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(Circulation. 1997;96:1930-1936.)
© 1997 American Heart Association, Inc.
Articles |
Enhanced Endothelin-Mediated Coronary Vasoconstriction and Attenuated Basal Nitric Oxide Activity in Experimental Hypercholesterolemia
From the Division of Cardiovascular Diseases and Internal Medicine, Mayo Foundation, Rochester, Minn.
Correspondence to Amir Lerman, MD, Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, 200 First St SW, Rochester, MN 55905.
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Abstract |
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Background Experimental hypercholesterolemia is associated with coronary vasomotor dysfunction. This study was designed to test the hypothesis that experimental hypercholesterolemia is characterized by altered coronary vasomotor responses to endothelin and inhibition of the endogenous NO pathway.
Methods and Results Endothelin-1 (ET-1) at 5 ng · kg-1 · min-1 or NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthase (NOS), at 50 µg · kg-1 · min-1 was infused into the left anterior descending coronary artery in pigs before and after 10 weeks of cholesterol diet. There was a significant increase in serum cholesterol. At 10 weeks, ET-1 resulted in an accentuated decrease in coronary blood flow (CBF) and coronary artery diameter (CAD) compared with baseline (-88±6% versus -45±9%, P<.05, and -77±14% versus -18±8%, P<.05, respectively) and an increase in coronary vascular resistance (CVR) (242±18% versus 110±17%, P<.05); ET receptor density and binding affinity in epicardial coronary arteries were unchanged. The effect of L-NMMA on CBF, CAD, and CVR was attenuated at 10 weeks (-7±8% versus -48±4%, -2±3% versus -17±5%, and 16±10% versus 125±32%; each P<.05). Immunohistochemistry staining for constitutive NOS revealed a decrease in immunoreactivity in the coronary arteries of hypercholesterolemic pigs.
Conclusions The present study demonstrates an enhanced coronary vasoconstrictive response to pathophysiological doses of endothelin and an attenuated response to the inhibition of endogenous NO activity, suggesting an alteration in coronary vascular reactivity in experimental hypercholesterolemia.
Key Words: endothelin • endothelium-derived factors • circulation • hypercholesterolemia
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Introduction |
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The endothelium functions as an important modulator of coronary vasomotor tone by synthesizing and releasing vasoactive substances. NO has been identified as an EDRF and is a potent vasodilator generated from the oxidation of the guanidino terminal nitrogen of L-arginine1 by eNOS. ET is an endothelium-derived 21-amino-acid peptide that is a potent vasoconstrictor 2 that causes calcium-dependent smooth muscle cell contraction via specific receptor binding.3 The balance between NO and ET vasoreactivity may be a major determinant that regulates coronary vasomotor function in physiological and pathophysiological states.
Alteration in coronary vascular tone has been found to exist in a number of pathophysiological states, including atherosclerosis and hypercholesterolemia.4 5 6 A porcine model of experimental hypercholesterolemia is characterized by abnormal coronary vasomotor function and elevated levels of circulating ET.7 However, the vasoactive properties of ET on the coronary circulation and the role of EDRF/NO in maintaining basal coronary tone in this pathophysiological state are not well defined. The present study was designed to test the hypothesis that, in a porcine model of experimental hypercholesterolemia, there is an enhanced coronary vasoconstrictive response to pathophysiological concentrations of ET and attenuated basal coronary EDRF activity.
