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Departments of 1 Surgery and 2 Physiology, Faculty of Medicine, Université de Montréal, Montréal H3C 3J7, and Institut de Cardiologie de Montréal, Montréal, Québec, Canada H1T 1C8
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Nitric oxide (NO) may normally impair endothelin
(ET) activity in epicardial coronary arteries. Lifting this inhibitory
feedback could reveal ET-dependent effects involving
ETA- and/or
ETB-receptor activation. In
conscious dogs, the blockade of
ETA receptors (intracoronary Ro-61-1790) increased external circumflex coronary artery diameter (CD) (sonomicrometry) by 0.10 ± 0.01 from 3.04 ± 0.12 mm
(P < 0.01) without altering coronary
blood flow (Doppler). Similarly, CD increased (0.09 ± 0.01 from
2.91 ± 0.14 mm; P < 0.01) when Ro-61-1790 was given after blockade of NO formation with
intracoronary N
-nitro-L-arginine
methyl ester (L-NAME). In
contrast, ETB-receptor blockade
(intracoronary Ro-46-8443) did not influence baseline CD with and
without L-NAME. In vitro,
increases in tension caused by
N-nitro-L-arginine
(L-NNA) or
PGF2
in arterial rings were reduced by ETA- but not
ETB-receptor blockade.
ETA-receptor blockade also reduced
the increase in tension caused by
L-NNA in human coronary arterial
rings. Thus ETA receptors, but not
ETB receptors, account for
ET-dependent constriction in canine epicardial coronary arteries in
vivo. ET-dependent effects were independent of the level of NO
formation in vitro and in vivo. In human epicardial coronary arterial
rings, ETA-receptor blockade also
caused significant relaxation.
endothelin; endothelium-derived factors
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THERE IS GROWING EVIDENCE suggesting that nitric oxide (NO) may normally impair endothelin (ET) production and action. In fact, pressor responses elicited by the blockade of NO formation in vivo are reversed by ET-receptor blockade and by ETA receptors in particular (1, 11, 12, 14, 22, 27). By lifting an inhibitory effect of NO on ET activity, ET-dependent constriction could become apparent after the blockade of NO formation. Consistent with these observations, thrombin-induced endothelium-dependent ET formation by the isolated porcine aorta is augmented after the blockade of NO formation or cGMP with methylene blue, whereas ET release was reduced by 8-bromo-cGMP, nitroglycerin, and 3-morpholinosydnonimine (2, 3, 18). ET itself may trigger NO formation through activation of ETB receptors, thereby limiting its vasoconstrictor effects (7, 10, 14, 23, 29). Removal of this negative feedback by the blockade of NO formation could magnify ET effects.
Our primary objective was to examine whether ET has significant
influence on the baseline caliber of large epicardial canine coronary
arteries in vivo through ETA
and/or ETB receptors. We also
examined the extent to which the basal level of NO formation influenced
the magnitude of ET effects in vivo, as well as in vitro, in coronary
arteries precontracted with
PGF2 or
N-nitro-L-arginine
(L-NNA) to suppress NO formation.
To rule out the possibility that ET activity in our conscious dogs may have been altered as a consequence of the instrumentation procedure, ET content of instrumented and noninstrumented large epicardial coronary arteries from the same animals was compared. Our in vitro studies also enabled us to exclude flow as a determinant of ET activity in vivo.
Finally, we examined if human coronary arteries from explanted atherosclerotic and idiopathic-cardiomyopathic hearts studied in vitro display significant relaxation to ETA-receptor blockade.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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All animal experimental procedures were approved by an ethical committee on animal care and performed in accordance with "Guide to the Care and Use of Experimental Animals" (Canadian Council on Animal Care publication No. [ISBN] 0-919087-18-3, Ottawa, 1993). Experiments involving human coronary vessels were reviewed and approved by an ethical committee on clinical research.
