endothelin stimulates Na+,K+-ATPase activity in rabbit aorta by a protein kinase C–dependent pathway,36 and the ability of Ang II to increase sodium pump activity, previously reported in rat VSMC cultures, is due to Na+ entry by Na+-H+ exchange stimulation.25 30 37 In the present experiments, the enhancement of Na+,K+-ATPase activity was significantly higher in SHR than in WKY cells, as observed by others.30 An increased signaling responsiveness to different stimuli has been reported in VSMCs from SHR, including the activation of phosphoinositide catabolism, protein kinase C, and Na+-H+ exchange,38 39 40 which are related to the actions of Ang II or endothelin.36 41 Additionally, a higher number of angiotensin receptors has been detected in SHR than in WKY VSMC cultures.40 Therefore, both the receptor and signaling abnormalities found in SHR cells may contribute to their increased response to vasoactive peptides in activating the vascular sodium pump.
We also investigated the effects of vasorelaxant endothelial compounds on Na+,K+-ATPase activity. It has been proposed that endothelium-derived NO and those drugs that mimic NO effects by enhancing intracellular levels of cGMP may exert part of their actions by stimulating vascular sodium pump activity.7 9 11 The relaxant effects of prostacyclin also have been related to the activation of vascular Na+,K+-ATPase.42 43 For this reason, in the present work we tested sodium nitroprusside, as an NO donor, the diffusible cGMP analogue 8-bromo-cGMP, and the prostacyclin analogue iloprost. None of these compounds modified the ouabain-sensitive 86Rb+ uptake in VSMCs from both WKY and SHR, indicating that at least in our experimental conditions, NO and prostacyclin do not play a key role in vascular sodium pump modulation.
Some previous reports indicate that the endothelium can modulate the vasocontractile responses evoked by ouabain by stimulating sodium pump activity in VSMCs and/or antagonizing the inhibitory effects of digitalis; this effect is mediated by a diffusible factor not related to NO, prostaglandins, or leukotrienes.14 15 In the present work, we tested the direct endothelial effects on Na+,K+-ATPase activity by coculture experiments or with the use of EC-derived CM. Both procedures increased ouabain-sensitive 86Rb+ uptake, which was significantly greater in SHR cells. Therefore, we can conclude that at least in the present experimental conditions, ECs are releasing a diffusible factor to the medium that can stimulate the activity of Na+,K+-ATPase in VSMCs. Analogous enhancement of sodium pump activity in VSMCs by cocultured endothelium has been previously reported by Berk.44 Although extrapolation from cell cultures to tissular conditions remains speculative, these data are consistent with previous contractility studies with isolated vascular segments that allow for a possible vasoactive role for such an endothelial factor.14 15
At present, we cannot explain the higher responses to this endothelial stimulatory effect observed in SHR VSMC cultures. As previously discussed, sodium pump activity and ouabain binding characteristics were similar in WKY and SHR cultures under baseline conditions, in agreement with data reported by others.30 45 Some authors even propose that the WKY may be an inadequate control for SHR; indeed, Hopp et al45 report similar binding characteristics in VSMC cultures from WKY and SHR, and cultures from normotensive Wistar rats exhibit a lower number of sodium pump sites and also a lower affinity to ouabain. They conclude that WKY may be closer genetically to SHR than to a normotensive strain such as the Wistar rat.45 Therefore, it seems unlikely that the differences observed in CM-induced effects in both cell types could be attributed to changes in the number and/or affinity for ouabain of the expressed sodium pump sites. An alternative hypothesis is that such differences could involve an enhanced signaling responsiveness to stimuli, analogous to that previously discussed for Ang II and endothelin. In agreement with this assumption, Orlov et al30 report similar baseline sodium pump activity in WKY and SHR cultured VSMCs, where the stimulation of sodium pump activity in response to Ang II is higher in the SHR cells.
On the other hand, coculture also increased the ouabain-insensitive 86Rb+ uptake in WKY VSMCs. This result agrees with that described by Berk,44 who cocultured VSMCs and ECs, and is explained by an increase in Na+-K+-2Cl- cotransporter activity induced by the endothelium. However, coculture had no effect on the ouabain-insensitive 86Rb+ uptake in SHR VSMCs. In addition, CM had no effect on the ouabain-insensitive 86Rb+ uptake in VSMCs from WKY and SHR. Although this finding has yet to be explained, it is possible that different factors may be involved in the endothelial activation of the Na+-K+ pump and Na+-K+-2Cl- cotransporter, which exert their effects in a different way according to cell type or experimental conditions.