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Methods |
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Animal Experiments
After approval by the Institutional Animal Care and Use Committee, experiments were conducted using 14 female juvenile domestic crossbred pigs weighing 25 to 37 kg. Each animal was fasted overnight before the day of the study but allowed ad libitum access to tap water. Animals were initially anesthetized with 30 mg/kg ketamine and 5 mg/kg xylazine IM, and additional anesthesia was given as a titrated infusion of a solution containing ketamine 5 g/L and xylazine 7.5 mg/L IV to maintain a constant level of anesthesia. The external or internal jugular vein was exposed by cutdown and cannulated with a 7F venous sheath. A flow-directed thermodilution catheter was advanced through the venous sheath for the measurement of RAP and CO. The external carotid artery was also exposed by cutdown and cannulated with an 8F arterial sheath. After injection of a bolus of 10 000 U heparin IV (followed by infusion of 1000 U heparin/h IV), an 8F Judkins left coronary guiding catheter was used to engage the left main coronary artery. A Doppler guidewire (FloWire, Cardiometrics Inc) 0.018 in. in diameter within a 2.2F coronary infusion catheter (Ultrafuse, SciMed Life System) was advanced and positioned into the proximal portion of the LAD. The Doppler guidewire was then positioned 3 to 5 mm distal to the tip of the infusion catheter. After instrumentation, the animals were allowed a 20-minute equilibration period to ensure stability, during which they received normal saline at a rate of 1 mL/min through the coronary infusion catheter. MAP and HR were monitored continuously throughout the procedure. Arterial blood samples for lipid profiles and circulating ET levels were obtained.
To verify that this model of experimental hypercholesterolemia would induce endothelial dysfunction, as defined by an abnormal epicardial coronary response to acetylcholine, a pilot group of 4 pigs was studied before and after 10 weeks of 2% cholesterol diet. Acetylcholine (Iolab Pharmaceuticals) at concentrations of 10-6, 10-5, and 10-4 mol/L (to achieve estimated final blood concentrations in the coronary bed of 10-8, 10-7, and 10-6 mol/L, assuming CBF of 80 mL/min) was infused for 3 minutes at each concentration into the LAD as previously described7 at baseline and again after 10 weeks of cholesterol diet by the catheterization procedural methodology described below. The effect of acetylcholine on the coronary vasculature was assessed by quantitative coronary angiography of the epicardial vessel.
For the study groups, after the initial equilibration period, ET
(Peninsula Laboratories) at 5 ng ·
kg-1 · min-1
was infused through the coronary infusion catheter in group 1
(n=5); a dose-response curve demonstrated no significant effect on CBF
at 2.5 ng · kg-1 ·
min-1 and severe coronary
vasoconstriction at 10 ng · kg-1
· min-1.8 In group 2 (n=5),
L-NMMA (Calbiochem) was infused at a rate of 50 µg ·
kg-1 · min-1
through the coronary infusion catheter. L-NMMA had previously
been administered at this dose in dogs, with significant effects on
CBF.8 In group 3 (n=4), ET and L-NMMA were coinfused at
the above doses; this group was designed to study the interaction
between the ET and EDRF pathways. Intracoronary infusions were
performed with an infusion pump (Harvard Apparatus) at a
rate of 1 mL/min. The following measurements were performed at baseline
(P1) and after 70 (P2) and 90 minutes (P3) of drug infusion: HR, MAP,
RAP, CO (average of three measurements), and average peak velocity
(obtained from on-line analysis of Doppler
parameters of intracoronary flow
velocity).9 At each time interval, selective
coronary angiography was also performed for the measurement of
CAD. The angles, skew rotation, and table height were kept constant
during the procedure. CAD within 5 mm distal to the tip of the
Doppler wire was measured by a blinded observer using a
computer-based image analysis system, as previously
described.10 CBF was calculated from the
Doppler-derived time-velocity integral and vessel diameter, as
previously described11 :
CBF=
(APV)(diameter/4)2, where APV is average peak
velocity. CBF, CAD, and CVR at P2 and P3 were also expressed as percent
change relative to baseline (%
CBF, %CAD, and %CVR,
respectively). SVR at each time interval was calculated from the
formula SVR=(MAP-RAP)/CO. CVR was determined from the formula
CVR=(MAP-PCWP)/CBF, where PCWP is pulmonary capillary wedge
pressure. After catheters and sheaths were removed, the incisions were
closed in three layers. The animals were allowed to recover from
anesthesia, and those in groups 1 and 2 were subsequently
placed on a diet of 2% cholesterol, 10% lard by weight
for a total of 10 weeks, after which the above procedure was repeated
in its entirety in both groups; the animals in group 3 were studied at
baseline only. After the 10-week procedure, the animals were killed,
and the coronary arteries were harvested, embedded in paraffin,
thin-sectioned, and mounted on glass slides. A separate group of 5
animals was killed to serve as normal controls for coronary
artery tissue staining and ET receptor analysis.