Instrumentation
Under general anesthesia with pentobarbital sodium (30 mg/kg iv), artificial ventilation, and sterile conditions, 21 mongrel dogs (29 ± 1 kg) underwent a left thoracotomy at the fifth intercostal space and were instrumented as previously described (19). Mean arterial pressure (MAP), left ventricular pressure (LVP) and its first derivative LV dP/dt, coronary blood flow (CBF), external circumflex coronary artery diameter (CD), and heart rate (HR) were recorded and measured as previously described (19). Pacing wires were sutured to the right ventricular (RV) outflow tract and connected to an external stimulator (model 5320, Medtronic, Minneapolis, MN). A Silastic (Dow Corning, Midland, MI) catheter for drug delivery was implanted in the proximal circumflex coronary artery. Analgesia was provided postoperatively with buprenorphine (0.3 mg im) (Temgesic, Reckitt and Colman Pharmaceuticals, Hull, UK). Prophylactic penicillin G procaine (300,000 U im) and penicillin G benzathine (300,000 U im) were administered for 10 days after the surgery.Protocols
In vivo.
Experiments were initiated 2-4 wk after surgery in conscious
healthy dogs laying quietly on their right side. Animals were pretreated with indomethacin (5.0 mg/kg iv) 30 min before the experiments. After a stable baseline was reached, RV pacing
(120-132 beats/min) was performed for 5-7 min until a steady
state was achieved.
N-nitro-L-arginine methyl ester
(L-NAME; 50.0 µg · kg
1 · min1
ic for 12 min) was then administered. This dose of
L-NAME blunts flow-dependent NO
formation in large epicardial arteries (19) and prevents CBF responses
to intracoronary infusions of ACh (19, 21). When a steady state was
reached after administration of L-NAME (15-20 min), RV
pacing was performed.
1 · min1
for 10 min followed by 0.25 µg · kg1 · min1
thereafter. In preliminary studies
(n = 5), this dose of Ro-61-1790 was adequate to reduce (P < 0.05)
the fall in CBF caused by intracoronary ET-1 (0.1 µg, American
Peptide, Sunnyvale, CA) from 21 ± 2 to 7 ± 2%. Baseline
measurements were made at steady state with and without RV pacing. At
least 48 h later and in the same animals, Ro-61-1790 was given
alone after indomethacin.
In separate experiments, the effects of selective
ETB-receptor blockade with
(R)-4-tert-butyl-N-[6-(2,3-dihydroxy-propyloxy)-5-(2-methoxy-phenoxy)-2-(4-methoxyphenyl)-pyrimidin-4-yl]-benzenesulfonamide (Ro-46-8443) (4) (HoffmannLa Roche) or combined
ETA-/ETB-receptor blockade with
4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2'-bipyrimidin-4-yl]-benzenesulfonamide sodium salt (bosentan) (8) were investigated. Ro-46-8443 or bosentan were infused at a dose of 30.0 µg · kg1 · min1
ic for 10 min followed by a continuous infusion of 1.0 µg · kg1 · min1
thereafter. This dose of Ro-46-8443 was adequate to prevent
(P < 0.05, n = 5) the early rise (63 ± 17 to 2 ± 2%) and the late decrease (24 ± 3 to 6 ± 2%) in CBF caused by intracoronary sarafotoxin 6c (0.3 µg, American
Peptide), an ETB-receptor agonist
(26). The dose of bosentan was adequate to impair
(P < 0.01, n = 6) the fall in CBF (23 ± 2 to 4 ± 1%) caused by intracoronary ET-1 (0.1 µg). The
effects of bosentan or Ro-46-8443 were examined with and without
prior L-NAME administration.
In six additional dogs not treated with indomethacin, the effects of
ETB-receptor blockade with
Ro-46-8443 were examined with and without
L-NAME treatment.
In vitro.