To determine whether known endothelial vasoactive factors are responsible for the activation of the vascular sodium pump by bovine aortic ECs, we designed several experiments. It is known that endothelium produces Ang II46 and endothelin-147 ; these compounds can stimulate the vascular Na+,K+-ATPase activity in our experimental conditions, as shown in Fig 2. We analyzed the possible involvement of these peptides in the CM-evoked effects using the angiotensin receptor antagonist saralasin and obtaining CM in the presence of the angiotensin-converting enzyme inhibitor captopril or with the endothelin-1 inhibitor phosphoramidon.29 None of these procedures had an effect on the CM-induced response, indicating that Ang II and endothelin-1 are not involved in the sodium pump activation by CM. On the other hand, to examine the possible role for NO11 13 or eicosanoids in the stimulation of Na+,K+-ATPase induced by endothelium, we obtained CM in the presence of the NO synthase inhibitor L-NAME, cyclooxygenase blocker indomethacin, or lipoxygenase and cyclooxygenase inhibitor ETYA.48 49 Again, none of these treatments modified the ability of CM to increase ouabain-sensitive 86Rb+ uptake in either WKY or SHR VSMCs. These data confirm our previous results with NO analogues and suggest that NO or metabolites derived via lipoxygenase and cyclooxygenase are unlikely to be involved in the vascular sodium pump activation produced by CM.
Previous works have proposed the possibility that endothelium could be antagonizing the inhibitory actions of ouabain on vascular Na+,K+-ATPase.14 15 In addition, it has been shown that bovine aortic endothelium can antagonize the inhibition of Na+,K+-ATPase induced by OLF in cocultured canine aortic smooth muscle.16 In the present experiments, ouabain induced parallel concentration-dependent reductions on the sodium pump activity in both cell types, either in control medium or in the presence of CM, SHR cultures being more resistant to ouabain effects. However, a possible biochemical antagonism between CM and ouabain can be discarded because [3H]ouabain binding experiments with commercially purified enzyme show that CM or control medium produce an identical displacement of labeled ouabain, probably because of the K+ content in both media. In addition, CM did not modify the activity of purified Na+,K+-ATPase preparations measured by 32P release according to well-known procedures.23 This group of results strongly indicates that the endothelium-derived compound that we are dealing with is not stimulating the Na+,K+-ATPase activity by a direct binding to this enzyme. The mechanisms of this stimulation are at present unknown; indeed, the results obtained in Na+-loaded cells suggest that CM activates the sodium pump in VSMC cultures even under conditions such that cell Na+ is not a limiting substrate for the pump.25
To identify some physicochemical characteristics of the possible endothelial factor or factors involved in sodium pump stimulation, we submitted CM to different treatments. Thus, heating at 100°C for 3 minutes or multiple freeze-thaw cycles did not affect CM effects, whereas heating for 10 minutes dramatically reduced the stimulatory effect of CM on sodium pump activity in VSMCs from both rat strains. Therefore, the endothelial factor or factors responsible for those effects appear to possess a stable structure in response to temperature changes, further discarding labile substances as prostaglandin-like molecules.48 In addition, the ability of CM to stimulate the Na+,K+-ATPase in cells from both rat strains was abolished by trypsin treatment for 150 minutes. Therefore, this endothelium-derived compound seems to be a peptide, or at least it has a peptidic structure essential for inducing vascular sodium pump activation. Finally, ultrafiltration assays indicate that the molecular weight of the compound involved in the endothelial effects is between 25 and 50 kD.
Although the chemical nature of this endothelial factor is still unknown, the data on heat stability, trypsin sensitivity, and molecular weight allow one to discard the known endothelial vasoactive agents. Moreover, these characteristics are consistent with other endothelium-derived substances that may be released from cultured ECs, namely, growth factors such as platelet-derived growth factor and transforming growth factor.44 50 Indeed, these endothelial mitogens can stimulate sodium pump activity in cultured VSMCs33 44 and may have vasoactive contractile or relaxant properties.51 52 Preliminary results from our laboratory indicate that in fact, CM possesses growth properties in both WKY and SHR VSMCs, SHR cultures being more sensitive than WKY cultures (unpublished results, 1995), which agrees with the higher sensitivity of SHR cells to growth factor effects.35 Therefore, from these data it is possible to speculate on the influence of endothelium-derived mitogens on the ionic homeostasis in VSMCs and subsequently in the regulation of vascular tone in either normotension or hypertension. The verification of this hypothesis, as well as its possible physiological or physiopathologic relevance, will of course require further experimental support.