ET Radioimmunoassay
Plasma ET was determined by the ET-1,2[125 I] assay from
Amersham as previously described by our laboratory.12
Briefly, blood samples were drawn into tubes containing chilled
potassium EDTA and immediately placed on ice until
centrifugation at 4°C. Plasma was separated and
frozen at -20°C until assay. Plasma was acidified with 0.5% TFA. C8
Bond Elut cartridges were washed in 4 mL methanol and 4 mL water. After
the plasma was applied, cartridges were washed with 2 mL normal saline
and 6 mL water. ET was eluted from the cartridges with 2 mL 90%
methanol in 1% TFA, dried, and reconstituted for the
radioimmunoassay.
Constitutive eNOS Immunostaining
Slides consisting of sections of right and left circumflex
coronary arteries (the LAD was not used because it was
instrumented during the acute experiments) embedded in paraffin were
deparaffinized with 95% ethanol. Endogenous peroxidase
activity was blocked by incubation of the slides for 10 minutes in 50%
absolute methanol/hydrogen peroxide. Nonspecific binding sites
were blocked with 5% goat serum/PBS/Tween 20 for 10 minutes.
Monoclonal antibody to eNOS was added to each slide (400 µL of 5
µg/mL dilution), incubated for 1 hour, and subsequently
incubated with biotinylated goat anti-mouse IgG (1/200 dilution) for 30
minutes and rinsed. The slides were subsequently incubated for 30
minutes with peroxidase-labeled streptavidin (1/500 dilution) and
rinsed. The slides were then stained for 15 minutes in
3-amino-9-ethylcarbazole solution, rinsed, and then counterstained for
1 minute in mercury-free hematoxylin. The slides were then rinsed for 5
minutes in running tap water and mounted in aqueous glycerol gelatin
media.
Immunohistochemical Staining Analysis
Five slides of hypercholesterolemic pigs and 5
slides of normal pigs were prepared as above and reviewed by five
independent observers in a blinded fashion. A scoring system to
quantify endothelial cell staining for eNOS was devised
as follows: 0, no staining; 1, positive staining in <25% of
endothelial cells per slide; 2, positive staining in
25% but <75% of endothelial cells per slide; and
3, positive staining in 75% of endothelial cells per
slide. The scores for each of the observers were averaged for each
slide, and the average scores for hypercholesterolemic
pigs versus normal pigs were calculated.
Membrane Preparation
The right and left circumflex coronary arteries were
dissected free of the myocardium. Arteries were immediately
frozen in liquid nitrogen and stored at -76°C. Preliminary studies
have shown that all three coronary arteries yielded similar
binding profiles; therefore, membranes were combined before
preparation. Frozen arteries were weighed, subsequently pulverized in
liquid nitrogen, and twice homogenized in 10 vol ice-cold
membrane buffer (in mmol/L: sucrose 25, MgCl2
3, EDTA 1, phenylmethylsulfonyl 0.5, and Tris-HCl 50; pH 7.4) for 10
seconds in a Tekmar tissue homogenizer. The
homogenate was centrifuged at 4000 rpm for 15
minutes at 4°C. The supernatant was filtered through a 212-µm nylon
screen and set aside on ice. The pellet was resuspended in 7 vol
membrane buffer and homogenized for 10 seconds. The
homogenate was centrifuged at 4000 rpm for 15
minutes at 4°C, and the resulting supernatant was filtered through a
212-µm nylon screen and added to the initial supernatant. The pellet
was discarded. The combined supernatant was centrifuged at
18 500 rpm for 30 minutes at 4°C and the supernatant discarded. The
pellet was resuspended in 1 vol plus 75 µL of membrane buffer
utilizing a sonic dismembranator (Fisher, model 300) for 
15 seconds.