A segment of canine proximal left anterior descending coronary artery
was carefully dissected and immediately immersed in ice-cold
physiological salt solution (PSS) containing indomethacin (105 mol/l, inhibitor of
cyclooxygenase) and of the following composition (103 mol/l): 130 NaCl, 4.7 KCl, 1.6 CaCl2, 1.18 KH2PO4,
1.17 MgSO4, 14.9 NaHCO3, 0.026 EDTA, 10 glucose,
aerated with 12% O2-5%
CO2-83% N2 (pH 7.4). Arterial rings 2 mm
long were mounted on 20-µm tungsten wires and connected to isometric
myographs (IMF, University of Vermont, VT). After a 1-h
stabilization period, arterial rings were repeatedly stretched and
challenged at each step with 40 × 103 mol/l KCl PSS until a
stable contractile response was obtained. Maximal tension
(Emax) averaged 4.6 ± 0.2 g (n = 87 rings) and was
obtained in each ring with 127 × 103 mol/l KCl PSS applied
at the end of the experiment. ACh
(106 mol/l) in arteries
precontracted with PGF2 (3 × 106 mol/l, 21 ± 6% of Emax) caused 87 ± 7% relaxation. In 12 vessels from 6 dogs, the endothelium was
removed mechanically by gentle rubbing of the intimal surface with a
wooden peg. Adequacy of endothelium removal was demonstrated by the
lack of relaxation to ACh
(106 mol/l) in arteries
precontracted with PGF2 (3 × 106 mol/l). In
these vessels, Emax
averaged 5.0 ± 0.2 g. K+-rich
solutions were prepared by replacing NaCl with equimolar amounts
of KCl.
4 mol/l) was applied,
and time was allowed for tension to reach a steady state (45 min).
Other rings were pretreated with either bosentan
(105 mol/l),
Ro-61-1790 (106
mol/l),
c(D-Trp-D-Asp-Pro-D-Val-Leu)
(BQ-123) (106 mol/l,
American Peptide), a selective
ETA-receptor blocker (15), or
Ro-46-8443 (107 mol/l)
20 min before L-NNA application.
The effects of L-NNA alone in
rings denuded of their endothelium were examined in parallel experiments. In additional experiments, Ro-61-1790 was applied after L-NNA treatment or after
precontraction with PGF2
(106 mol/l).
Adequacy and selectivity of ET-receptor blockade was verified in
separate experiments. Dose-response curves to ET-1 were examined before
and after Ro-61-1790
(106 and
107 mol/l), Ro-46-8443
(106 mol/l), or bosentan
(106 mol/l). The effects of
sarafotoxin 6c were examined before and after Ro-46-8443
(107 mol/l) or
Ro-61-1790 (106
mol/l). In vessels precontracted with
PGF2 (3 × 106 mol/l), the effects of
sarafotoxin 6c were examined before and after Ro-46-8443
(107 mol/l). The effects of
sarafotoxin 6c were also examined without indomethacin pretreatment.
Human epicardial coronary arteries were obtained from explanted hearts.
Because calcified lesions were present in proximal coronary arteries,
second- or third-order branches (300-500 µm) of the right
coronary artery free of macroscopic lesions were obtained from
atherosclerotic hearts (n = 4).
Vessels of the same caliber were obtained from explanted idiopathic
cardiomyopathic hearts (n = 4). First,
in vessels precontracted with angiotensin II
(107 mol/l), the effects of
ACh (106 mol/l) were
examined. Then, the effects of
L-NNA
(104 mol/l) were examined
with and without prior treatment with BQ-123 (106 mol/l). This dose of
BQ-123 has been reported to block ET-1-induced contractions in human
coronary arteries (9). The effects of L-NNA were also examined in
vessels without endothelium. Adequacy of endothelial denudation was
confirmed by the suppression of substance P
(107 mol/l)-induced
relaxation in vessels precontracted with angiotensin II
(107 mol/l).
Immunoreactive (ir) ET Measurements.
Pairs of canine arterial segments of ~1.0 cm long of both the circumflex and left anterior coronary vessels were obtained to measure ET content. In one sample from each vessel endothelium removal was performed. After the vessels were cleaned of all tissue adherent to the adventitia, normal and denuded vessels were stored at
70°C
until further processing.
Tissue (ir)-ET levels were measured with a RIA after extraction and purification of ET, according to the method described earlier (28). The RIA procedure was carried out according to the procedure described by the supplier of the ET-1 antibody (Peninsula, Belmont, CA). Total protein content was measured before extraction using the fluorescamin assay (28). Data are reported as ir-ET in picograms per milligram of protein. The cross-reactivity of ET-2, ET-3, and proendothelin in this assay was <7%, <7%, and <17%, respectively.
