(Circulation. 1996;94:44-51.)
© 1996 American Heart Association, Inc.
Articles |
Regulation of Nitric Oxide Production in Human Coronary Microvessels and the Contribution of Local Kinin Formation
Presented in abstract form at FASEB 1994, Anaheim, Calif, and at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form (Circulation. 1994;90:I-295).
From Columbia University, College of Physicians and Surgeons, New York, NY (M.R.K., R.E.M., L.J.A.); New York Medical College, Valhalla (N.S., X.Z., G.K., A.N., T.H.H.); and University of Southern California, Los Angeles (C.C.M.).
Correspondence to Thomas H. Hintze, PhD, Professor, Department of Physiology, New York Medical College, Valhalla, NY 10595.
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Abstract |
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Background The goal of this study was to define the regulation of nitric oxide release by coronary microvessels from the failing and nonfailing human heart and to determine the role of local kinin production in the elaboration of nitric oxide by human coronary microvascular endothelium.
Methods and Results Ten hearts from humans with
end-stage heart failure and two hearts from patients without heart
failure were harvested at the time of orthotopic cardiac
transplantation. Microvessels were sieved and the production of
nitrite was determined by the Griess reaction. Microvessels were
incubated in the presence of agonists for nitric oxide
production (acetylcholine and bradykinin), which caused
dose-dependent increases in nitrite, a response that was blocked by
NG-nitro-L-arginine methyl
ester and receptor-specific antagonists (atropine and
HOE 140, respectively). In addition, the production of nitrite
by microvessels from the failing heart appeared to be less than that
produced by microvessels from the nonfailing heart. Incubation with
norepinephrine or the 
2-adrenergic agonist
BHT 920 also caused dose-dependent increases in nitrite
production, which were blocked by the B2-receptor
antagonist HOE 140. This implicated local kinin synthesis
as an intermediate step in the production of nitric oxide in
response to 2-adrenoceptor stimulation. The
production of nitric oxide was also prevented by the addition
of serine protease inhibitors, which blocked the action of
local kallikrein, again suggesting a role for local kinin synthesis.
Conclusions Our results indicate that nitric oxide is produced by human coronary microvessels, that nitric oxide production may be reduced but certainly not increased in microvessels from the failing human heart, and that there is active local kinin generation in these blood vessels.
Key Words: endothelium-derived factors • bradykinin • enzymes • norepinephrine • receptors
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Introduction |
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The coronary microcirculation is a dynamic vascular bed that responds through endothelium-dependent and -independent mechanisms to metabolic tissue demands, changes in blood flow, circulating humoral factors, and direct neuronal activation.1 2 The vascular endothelium produces vasodilators such as NO and prostanoids, the synthesis of which may be defective in disease states such as heart failure. For example, we3 recently showed that endothelium-mediated control of the canine coronary circulation is impaired in dogs with pacing-induced dilated cardiomyopathy and results in diminished vasodilator responses to coronary occlusion and ACH. Studies4 have shown abnormalities in endothelium-dependent dilation of the coronary microvasculature in patients with dilated cardiomyopathy who were administered ACH but a near-normal response to administration of the endothelium-independent vasodilator adenosine. Peripheral endothelial cell dysfunction, as exhibited by a blunted vasodilator response to ACH, of hindquarter resistance vessels has been reported as a consequence of an experimental form of heart failure in rats,5 and similarly, in forearm resistance vessels of humans with dilated cardiomyopathy.6
2-Adrenoceptors, which are present on intact
endothelial cells, have been shown to stimulate the
production of EDRF or NO,7 8 9 10 perhaps to modulate
the constriction caused by stimulation of these receptors.
-Adrenoceptors are present in the coronary
microcirculation, especially on endothelial cells, and
in cat and dog heart, it appears that 2-adrenoceptors
are located preferentially in the small coronary arterioles,
those <100 µm in diameter.11 Although
-adrenoceptors are present in the human coronary
circulation,9 -adrenergic stimulation of NO release
has not been fully described in humans, nor has the mechanism through
which NO release occurs been fully elucidated.