A 50-µL aliquot was immediately used for determining protein
concentration with BCA protein assay reagent (Pierce) with BSA as
standard. The remaining membrane suspension was stored at -76°C
before dilution for use in binding experiments.
Receptor Binding Assay
Experiments were carried out in a final volume of 200 µL with
binding buffer of the following composition: BSA 0.4%, bacitracin
0.1%, MnCl2 25 mmol/L, MgCl2
3 mmol/L, EDTA 1 mmol/L, phenylmethylsulfonyl
0.5 mmol/L, and Tris-HCl 50 mmol/L; pH 7.4.
Bound and free ligands were separated with a Skatron cell harvester
(Skatron Instruments Inc). With this device, the contents of the
reaction chamber are rapidly rinsed (50 mmol/L Tris-HCl
buffer, pH 7.4) and immediately vacuum-filtered through a 1-µm
retention glass filter mat. Radioactivity retained on the filters was
counted on a gamma counter (Beckman Gamma 4000). The specific activity
of the label (125I-ET-1) was 2000 Ci/nmol and was diluted
such that 50 µL yielded 20 000 cpm (23 pmol/L final
concentration). An incubation time of 3 hours at 25°C was determined
in preliminary studies to be a time of stable equilibrium of
125I-ET-1 binding (data not shown). On the basis of
preliminary studies, 2 µg of membrane protein was selected to be the
appropriate amount for binding studies, because this concentration was
on the linear portion of the membrane concentration versus the
125I-ET-1 binding curve for membranes from all three
coronary arteries.
Competition binding experiments were performed in incubations containing membrane (2 µg) and 125I-ET-1 plus increasing concentrations of unlabeled ET-3. Nonspecific binding was determined from nonlinear curve fitting but was originally estimated in parallel incubations containing the same amount of buffer without competitor plus 1 µmol/L unlabeled ET-1.
Statistics
Data are expressed as mean±SEM. Comparison of data within
groups was performed by repeated-measures ANOVA and Student's paired
t test; comparison between groups was performed by an
unpaired t test. Competition binding data were
analyzed by use of mean values for each group with the
nonlinear curve-fitting program Ligand.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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After 10 weeks of cholesterol feeding, acetylcholine infusion at the maximum dose of 10-4 mol/L resulted in an epicardial vasoconstrictive response compared with baseline (-23±12% versus 5±2%, P<.05).
Table 1
summarizes the basal systemic and
coronary hemodynamic parameters,
lipid profiles, and ET-1 levels of the pigs in groups 1 and 2 (n=10) in
the anesthetized, postabsorptive state before and after 10
weeks of cholesterol feeding. There was no change between
the two time periods in HR, CO, SVR, CBF, or CAD. CVR increased
significantly, as did MAP. There was a significant increase in total
cholesterol, LDL cholesterol, and HDL
cholesterol after 10 weeks of cholesterol
feeding. Circulating ET levels increased significantly, from 6.1±0.8
to 12.5±1.1 pg/mL (P<.05).
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Table 2 summarizes the systemic
hemodynamic data in response to intracoronary
administration of ET-1 and L-NMMA before and after 10 weeks of
cholesterol feeding. Before cholesterol
feeding, intracoronary infusion of ET-1 did not have a
significant effect on HR, systemic MAP, CO, or SVR (P3 versus P1).
After 10 weeks of 2% cholesterol diet,
intracoronary ET infusion caused a significant decrease in CO
(P3 versus P1) and a significant increase in SVR (P2 versus P1; P3
versus P1). Before cholesterol feeding,
intracoronary infusion of L-NMMA resulted in a decrease in HR
along with an increase in MAP and SVR (P3 versus P1). After 10 weeks of
2% cholesterol diet, there was no significant effect of
L-NMMA on CO, HR, or MAP; SVR increased in response to L-NMMA (P2
versus P1; P3 versus P1), although the increase was somewhat attenuated
compared with the increase before cholesterol feeding.