Except when specifically mentioned, drugs were obtained from Sigma Chemical (St. Louis, MO).
Data Analysis
Data are reported as means ± SE. Data were read directly from the strip charts under steady-state conditions with and without RV pacing. Simultaneous comparisons of baselines with or without RV pacing were made with one-way ANOVA for repeated measurements followed by a Bonferroni post hoc test to isolate specific contrasts (13). When appropriate, paired comparisons were made with t-tests.For all experiments conducted in vitro,
n refers to the number of dogs or
human donors. Contractions are expressed as percentage of
Emax achieved in the
presence of 127 × 103
mol/l KCl PSS at the end of each individual experiment. In vessels precontracted with PGF2 or
angiotensin II, relaxations are reported as percent inhibition of
contraction induced by the agonist. EC50 of agonists were measured on
each dose-response curve using a logistic curve-fitting approach.
pD2 values are the negative log of
EC50. Student's
t-tests (unpaired) were used to
compare pD2 values and ET-1 tissue levels.
Statistical significance was reached when P < 0.05 in all cases.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In Vivo
Blockade of ETA receptors.
Except for an increase in CD and a slight decrease
(P < 0.05) in MAP (97 ± 4 to 90 ± 5 mmHg), Ro-61-1790 had no other significant hemodynamic
effects, as reported in Fig. 1. With RV
pacing, a similar pattern of responses to Ro-61-1790 was
displayed, as reported in Fig. 1.
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Blockade of ETB receptors.
Ro-46-8443 alone failed to influence baseline CD. CBF and HR fell
(P < 0.01) whereas LVP and MAP
increased (P < 0.05) after Ro-46-8443, as reported in Fig. 3 and
Table 2. Under RV pacing, hemodynamic
responses elicited by Ro-46-8443 displayed a similar pattern, as
reported in Fig. 3 and Table 2. Ro-46-8443 given after
L-NAME with and without RV
pacing failed to alter CD and did not cause further hemodynamic
effects, as reported in Fig. 3. In animals not treated with
indomethacin, Ro-46-8443 failed to alter baseline CD before or
after L-NAME. Thus, with and
without normal NO formation, blockade of
ETB receptors had no significant influence on baseline CD.
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Combined blockade of ETA and ETB receptors. Except for an increase (P < 0.01) in CD (0.08 ± 0.02 from 2.98 ± 0.10 mm), bosentan alone had no other hemodynamic effects. Bosentan given after L-NAME also increased (P < 0.01) CD (0.10 ± 0.01 from 2.77 ± 0.08 mm) without other hemodynamic effects. Changes in CD elicited by bosentan did not statistically differ with and without L-NAME. Thus, with and without normal NO formation, combined ETA-/ETB-receptor blockade selectively increased CD.
In Vitro
Canine coronary arteries.
In rings stretched to their optimal baseline tension, none of the ET
blockers had significant effects on resting tension. L-NNA increased tension, as
reported in Fig. 4.
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6 mol/l)-induced
relaxation (87 ± 7%) in vessels precontracted with
PGF2. More importantly, L-NNA-induced contractions were
absent in rings without endothelium, consistent with an
endothelium-dependent effect of
L-NNA.
Pretreatment of the rings with Ro-61-1790
(106 mol/l) reduced
L-NNA-induced contractions
thereafter, as reported in Fig. 4A. In
rings precontracted with L-NNA
(3.3 ± 0.7% Emax,
n = 6) or PGF2 (2.7 ± 0.5%
Emax,
n = 7), Ro-61-1790 also reduced
(P < 0.05) tension to 1.9 ± 0.4 and 1.4 ± 0.3% Emax,
respectively. Ro-61-1790
(107 mol/l) shifted the
dose-response curve to ET-1 (pD2:
8.01 ± 0.13 to 7.14 ± 0.07, P < 0.01, n = 8), and Ro-61-1790
(106 mol/l) abolished
(n = 8) ET-1-induced contractions.
In contrast, pretreatment of the rings with Ro-46-8443 did not
significantly alter the magnitude of
L-NNA-induced contractions, as
reported in Fig. 4B.