The purpose of the current study was to use direct measurement of the hydration product of NO, nitrite, in vitro to determine whether human coronary microvessels produce NO; to compare the regulation of NO release by the coronary microcirculation in the failing and nonfailing human heart; and to determine the role of local kinin production in the elaboration of NO by the coronary microvascular endothelium. A preliminary report was presented previously.12
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Methods |
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Procurement of Human Hearts
Explanted hearts were harvested from 12 patients at the time of orthotopic cardiac transplantation and immediately placed in iced normal saline in the operating room. The apical portion of each heart was removed for study; this included the left ventricular free wall, the septum, and a portion of the right ventricular free wall. In addition, hemodynamic information was collected on all patients. Ten patients had severe, end-stage cardiac failure at the time of cardiac transplantation. Two patients had intact ventricular function and underwent transplantation for reasons other than ventricular failure.
Preparation of Coronary Microvessels
Hearts were transported on ice to the laboratory, where
microvessels were isolated by use of the method of Gerritsen and
Printz13 as described by us recently.3 14 15
The hearts were received in the laboratory in less than 30 minutes in
all but one instance; one heart was received 45 minutes after removal.
Each heart was placed in iced PBS that contained 1% bovine serum
albumin, pH 7.40. Left ventricular free-wall,
midmyocardial tissue was dissected free of epicardium, endocardium,
large coronary arteries, and fat. The remaining
myocardium was cut with scissors into small pieces and
suspended in ice-cold PBS. From this sample, microvessels were
collected by use of sequential sieving and glass-bead separations
as described previously.3 13 14 15 16 This yielded a preparation
that was virtually free of contaminating myocytes and that consisted
only of arterioles, venules, and capillaries <80 µm in diameter;
approximately 850 mg of microvessels was collected per heart.
Microvessels were placed in a small packet made of 80-µm nylon mesh and placed in a constant-temperature (37°C) tissue bath that contained PBS. A gas mixture of 95% oxygen and 5% carbon dioxide was bubbled through the bath for 30 minutes. Twenty-milligram aliquots of tissue were placed in plastic tubes that contained 500 mL of PBS alone or 450 mL of PBS with the addition of 50 mL of drug to stimulate or inhibit NO production and were incubated in the bath for 20 minutes. All nitrite production is presented as pmol/mg wet weight per 20 minutes.
Measurement of Nitrite Release
Nitrite (NO2) is the hydration product of NO.
With use of the Griess reaction, sulfanilic acid is diazotized by NO,
and the resultant pink color is proportional to the amount of nitrite
(hence, NO) present. Sulfanilamide (450 mL of 0.1% solution) and
N-(1-naphthyl)ethylenediamine (50 mL of 0.2%) were
added to each tube of microvessels after incubation. After a 10-minute
period at room temperature, the supernatant was removed from each tube
and the optical density measured at a wavelength of 540-µm absorbance
(Uvikon 930 spectrophotometer, Kontron Instruments). Standard curves
were generated each day by the addition of known amounts of nitrite to
buffer.
NO/Nitrite Release From Human Coronary
Microvessels
Microvessels from all hearts were incubated with an
antagonist for 10 minutes before the addition of the
maximal stimulatory dose of agonist used. Because of the limited amount
of tissue obtained from each heart, complete dose-response curves
to agonist with antagonist could not be generated, so that
only the highest dose of each agonist was studied in the presence of an
antagonist. We also specifically examined the potential
role of local kinin formation in NO production. Microvessels
were incubated with stimulators of local kinin production, and
preincubation of microvessels with several inhibitors of
enzymes that could generate kinins (including aprotinin, soybean
trypsin inhibitor, and dichloroisocoumarin17 )
was performed to determine the potential role of local kinin formation
in the production of NO.