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Fig 1 illustrates the percent change in
CBF, CAD, and CVR between P3 and P1 for Groups 1 and 2 before and after
10 weeks of experimental hypercholesterolemia.
At baseline, before cholesterol feeding, administration of
pathophysiological doses of ET resulted in a
significant percent decrease in CBF (-45±9%, P<.05) and
CAD (-18±8%, P<.05) and a significant increase in CVR
(110±17%, P<.05). After 10 weeks of 2%
cholesterol diet, intracoronary administration of
ET resulted in a significant percent decrease in CBF and CAD at P3
compared with values before cholesterol feeding (-88±6%
versus -45±9%, P<.05, and -77±14% versus -18±8%,
P<.05, respectively) and enhanced the increase in CVR
(242±18% versus 110±17%, P<.05). There was no
significant change in circulating ET concentrations after the
administration of intracoronary ET-1.
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Before cholesterol feeding, intracoronary L-NMMA resulted in a significant decrease in CBF (-48±4%, P<.05) and CAD (-17±5%, P<.05) and a significant increase in CVR (125±32%, P<.05). After 10 weeks of 2% cholesterol diet, the administration of intracoronary L-NMMA had a significantly smaller effect on CBF, CAD, and CVR; compared with baseline, the decreases in CBF and CAD were significantly attenuated (-7±8% versus -48±4%, P<.05, and -2±3% versus -17±5%, P<.05, respectively), as was the increase in CVR (16±10% versus 125±32%, P<.05).
In group 3, the decreases in CBF (-61±10%) (Fig 2), CAD (-38±11%), and CVR (206±50%)
were intermediate between those values observed in group 1 at baseline
and 10 weeks.
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Competition binding assays demonstrated that ET-3 binds to both a
high-affinity binding site, representing the ET-B receptor,
and a low-affinity binding site, representing the ET-A
receptor. The inhibition constant (Ki) as well
as the quantity (Bmax) of both the high-affinity and the
low-affinity binding sites did not change significantly in response to
experimentally induced hypercholesterolemia,
signifying that neither the binding affinity nor the receptor density
of either ET receptor subtype is affected in this model (Table 3).
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Hematoxylin-eosin staining of hypercholesterolemic
animals revealed intact endothelial structure, with
occasional areas of intimal thickening. Staining for constitutive eNOS
revealed an overall decrease in the presence of enzyme immunoreactivity
after 10 weeks of high-cholesterol diet in comparison with
normal animals; this was particularly apparent in areas of abnormal
subendothelial architecture (Fig 3). According to the scoring system
assigned for constitutive eNOS staining,
hypercholesterolemic pigs had a score of 0.52±0.3,
whereas normal pigs had a score of 2.48±0.3 (P=.002).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study demonstrates that experimental hypercholesterolemia is characterized by an enhanced coronary vasoconstrictive response to pathophysiological doses of intracoronary ET-1 without a change in ET receptor density or binding affinity, as well as an attenuated response to the inhibition of endogenous coronary EDRF activity associated with decreased coronary eNOS immunoreactivity. In addition, it confirms that this experimental model induces endothelial dysfunction, as demonstrated by epicardial coronary arterial response to acetylcholine.