In separate experiments, sarafotoxin 6c at the highest concentration
examined (3 × 106
mol/l) caused slight contractions (10 ± 2%
Emax,
n = 6) only after
L-NNA, which were abolished by
Ro-46-8443 (107 mol/l)
and unaltered (9 ± 2%
Emax,
n = 6) by Ro-61-1790
(106 mol/l). ET-1-induced
contractions were maintained after
106 mol/l Ro-46-8443
(pD2: 8.05 ± 0.23 to 7.98 ± 0.05, n = 6). Conversely, in
vessels precontracted with PGF2
(3 × 106 mol/l),
sarafotoxin 6c (109 to 3 × 106 mol/l) had no
significant relaxant effect with and without indomethacin. Thus
ETB-dependent effects were trivial
in those large canine coronary arteries.
Consistent with the data reported with Ro-61-1790, bosentan
(105 mol/l) also reduced
L-NNA-induced contractions, as
reported in Fig. 4C. Bosentan
(105 mol/l) abolished
ET-1-induced contractions. Pretreatment
(n = 5) with BQ-123
(106 mol/l), an
ETA-receptor blocker, reduced
(P < 0.01)
L-NNA-induced tension from 4.6 ± 0.4 to 2.1 ± 0.6%
Emax.
ir-ET tissue levels from noninstrumented left anterior descending and
from instrumented circumflex coronary arteries did not differ, as
reported in Fig. 5. In both vessels removal
of the endothelium led to significant reductions of ir-ET tissue
content.
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Human coronary arteries.
After precontraction with angiotensin II
(107 mol/l), ACh
(106 mol/l) caused
relaxation (78 ± 13%) in rings from idiopathic cardiomyopathic
hearts but constriction (38 ± 4%
Emax) in rings from
atherosclerotic hearts. Without precontraction, BQ-123
(106 mol/l) had no effect
on baseline tension in vessels from atherosclerotic or idiopathic
cardiomyopathic hearts. As expected,
L-NNA
(104 mol/l) increased
tension in both groups, as reported in Fig. 6. This effect was greater
(P < 0.05) in vessels obtained from atherosclerotic than from idiopathic cardiomyopathic hearts. In both
groups, pretreatment with BQ-123
(106 mol/l) significantly
reduced (P < 0.05)
L-NNA-induced tension. In
vessels without endothelium,
L-NNA
(104 mol/l) failed to
increase baseline tension.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present data highlight important features of ET activity in
coronary vessels. Significant ET-dependent constriction involving ETA receptors was demonstrated in
conductance coronary vessels of conscious dogs under baseline
conditions. In contrast,
ETB-receptor blockade had no
influence on baseline caliber of large epicardial coronary arteries. In
fact, in canine vessels, direct
ETB-receptor stimulation with
sarafotoxin 6c resulted in only weak contractions at the highest doses
examined (3 × 106
mol/l) and inconsistent relaxations in precontracted vessels. Suppression of prostacyclin formation with indomethacin did not account
for the failure of ETB-receptor
blockade to alter baseline CD. A significant interaction between NO and
ET under baseline conditions was not apparent because
ETA- or combined
ETA-/ETB-receptor blockade increased the diameter of large epicardial to a similar extent
in the face of normal or suppressed NO formation. Our in vitro data on
canine and human coronary arteries agree with the general conclusion
that ETA-receptor blockade
revealed significant ET-dependent constriction in large epicardial
coronary arteries. These in vitro data also imply that flow was not
primarily involved in controlling ET production under baseline conditions.
Several studies have now reported that pressor responses caused by arginine analogs may not solely reflect the suppression of NO-dependent effects (1, 11, 12, 14, 22, 27). In fact, responses elicited by blockers of NO formation are partially reversed by ET-receptor blockers. Together with the observation that NO/cGMP blunts ET-dependent responses in vitro (2, 3, 18), we were led to hypothesize that a functional interaction between NO and ET played a significant role in vascular regulation. If NO has inhibitory effects on ET formation/action, greater dilator responses to blockade of ET receptors would be expected after L-NAME in vivo. Our data in conscious dogs do not support this hypothesis since selective ETA- or combined ETA-/ETB-receptor blockade led to similar increases in CD when NO formation is normal or impaired.