Agonists used to stimulate NO production included ACH (10-8 to 10-5 mol/L) and BK (10-8 to 10-5 mol/L). The highest dose of BK was also studied after preincubation with the bradykinin-2 (B2) receptor blocker HOE 140 (10 µmol/L). Likewise, the highest dose of ACH was studied after preincubation with the muscarinic receptor–antagonist atropine methyl bromide (10 µmol/L) or HOE 140. To ensure that the measured nitrite truly reflected NO formation, preincubation with the NO synthase inhibitor L-NAME (100 µmol/L) was performed before the addition of the maximal dose of each agonist.
Contribution of Local Kinin Production in Human
Coronary Microvessels
To test the response to -adrenergic stimulation,
microvessels were incubated with norepinephrine, the
nonselective adrenergic agonist, in increasing doses
(10-8 to
10-5 mol/L) alone, with L-NAME, or with
the addition of the B2 receptor antagonist HOE
140 (10 µmol/L). Increasing doses of the selective
2-adrenergic receptor agonist BHT 920
(10-8 to
10-5 mol/L) were also used alone and with
the addition of HOE 140 (10 µmol/L). To determine the role of local
kinin synthesis as a mediator of the release of NO in response to
2-adrenergic receptor stimulation, serine protease
inhibitors were used to block kallikrein-mediated local
kinin formation. Microvessels were preincubated with soybean trypsin
inhibitor (10 µmol/L), dichloroisocoumarin (10 µmol/L),
or aprotinin (10 µmol/L); the maximal dose of
norepinephrine (10-5 mol/L)
or BHT 920 (10-5 mol/L) was then added.
At the end of each incubation, the supernatant was separated from the
microvessels and nitrite production was determined.
Potential for NO Production by Myocytes
As determined by phase-contrast microscopy, the eluted
tissue obtained from human hearts during the last sieving step
primarily contained myocytes. In two human heart preparations, these
myocytes were collected, centrifuged, and incubated with the
maximal stimulatory dose of BK or ACH, and nitrite was measured.
Statistical Analysis
Differences from control for each dose of agonist were
determined by use of a Student's t test and ANOVA.
Differences between nitrite production of microvessels from
normal and failing canine hearts were determined by unpaired
t test, and a Bonferroni correction was used when multiple
comparisons were performed. Standard curves for nitrite
production as determined by spectrophotometry were constructed
each day; best fit was determined by use of a linear least-squares
analysis. Only standard curves with r>.90 were
used. We calculated the amount of nitrite by substituting the measured
absorbance of each sample into the equation of the line. All of these
techniques have been used by us previously.3 14 15 18
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Patient Population
Pertinent clinical and hemodynamic data are listed in the Table
. Of the 12 patients from whom hearts were
harvested, 10 were male and 2 were female. The age range was 6.5 months
to 63 years, with a median age of 12 years. The most common clinical
diagnosis was idiopathic dilated cardiomyopathy (6
of 12 patients). Ten hearts were designated as failing (patients F-1 to
F-10 in the Table); these were obtained from patients whose main
indication for transplantation was low cardiac output and failure of
the left ventricle. Two hearts were nonfailing (patients NF-1 and NF-2
in the Table); 1 of these was obtained from a patient who was
transplanted for cyanotic congenital heart disease not amenable to
conventional surgery and the other from a patient who underwent
retransplantation because of accelerated coronary vasculopathy
of his first cardiac allograft. All patients were considered NYHA class
4. Seven of the 10 patients with heart failure required
intravenous inotropic support before transplantation.
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On pathological examination, all hearts exhibited some degree of myocardial hypertrophy. This was quantified as the percent above estimated normal heart weight for age and sex, which was more useful than absolute weight in grams because the population from which the explanted hearts were obtained included both pediatric and adult patients of both sexes. The mean percent above normal heart weight was 190±20% (mean±SE).
NO/Nitrite Release From Human Coronary
Microvessels
Increasing doses of BK or ACH caused dose-related increases in
nitrite production in all human hearts. The response to BK by
microvessels isolated from all failing and nonfailing human hearts is
shown in Fig 1. The maximal dose of BK
(10-5 mol/L) yielded a mean percent
change from control of 157±28% (n=10) in microvessels from failing
hearts. The magnitude of the response to ACH was similar.