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Acknowledgments |
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This work was supported by grants from FISS (93/0916E), DGICYT (FAR 91-0205 and PM 92-0037), Biomed Project (PL93 1375), and Bayer España.
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Footnotes |
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Reprint requests to Dr Carlos F. Sánchez-Ferrer, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma, c/Arzobispo Morcillo, 4, 28029-Madrid, Spain.
Received July 5, 1994; first decision October 26, 1994; accepted March 2, 1995.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Goto A, Yamada K, Yagi N, Yoshioka M, Sugimoto T. Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev. 1992;44:377-397. [Medline] [Order article via Infotrieve]
2.
Hamlyn JM, Blaustein MP, Bova S, Ducharme DW, Harris
DW, Mandel F, Mathews WR, Ludens JH. Identification and
characterization of a ouabain-like compound from human plasma.
Proc Natl Acad Sci U S A. 1991;88:6259-6263.
3.
Huang BS, Leenen FHH. Brain ouabain and central
effects of dietary sodium in spontaneously hypertensive rats.
Circ Res. 1992;70:430-437.
4. Mulvany MJ. Changes in sodium pump activity and vascular contraction. J Hypertens. 1985;3:429-436. [Medline] [Order article via Infotrieve]
5. Shibata R, Morita S, Nagai K, Miyata S, Iwasaki T. Calcium dependence of ouabain-induced contraction in aortas from hypertensive rats. Eur J Pharmacol. 1990;190:147-157. [Medline] [Order article via Infotrieve]
6. Sakai Y, Inazu M. Sodium pump activity and contractions of renal arteries from spontaneously hypertensive rats. Eur J Pharmacol. 1991;200:227-231. [Medline] [Order article via Infotrieve]
7. Rapoport RM, Schwartz K, Murad F. Effects of Na+,K+ pump inhibitors and membrane depolarizing agents on acetylcholine-induced endothelium-dependent relaxation and cyclic GMP accumulation in rat aorta. Eur J Pharmacol. 1985;110:203-209. [Medline] [Order article via Infotrieve]
8.
Alonso MJ, Salaices M, Sánchez-Ferrer CF,
Marín J. Predominant role for nitric oxide in the
relaxations induced by acetylcholine in cat cerebral arteries.
J Pharmacol Exp Ther. 1992;261:12-20.
9. Alonso MJ, Salaices M, Sánchez-Ferrer CF, Ponte A, López-Rico M, Marín J. Nitric oxide related and non related mechanisms in the acetylcholine-evoked relaxations in cat femoral arteries. J Vasc Res. 1993;30:339-347. [Medline] [Order article via Infotrieve]
10.
Boulanger C, Hendrickson H, Lorenz RR, Vanhoutte PM.
Release of different relaxing factors by cultured endothelial
cells. Circ Res. 1989;64:1070-1078.
11. Gupta S, Sussman I, McArthur CS, Tornheim K, Cohen RA, Ruderman NB. Endothelium-dependent inhibition of Na+-K+ ATPase activity in rabbit aorta by hyperglycemia: possible role of endothelium-derived nitric oxide. J Clin Invest. 1992;90:727-732.
12. Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol. 1988;93:515-524. [Medline] [Order article via Infotrieve]
13. Arvola P, Pörsti I, Vuorinen P, Pekki A, Vapaatalo H. Contractions induced by potassium-free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats. Br J Pharmacol. 1992;106:157-165. [Medline] [Order article via Infotrieve]
14.
Sánchez-Ferrer CF, Fernández-Alfonso MS,
Ponte A, Casado MA, González R, Rodríguez-Mañas L,
Pareja A, Marín J. Endothelial modulation of the
ouabain-induced contraction in human placental vessels.
Circ Res. 1992;71:943-950.
15.
Rodríguez-Mañas L, Pareja A,
Sánchez-Ferrer CF, Casado MA, Salaices M, Marín J.