There is an apparent discrepancy between our in vitro and in vivo data
concerning the effects of ET-receptor blockade on coronary arteries.
The failure of ET-receptor blockade to influence basal tension in
isolated human and canine coronary vessels is most certainly related to
the lack of spontaneous active tone in stretched arterial rings. In
fact, when basal tension was increased with PGF2, significant ET-dependent
effects involving ETA receptors became apparent. Thus the increase in tension rather than the selective
blockade of NO formation explained why significant ET-dependent effects
were displayed after L-NNA.
ETB receptors located on endothelial cells have been reported to stimulate NO and prostacyclin formation (7, 10, 14, 23), whereas those located on smooth muscle cells mediate constriction (26). A blockade of endothelial ETB receptors should lead to constrictor responses displayed only with normal NO formation. In contrast, blockade of smooth muscle ETB receptors should result in dilation of coronary vessels. In our hands, blockade of ETB receptors failed to influence large epicardial coronary artery caliber with normal or impaired NO formation. A functional interaction between ET and prostacyclin was not involved since ETB-receptor blockade failed to influence CD with and without indomethacin pretreatment in our conscious dogs. In the same connection, sarafotoxin 6c had trivial effects in vitro in rings treated or not treated with indomethacin. Thus large epicardial canine coronary arteries may be lacking ETB receptors, consistent with the trivial effects of sarafotoxin 6c in our in vitro experiments. This conclusion is in line with reports showing little effects of selective ETB agonists on large epicardial coronary arteries in anesthetized dogs (6, 23). In contrast, Teerlink et al. (26) showed significant constriction in response to high doses of sarafotoxin 6c. Conceivably the threshold of vascular responses caused by ETB-receptor activation may be higher than for responses triggered through ETA receptors. This may account for a limited contribution of ETB receptors to baseline vascular tone in vivo when endogenous ET production is normal.
We were concerned about the possibility that instrumentation of the vessels influenced our measurements of vascular reactivity to ET blockers in conscious dogs by altering normal ET production. To rule out this possibility, noninstrumented left anterior descending coronary arteries from the same animals were studied in vitro. Consistent with our data in conscious dogs, pretreatment of the rings with a selective ETA- or a combined ETA-/ETB-receptor blocker before L-NNA application led to smaller increases in tension. In contrast, blockade of ETB receptors did not influence L-NNA-induced tension. Comparison of ir-ET levels in the instrumented circumflex and in the noninstrumented left anterior coronary arteries reveals similar ir-ET content. Thus the significant ET-dependent constriction demonstrated in vivo was not causally related to the instrumentation of the vessels. Reporting our data on ir-ET per milligram of protein did not reflect the fact that a major fraction of total ir-ET was localized into the endothelium, which contributes little to total protein content of the vessel wall. Nevertheless, a significant portion of total ir-ET found in coronary arteries was localized outside the endothelium. Given that ET, and ET-1 in particular, has a distribution normally restricted to the endothelium (20), extraendothelial ir-ET may reflect tissue-bound ET rather than locally formed ET in our experiments. It is also conceivable that ir-ET localized into the endothelium of vasa vasorum influenced our measurements.