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The maximal dose of BK was also studied after preincubation with HOE
140 (10 µmol/L; n=12), which abolished any significant increase in
nitrite production. Likewise, the maximal dose of ACH was
studied after preincubation with atropine or HOE 140. Atropine blocked
the increases in nitrite production in response to ACH (n=7;
P<.05), whereas nitrite production was unaffected
by HOE 140 (data not shown). Preincubation of microvessels with the NO
synthase inhibitor L-NAME before the addition of the
maximal dose of each agonist significantly diminished the response to
either BK or ACH, as shown in Fig 2.
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compared with maximal dose of agonist alone.
Contribution of Local Kinin Production in Human
Coronary Microvessels
Nonspecific -adrenergic stimulation with
norepinephrine or 2-adrenergic receptor
selective stimulation with BHT 920 caused dose-dependent increases
in nitrite release from human coronary microvessels. Results
from studies in microvessels from failing and nonfailing human hearts
are shown in Fig 1
.
The addition of the B2-receptor blocker HOE 140 to the
maximal dose of each agonist eliminated the increases in nitrite
production (n=12). Incubation with L-NAME before the addition
of agonist had a similar effect. Data are shown in Fig 3.
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Preincubation of microvessels isolated from all human hearts with the
serine protease inhibitors soybean trypsin
inhibitor (10 µmol/L), dichloroisocoumarin (10 µmol/L),
or aprotinin (10 µmol/L) was performed to block the action of
kallikrein. The maximal dose of norepinephrine
(10-5 mol/L) or BHT 920
(10-5 mol/L) was then added. No
significant difference was observed in nitrite production by
microvessels compared with basal levels. These data are shown in Fig 4. Preincubation with the serine protease
inhibitors had no effect on the stimulatory action of the
maximal dose of ACH or BK.
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Potential for NO Production by Myocytes
Human myocytes, collected from the final elution step in
microvessel preparations, were centrifuged and incubated with
the maximal stimulatory dose of BK or ACH. None of the tissue that was
tested generated measurable amounts of nitrite in the basal state or in
the presence of these agonists.
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Discussion |
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There were several significant findings of this study. First, BK, ACH, norepinephrine, and BHT 920 caused dose-dependent increases in nitrite release from coronary microvessels isolated from failing and nonfailing human hearts. When these two groups of hearts were compared, we found that nitrite release by failing human hearts appeared to be less than the nitrite release measured in nonfailing human hearts. However, it was not possible to procure a sufficient number of nonfailing human hearts for statistical comparisons. Our studies with human microvessels indicated that nitrite release in response to all agents that stimulated nitrite production was markedly reduced by L-NAME, an inhibitor of NO synthase, which confirmed that nitrite production was an indication of NO metabolism. As expected, HOE 140 specifically inhibited nitrite release in response to BK; similarly, atropine selectively blocked the effects of ACH. Surprisingly and perhaps most interestingly, nitrite release in response to
2-adrenergic receptor stimulation with
norepinephrine or BHT 920 was inhibited by the
B2-receptor antagonist HOE 140. To test the
involvement of local kinin production in this process, the
serine protease inhibitors soybean trypsin
inhibitor, aprotinin, or dichloroisocoumarin were used to
block the action of local kallikrein. All three agents caused
significant decreases in nitrite release during
2-receptor stimulation.