Endothelial role in ouabain-induced contractions in guinea pig
carotid arteries. Hypertension. 1992;20:674-681.
16.
Overbeck HW, Wallick ET, Shikuma R, Magargal WW.
Hypertensive dog plasma inhibits the
Na+-K+ pump of cultured vascular smooth
muscle. Hypertension. 1988;12:32-38.
17. Ramani P, Bradley NJ, Fletcher CDM. QBEnd/10, a new monoclonal antibody to endothelium: assessment of its diagnostic utility in paraffin sections. Histopathology. 1990;17:237-242. [Medline] [Order article via Infotrieve]
18. Magargal WW, Overbeck HW. Effect of hypertensive rat plasma on ion transport of cultured vascular smooth muscle. Am J Physiol. 1986;251:H984-H990.
19.
Longo N, Griffin LD, Elsas LJ. A simple method
for evaluation of Rb+ transport and
Na+-K+ pump stoichiometry in adherent
cells. Am J Physiol. 1991;260:C1341-C1346.
20. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
21.
Khalil F, Hopp L, Searle BM, Tokushige A, Tamura H,
Kino M, Aviv A. [3H]ouabain binding to cultured
rat vascular smooth muscle cells. Am J Physiol. 1984;246:C551-C557.
22. Dixon M. The determination of enzyme inhibitor constants. Biochem J. 1953;55:170-171. [Medline] [Order article via Infotrieve]
23.
Kelly RA, O'Hara DS, Canessa M, Mitch WE, Smith TW.
Characterization of digitalis-like factors in human
plasma. J Biol Chem. 1985;260:11396-11405.
24. Fleming WW, Westfall DP, De La Lande IS, Jellet LB. Long-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. J Pharmacol Exp Ther. 1972;181:330-345.
25.
Brock TA, Lewis JA, Smith JB. Angiotensin
increases Na+ entry and
Na+/K+ pump activity in cultures of
smooth muscle from rat aorta. Proc Natl Acad Sci
U S A. 1982;79:1438-1442.
26. Peiró C, Sagarra MR, Redondo J, Sánchez-Ferrer CF, Marín J. Vascular smooth muscle proliferation in hypertensive transgenic rats. J Cardiovasc Pharmacol. 1992;20(suppl 12):S128-S131.
27. Sánchez-Ferrer CF, Ponte A, Casado MA, Rodríguez-Mañas L, Pareja A, González R, Fernández-Alfonso MS, Redondo J, Marín J. Endothelial modulation of the vascular sodium pump. J Cardiovasc Pharmacol. 1993;22(suppl 2):S99-S101.
28. Arribas S, Sánchez-Ferrer CF, Peiró C, Ponte A, Salaices M, Marín J. Functional vascular renin-angiotensin system in hypertensive transgenic rats for the mouse renin gene Ren-2. Gen Pharmacol. 1994;25:1163-1170. [Medline] [Order article via Infotrieve]
29. Ikegawa R, Matsumura Y, Tsukahara Y, Takaoka M, Morimoto S. Phosphoramidon, a metalloproteinase inhibitor, suppresses the secretion of endothelin-1 from cultured endothelial cells by inhibiting a big endothelin-1 converting enzyme. Biochem Biophys Res Commun. 1990;171:669-675. [Medline] [Order article via Infotrieve]
30. Orlov SN, Resink TJ, Bernhardt J, Bühler FR. Na+-K+ pump and Na+-K+ co-transport in cultured vascular smooth muscle cells from spontaneously hypertensive and normotensive rats: baseline activity and regulation. J Hypertens. 1992;10:733-740. [Medline] [Order article via Infotrieve]
31.
Tamura H, Hopp L, Kino M, Tokushige A, Searle BM,
Khalil F, Aviv A. Na+,K+
regulation in cultured vascular smooth muscle cells of the
spontaneously hypertensive rats. Am J Physiol. 1986;250:C939-C947.
32. Kuriyama S, Kaguchi Y, Nakamura K, Hashimoto T, Sakai O. Effect of serum on cell membrane Na-K transport of vascular smooth muscle in culture: a comparative study between normotensive and hypertensive rats. Pharmacol Res. 1992;25:155-165. [Medline] [Order article via Infotrieve]
33.