The finding of elevated circulating ET levels in association with hypercholesterolemia in this study is in accord with a previous investigation.7 The production and activity of ET are enhanced by known atherogenic risk factors13 ; in addition, ET plays an important role in the atherogenic process, with effects on vasoreactivity and cellular proliferation.13 14 ET production and secretion are enhanced in the presence of elevated serum lipids7 15 16 and oxidized LDL.17 18 Circulating and tissue ET immunoreactivity are also enhanced in advanced atherosclerosis in humans and correlate with the severity of the disease.19
Previous in vitro studies suggest that hypercholesterolemia in the absence of atherosclerotic lesions increases vascular reactivity to ET.20 Intracoronary administration of exogenous ET in this study resulted in a significant decrease in CBF and an increase in CVR in hypercholesterolemic pigs compared with normal pigs. The mechanism for the enhanced coronary arterial sensitivity to ET in hypercholesterolemia may be multifactorial and has been postulated to be due in part to enhanced ET effect via upregulation of ET receptors, altered postreceptor mechanisms, and/or impaired EDRF activity. Although it has been demonstrated that in experimental hypercholesterolemia in marmosets21 and in atherosclerotic human coronary arteries,22 23 the ET-B receptor subtype is upregulated, this has not been demonstrated in the present porcine model. Competition binding assays using 125I-ET-1 and ET-3 in the present study did not demonstrate a change in ET receptor binding affinity or density in this model for either the ET-A or ET-B subtype. It is also conceivable that in experimental hypercholesterolemia, there may be a supersensitization of postreceptor mechanisms leading to enhanced vasoconstriction to pathophysiological doses of ET; postreceptor changes, specifically postreceptor desensitization, has been proposed as a mechanism to explain the attenuated coronary vasoconstrictive response to ET in an experimental model of heart failure.24 The inter- action between the ET and EDRF pathways should also be considered as a mechanism to explain the enhanced ET sensitivity observed in this study; for example, it has previously been demonstrated that the inhibition of endogenous NO generation by administration of L-NMMA enhances the coronary vasoconstriction caused by pathophysiological concentrations of ET.13 In addition, it has been demonstrated that chronic inhibition of NO bioavailability leads to elevated plasma immunoreactive ET concentrations25 ; insofar as the present model of experimental hypercholesterolemia appears to be associated with decreased NO bioavailability as well, such an interaction may also be operational here. The observation that the coronary hemodynamic response to the concomitant administration of ET-1 and L-NMMA (group 3) was intermediate between that observed for ET-1 alone at baseline and ET-1 alone at 10 weeks (group 1) supports the notion that the enhanced vasoconstrictive effect of ET-1 in this model of experimental hypercholesterolemia is in part a result of attenuated EDRF activity. The reciprocal regulation of ET-1 and constitutive eNOS in in vitro studies of proliferating endothelial cells has also been demonstrated,26 which lends further support to the notion that pathophysiological states may be characterized by an imbalance between these two systems. It appears that EDRF inhibition results in a hypersensitivity to ET in the coronary circulation in this model of experimental hypercholesterolemia, although enhanced ET postreceptor activity and additional vasoconstrictors may also be operational in these observations. For example, patients with endothelial dysfunction, defined as an abnormal response to acetylcholine, have been demonstrated to have enhanced sensitivity to intracoronary administration of catecholamines.27 Furthermore, in spontaneously hypertensive rats, angiotensin II has been demonstrated to cause endothelial production of ET in situ, and ET in turn potentiated contractions induced by norepinephrine, suggesting that ET may act to amplify the pressor effects of the renin-angiotensin system.28 Therefore, a more complex interaction between ET and other circulating vasoconstrictors contributing to our findings cannot be ruled out.