The patient population from which the explanted hearts were obtained
included 10 patients with symptomatic severe congestive
heart failure and hemodynamics consistent with
ventricular dysfunction and 2 patients with no symptoms of
heart failure and hemodynamics that reflected preserved
ventricular function. The population included both adults
and children. It is evident that the first 10 patients were
"failing," as evidenced by elevated pulmonary capillary
wedge pressures, low mixed venous oxygen saturations, low cardiac
indexes, and high resting heart rates. This observation was supported
by measures of ventricular function, which were obtained
routinely in adults as left ventricular ejection fraction
by multigated acquisition scan and in children as percent fractional
shortening of the left ventricle from transthoracic M-mode
echocardiogram. In the 10 patients with failing hearts, these values
were well below normal. Of the 2 patients with nonfailing hearts, 1
(designated as NF-1 in the Table) had congenital heart disease and
persistent cyanosis but low pulmonary capillary wedge pressure
and qualitatively normal ventricular function by
transthoracic M-mode echocardiogram. The other patient
(designated as NF-2 in the Table) underwent retransplantation for
posttransplant coronary vasculopathy; this patient had normal
hemodynamics and qualitatively normal
ventricular function by echocardiogram. Although we could
not designate these patients as "failing," we also could not call
these patients truly "normal," given their anatomic or
pathological indications for cardiac transplantation.
The patients with symptomatic heart failure were all treated with an oral regimen that included digoxin and diuretics. Six of the 10 patients were treated with ACE inhibitors. Seven of the 10 patients were treated chronically in the hospital with intravenous dobutamine until the time of transplantation. There is some question as to the effect of these medications on endogenous NO production. Dobutamine primarily stimulates ß1-adrenergic receptors, and therapy before transplantation and isolation of coronary microvessels appears to have little or no effect on NO/nitrite production in vitro, ie, this did not cause increased nitrite production in the basal or stimulated state in microvessels from failing hearts compared with normal. Of note, direct application of specific ß-adrenergic receptor agonists has been shown to have no effect on stimulation of EDRF production or release in isolated canine coronary artery rings.19 The vasodilator properties of ACE inhibitors and their modulation of the vascular response to BK have been well described.20 21 22 Although it is possible that ACE-inhibitor therapy may alter nitrite production in isolated coronary microvessels, we have not found this to be true.
Our data support work that previously demonstrated that endothelium-dependent dilation is abnormal in congestive heart failure and that this impairment extends to the coronary microcirculation. In studies in humans, Drexler et al6 demonstrated the consequences of impaired NO production by showing that patients with congestive heart failure exhibit a blunted increase in forearm blood flow to endothelium-dependent dilating agents, such as ACH, compared with healthy patients. Those data also suggest that the basal release of NO is preserved or may be enhanced in forearm resistance vessels in patients with heart failure, which implies a compensatory role in the modulation of tissue perfusion in response to humoral agents after the development of heart failure. The probability that intrinsic microvascular disease exists in the coronary circulation has been explored by Treasure et al,4 who demonstrated a differential response in small-vessel resistance to endothelium-dependent (ACH) and -independent (adenosine) dilators in normal patients and those with cardiomyopathy. Although coronary blood flow was increased in both groups with adenosine, ACH caused significant increases in coronary blood flow in the control group only; blood flow was not altered in patients with cardiomyopathy.
Data from our laboratory3 showed attenuated dilation of the large coronary artery and increases in coronary blood flow in conscious dogs with pacing-induced cardiomyopathy after brief occlusion of the left circumflex coronary artery, intravenous administration of ACH, or administration of arachidonic acid. The diminished response to these vasodilators suggests a defect in endothelial function of both the large coronary arteries and the coronary microvasculature of failing hearts. Recently, in our laboratory, Zhao et al18 showed that reflex cholinergic coronary vasodilation is almost abolished during pacing-induced heart failure in the dog because of the disappearance of NO. Both isolated large coronary arteries and microvessels of failing hearts produced significantly less nitrite in vitro in response to all agonists in our previous studies,3 16 and this effect was blocked by the addition of L-NAME. These data support the contention that endothelium-mediated control of the coronary circulation may be depressed in the failing heart.
It has also been postulated that NO modulates -adrenergic
vasoconstriction in a number of blood vessels. Jones et
al7 showed that -adrenergic receptors are
present throughout the canine coronary microcirculation,
with 1-receptors predominating in small arteries and
2-receptors in arterioles. When
norepinephrine, ß-adrenergic receptor blockade, and
selective inhibition of either 1-receptors with prazosin
or 2-receptors with rauwolscine were used, constriction
caused by 1- or 2-adrenergic receptor
activation was potentiated during inhibition of NO synthase.