Allen JC, Navran SS, Seidel CL, Dennison DK, Amann JM,
Jemelka SK. Intracellular Na+ regulation of
Na+ pump sites in cultured vascular smooth muscle
cells. Am J Physiol. 1989;256:C786-C792.
34.
Feltes TF, Seidel CL, Dennison DK, Amick S, Allen JC.
Relationship between functional Na+ pumps and
mitogenesis in cultured coronary artery smooth muscle cells.
Am J Physiol. 1993;264:C169-C178.
35. Scott-Burden T, Resink TJ, Bühler FR. Enhanced growth and growth factor responsiveness of vascular smooth muscle cells from hypertensive rats. J Cardiovasc Pharmacol. 1989;14:S16-S21.
36.
Gupta S, Rudermann NB, Cragoe EJ, Sussman I.
Endothelin stimulates Na+-K+-ATPase
activity by a protein kinase-C pathway in rabbit aorta.
Am J Physiol. 1991;261:H38-H45.
37.
Berk BC, Aronow MS, Brock TA, Cragoe E Jr, Gimbrone MA,
Alexander RW. Angiotensin-II stimulated
Na+/H+ exchange in cultured vascular
smooth muscle cells. J Biol Chem. 1987;262:5057-5064.
38. Murakawa K, Kohno M, Yasunari K, Yokokawa K, Hori T, Takeda T. Possible involvement of protein kinase C in the maintenance of hypertension in spontaneously hypertensive rats. J Hypertens. 1988;6(suppl 4):S157-S159.
39. Berk BC, Vallega G, Muslin AJ, Gordon HM, Canessa M, Alexander RW. Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange. J Clin Invest. 1989;83:822-829.
40. Paquet JL, Baudoin-Legros M, Brunelle G, Meyer P. Angiotensin II-induced proliferation of aortic myocytes in spontaneously hypertensive rats. J Hypertens. 1990;8:565-572. [Medline] [Order article via Infotrieve]
41. Griendling KK, Terutaka T, Berk BC, Alexander RW. Angiotensin II stimulation of vascular smooth muscle. J Cardiovasc Pharmacol. 1989;14(suppl 6):S27-S33.
42.
Lockette WE, Webb RC, Bohr DF. Prostaglandins
and potassium relaxation in vascular smooth muscle of the rat: the role
of Na,K-ATPase. Circ Res. 1980;46:714-720.
43.
Fukuda S, Horioka M, Tanaka T, Shimoji K.
Prostaglandin E1-induced vasorelaxation in porcine
coronary arteries. J Pharmacol Exp Ther. 1992;260:1128-1132.
44. Berk BC. Endothelium stimulates Na+,K+-ATPase and Na+-K+-Cl- cotransport activity in co-cultured vascular smooth muscle. Circulation. 1989;80(suppl II):II-481. Abstract.
45.
Hopp L, Khalil F, Tamura H, Kino M, Searle BM,
Tokushige A, Aviv A. Ouabain binding to cultured vascular smooth
muscle cells of the spontaneously hypertensive rat. Am J
Physiol. 1986;250:C948-C954.
46.
Kifor I, Dzau VJ. Endothelial renin-angiotensin
pathway: evidence for intracellular synthesis and secretion of
angiotensin. Circ Res. 1987;60:422-428.
47. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988;332:411-415. [Medline] [Order article via Infotrieve]
48. Dusting GJ, MacDonald PS. Prostacyclin and vascular function: implications for hypertension and atherosclerosis. Pharmacol Ther. 1990;48:323-344. [Medline] [Order article via Infotrieve]
49. Marín J. Mechanisms involved in the increased vascular resistance in hypertension. J Auton Pharmacol. 1993;13:127-176. [Medline] [Order article via Infotrieve]
50. Bobik A, Campbell JH. Vascular derived growth factors: cell biology pathophysiology, and pharmacology. Pharmacol Rev. 1993;45:1-42. [Medline] [Order article via Infotrieve]
51. Berk BC, Brock TA, Webb RC, Taubman MB, Atkinson WJ, Gimbrone MA, Alexander RW. Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aortic contractions. J Clin Invest. 1985;75:1083-1086.
52. Cunningham LD, Brecher P, Cohen RA. Platelet-derived growth factor receptors on macrovascular endothelial cells mediate relaxation via nitric oxide in rat aorta. J Clin Invest. 1992;89:878-882.
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