The intracoronary administration of L-NMMA in hypercholesterolemia resulted in a significant attenuation of the coronary vasomotor effect observed in the normal animals. L-NMMA mediates vasoconstriction in vitro and has been shown to produce a hypertensive response in animals and cause forearm arterial vasoconstriction in humans.29 It has no intrinsic constrictor effect on vascular smooth muscle; rather, its effects are entirely endothelium dependent, and its vasoconstrictor properties result from an inhibition of an endogenous endothelium-dependent vasodilator mechanism.29 Net NO-dependent vasodilator tone is maintained in the basal physiological state, most likely through the physical activation of endothelial cells by factors such as shear stress and pulsatile flow.29
Possible mechanisms for the attenuated effect of L-NMMA on the coronary circulation in hypercholesterolemia include a decreased basal release and/or enhanced breakdown of EDRF, substrate (L-arginine) deficiency, and the direct effects of hypercholesterolemia. The present study is supported by previous in vitro studies that have shown that the vasoconstrictor effect of L-NMMA is attenuated in atherosclerotic arteries.30 Moreover, this study is in accord with recent in vivo studies that demonstrate that EDRF contributes significantly to basal coronary tone in normal patients but less so in patients with coronary risk factors.31 Furthermore, it has been suggested that basal and stimulated EDRF synthesis may be differentially regulated.32 The acute administration of L-arginine has been shown to improve forearm blood flow33 as well as CBF34 in hypercholesterolemic subjects in response to the endothelium-dependent vasodilator acetylcholine; in addition, acute and chronic administration of L-arginine improves vasomotor function in animal models of hypercholesterolemia,35 36 suggesting an absolute L-arginine deficiency or, alternatively, a relative substrate deficiency resulting in a functional decrease in eNOS activity. Stainable eNOS in hypercholesterolemic porcine coronary arteries was decreased in this study, which supports the concept that decreased EDRF/NO bioavailability as a result of a deficiency of eNOS may in part explain the basal reduction of EDRF-dependent coronary tone in experimental hypercholesterolemia. The direct effects of hypercholesterolemia on the EDRF/NO pathway also need to be considered. Lipoproteins can specifically interfere with the L-arginine–NO pathway37 ; a direct inactivation of EDRF by native LDL has been postulated, without cellular impairment of EDRF synthesis.38 NO, which is approximately eight times more soluble in hydrophobic than hydrophilic media, could dissolve in the hydrophobic core of the LDL molecule and therefore be unavailable to exert its target effects on smooth muscle cells. In addition, oxidized lipoproteins have been shown in cell culture to inhibit the production of NO by eNOS.39 Furthermore, ET downregulates the expression of inducible NOS in cultured rat glomerular mesangial cells,40 further supporting the interaction between the ET and EDRF pathways in hypercholesterolemia.
In the present study, there appeared to be a net increase in CVR in the basal state in hypercholesterolemic animals compared with CVR before cholesterol feeding. This may result from attenuated coronary EDRF activity and/or enhanced circulating ET concentrations, along with the effects of other circulating vasoconstrictor substances.
The enhanced response to ET and attenuated EDRF activity observed in experimental hypercholesterolemia may have clinical implications. Abnormalities in vasomotor regulation have been demonstrated in pathophysiological states such as atherosclerosis and heart failure. It has been proposed that such abnormalities in vasomotor function precede overt manifestations of these pathophysiological states; for example, we have recently demonstrated in human subjects that endothelial dysfunction in the absence of angiographically significant coronary disease is characterized by increased circulating levels of ET and decreased coronary guanosine 3',5'-cGMP, the second messenger of NO.12 Therefore, the relative imbalance of these vasomotor pathways may conceivably cause myocardial ischemia in the absence of significant epicardial coronary artery stenoses, a so-called state of early atherosclerosis. It is interesting to postulate that therapeutic interventions such as ET receptor blockers or supplementation with NO donors at an early stage may slow or even prevent the progression to overt disease.
In summary, the present study demonstrates that there is an enhanced vasoconstrictive effect on the coronary circulation in response to exogenously administered intracoronary ET and a reduction in the role of EDRF-dependent vasodilator tone in maintaining basal CBF in experimental hypercholesterolemia that is associated with a decrease in eNOS immunoreactivity without significant changes in ET receptor density or binding affinity. These data support a role for the balance between ET and EDRF in the regulation of coronary vascular tone.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the National Institutes of Health (HL-03180-01), the Mayo Foundation, and the Miami Heart Research Institute.
Received January 2, 1997; revision received February 26, 1997; accepted April 2, 1997.
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