In comparing the response to norepinephrine of
carotid, mesenteric, renal, and femoral large arteries of the pig,
greyhound, and mongrel dog, Angus et al8 have shown that
there is a nonuniformity of distribution of
2-adrenoceptors on endothelium and
smooth muscle. They also demonstrated that 2-receptors
on endothelium mediate the release of EDRF, as
evidenced by an enhanced concentration-contraction curve to
norepinephrine in vessels denuded of
endothelium. More specifically,
norepinephrine relaxed precontracted arteries in the
presence of ß-adrenergic receptor blockade, and this was
inhibited by 2-adrenoceptor
antagonists.8
Studies in humans conducted by Indolfi et al9 showed that
in vivo administration of the selective 2-receptor
agonist BHT 933 to patients with normal coronary arteries
induced a reduction in coronary artery diameter and
coronary blood flow velocity. 2-Adrenergic
blockade did not change coronary blood flow in normal subjects;
however, in patients with atherosclerosis, regional
coronary blood flow decreased after receptor blockade. This
supports our data as well in that 2-receptors
participate in the modulation of vascular resistance in the human
heart.
It has not been shown before in blood vessels from humans, however,
that the stimulatory effects of 2-adrenergic receptor
activation on NO release occur via activation of local kinin synthesis.
In the present study, nonselective -stimulation with
norepinephrine and the selective
2-adrenergic stimulator BHT 920 caused
dose-dependent increases in nitrite release from coronary
microvessels isolated from both failing and nonfailing human hearts.
The addition of the B2-receptor antagonist HOE
140 prevented the increase in nitrite, and given this evidence, we
postulated a novel potential mechanism of NO release in response to
2-adrenoceptor activation, ie, local kinin
production was the intermediate step responsible for the
ultimate release of EDRF. To further investigate this hypothesis,
inhibition of kallikrein activity was achieved by use of three
different serine protease inhibitors. As demonstrated by
others, aprotinin can block local kinin
production23 24 25 26 and in fact is used currently to
improve hemostasis during open-heart surgery that requires
cardiopulmonary bypass. Aprotinin inhibits the contact
activation of tissue kallikrein and thereby blocks the intrinsic
coagulation cascade and prevents the total body inflammatory response
that is seen with cardiopulmonary
bypass.27
Kallikrein or a kallikrein-like enzyme appears to be present
ubiquitously in human vascular tissue and in circulating
plasma.26 28 Kinin precursors, such as
high-molecular-weight kininogen, also circulate in plasma and
are taken up by high-affinity binding sites present on the
surface of endothelial cells.29 BK is then
formed and binds to B2-receptors on the
endothelial cell surface to release NO and
prostacyclin. In the current study, we have shown that activation of
the kallikrein-kinin system can be initiated by
2-adrenoceptor stimulation, and the end product of
this is nitrite (NO2). The importance of this is clear:
local kinin production, and hence NO, may modulate
coronary vasoconstriction after -adrenergic receptor
stimulation in the human coronary circulation.
Many authors20 21 22 28 have speculated that the blood pressure–lowering effects of ACE-inhibitor therapy are not just a manifestation of angiotensin II inhibition but are also due to diminished BK degradation. One study30 showed that plasma kinin levels of hypertensive patients who received ACE-inhibitor therapy were not significantly altered; however, enhanced local synthesis or storage of kinins within the vascular endothelium could be responsible for the hypotensive effects of the drug. It is also possible that in pathological states such as heart failure, endogenous BK may be present in subthreshold amounts and its vasodilator capacity may only be unmasked by ACE-inhibitor therapy. In addition, these compounds can potentiate other in vitro endothelium-dependent relaxations, such as the vascular response to ACH and shear stress.22
The systemic benefit of EDRF production rests not only in its action as a vasodilator but also in its ability to diminish total tissue oxygen consumption and limit tissue metabolism, as we have recently shown31 ; myocardial tissue oxygen consumption may be regulated in part by NO synthesized by the coronary microvessel endothelium. It is interesting and perhaps only coincidental that many patients with severe heart failure are treated with ACE inhibitors. In fact, 6 of 10 patients in our study received ACE inhibition. Perhaps at least part of the beneficial effect is to preserve NO production by extending the half-life of locally formed kinins.