Our data from human coronary vessels are qualitatively similar to those obtained in canine vessels, i.e., ETA-receptor blockade relaxed vessels precontracted by the blockade of NO formation, an endothelium-dependent phenomenon. Several important differences between canine and human arterial rings should be kept in mind when considering the present data. Human coronary rings were only available from the right coronary artery and the sampling site corresponded to second- and third-order vessels because of atherosclerotic lesions in proximal coronary arteries. Vessels of the same caliber were obtained from idiopathic cardiomyopathic hearts devoid of proximal coronary arterial lesions. Vessels from atherosclerotic and idiopathic cardiomyopathic hearts differed with respect to their responses to an ACh challenge following precontraction with angiotensin II. Relaxation was displayed by vessels from idiopathic cardiomyopathic hearts whereas ACh caused contraction in vessels from atherosclerotic hearts, consistent with an altered endothelial function. In both groups, L-NNA induced significant increases in baseline tension that were magnified in atherosclerotic vessels. An augmented ET activity in atherosclerotic vessels presumably accounts for these responses that were sensitive to the selective blockade of ETA receptors (BQ-123). This conclusion is consistent with the data of Lerman et al. (17), who showed elevated plasma ET levels in patients displaying vasoconstrictor instead of vasodilator responses to intracoronary ACh. In the same connection, it is interesting to note that the severity of atherosclerotic lesions in patients is closely related to their circulating ET levels (16). Our data and these earlier observations suggest that in human coronary arteries, ET-dependent constriction may become more important when endothelial function is altered. When trying to extend our data to normal human coronary vessels it should be kept in mind that "control" vessels were obtained from idiopathic cardiomyopathic hearts, which may substantially differ from normal blood vessels. It is also important to note that although our human and canine vessels displayed substantial vasomotor responses to the various interventions, the observations made under in vitro experimental conditions may not mirror the functional status of in situ human and canine coronary vessels.
There are some specific features of our in vivo experiments that deserve to be emphasized. In particular, the use of intracoronary drug delivery in our conscious dogs allows us to specifically examine coronary responses and to minimize confounding hemodynamic changes expected from systemic drug delivery. In our in vivo experiments, L-NAME was used as a blocker of NO formation that blunts flow-dependent responses, an endothelium- and NO-dependent process (19). In vitro, L-NNA was preferred over L-NAME to exclude potentially nonspecific effects of L-NAME described in vitro (5) but not observed in vivo (25). By continuously monitoring CBF, we were able to exclude the influence of flow-dependent effects on our measurements of CD, in particular when ET-receptor blockade was achieved before blockade of NO formation. To account for changes in HR, hemodynamic measurements were made under spontaneous HR as well as during RV pacing. We cannot exclude the possibility that a decrease in coronary distending pressure resulting from a fall in MAP could have influenced our measurements of CD in vivo. In this situation, the effects of ETA-receptor blockade on CD could only have been underestimated in the present experiments. The small amplitude of MAP decreases caused by Ro-61-1790 and, more importantly, the fact that bosentan caused similar increases in CD but failed to significantly alter MAP suggest that changes in MAP had little influence on CD responses elicited by ET-receptor blockers. Taken together, the above observations indicate that changes in CD caused by ET-receptor blockade were causally related to the suppression of ET-dependent effects in large coronary arteries rather than being secondary to hemodynamic changes. In further support of this contention, our in vivo data were confirmed in vitro.
In conclusion, blockade of ETA receptors revealed significant baseline ET-dependent constriction in large epicardial canine coronary arteries in vivo. In precontracted arterial rings, ETA-receptor blockade caused relaxation. The amplitude of ET-dependent effects was independent of the level of NO formation. In contrast, ETB-receptor blockade had no significant influence on the caliber of large epicardial arteries in vivo. ET content of instrumented and noninstrumented vessels was similar, thereby ruling out the possibility that in vivo ET activity was altered as a consequence of the instrumentation. Isolated human coronary arteries also displayed ET-dependent constriction involving ETA receptors, magnified in vessels from atherosclerotic hearts.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Martine Clozel and Sebastien Roux (F. Hoffmann-La Roche Ltd., Basel, Switzerland) for generously providing bosentan, Ro-61-1790, and Ro-46-8443. The authors also thank Claude Mousseau, Jhésabelle Voyer, and Thanh-Dung Nguyen for expert technical assistance.
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FOOTNOTES |
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This work was supported through grants from the Medical Research Council of Canada, Canadian Heart and Stroke Foundation, Fonds de la Recherche en Santé du Québec, and Fonds de la Recherche de l'Institut de Cardiologie de Montréal.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Lavallée, Institut de Cardiologie de Montréal, 5000 East Bélanger St., Montréal, Québec, Canada H1T 1C8 (E-mail: lavallem{at}icm.umontreal.ca).
Received 16 November 1998; accepted in final form 23 March 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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P < 0.01 vs. L-NNA alone.