Recent studies indicated that myocytes may produce NO. Pinsky et
al32 demonstrated increased inducible NO synthase
expression in rat myocytes in response to cytokines such as
interleukin-1 and tumor necrosis factor-. Cytokines may be
elaborated in response to immunologic challenge and inflammatory
states, such as cardiac allograft rejection and
cardiomyopathy. The end result of enhanced
myocardial inducible NO synthase activity may be autotoxicity, reduced
contractility (myocyte shortening) that contributes to
myocyte necrosis, and eventual ventricular dysfunction. It
is unclear, however, whether myocyte inducible NO synthase is active in
basal conditions and whether the enhanced synthesis observed in the rat
model is also seen in healthy humans and in disease states. In the
present studies in human coronary microvessels, the
preparations were virtually free of contaminating myocytes.
There are a number of potential limitations of the present study that should be discussed. First, our vessel preparation was a mixture of small coronary blood vessels, and reduced nitrite production may have been present in some or all of the segments, including arterioles, capillaries, and venules. Because some of our recent studies33 indicated reduced NO synthase gene expression in aorta from the dog with overt congestive heart failure, it is most likely that this reduced NO production extends throughout the vascular tree. Another potential criticism is that human microvessel nitrite production does not reflect the magnitude of the actual defect in heart failure, since the patients were medicated to preserve cardiac function and since even the microvessels from the nonfailing hearts were from "sick hearts." Be that as it may, microvessels from the hearts of dogs with pacing-induced heart failure that were not medicated for the treatment of heart failure may be indicative of the true magnitude of the blood vessel defect, ie, they have even lower nitrite production.3 If vessels from the nonfailing human heart do not reflect normal human coronary microvessels and hypothetically also exhibit altered NO production, then the logical explanation from all of our data, human and canine, is that NO production from normal human coronary microvessels should be even higher than we measured in the nonfailing group. Finally, because of medication of these patients, the drugs may have altered NO production even after the microvessels were isolated. The microvessels were extensively washed during preparation, which took 4 to 5 hours, and this should have removed water-soluble drugs. However, if drugs such as the ACE inhibitors altered endothelial gene expression, then an altered NO production may have persisted. Even so, nitrite production from human coronary microvessels appeared to be low.
In summary, viable coronary microvessels were isolated
successfully from failing and nonfailing human hearts. These
microvessels produced NO in response to known agonists for NO
production (ACH, BK, norepinephrine, and BHT 920),
and the stimulatory action of these substances was blocked by L-NAME.
The nonselective -adrenergic receptor agonist
norepinephrine and the selective
2-adrenergic receptor agonist BHT 920 also caused NO
production; their action was blocked by the
B2-receptor antagonist HOE 140, which
implicates local kinin production as an intermediate step.
Inhibitors of kallikrein activity (the serine protease
inhibitors aprotinin, soybean trypsin
inhibitor, and dichloroisocoumarin) all prevented NO
production in response to norepinephrine and BHT
920.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grant PO-1-HL43012 from the National Heart, Lung, and Blood Institute.
Received October 5, 1995; revision received December 19, 1995; accepted December 21, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Moncada S, Palmer RM, Higgs EA. The discovery of nitric oxide as the endogenous nitrovasodilator. Hypertension. 1988;12:365-372.
2.
Miller VM, Vanhoutte P. Enhanced release of
endothelium-derived factor(s) by chronic increase
in blood flow. Am J Physiol. 1988;255:H446-H451.
3.
Wang J, Seyedi N, Xu XB, Wolin MS, Hintze TH.
Defective endothelium-mediated control of
coronary circulation in conscious dogs after heart
failure. Am J Physiol. 1994;266:H670-H680.
4.
Treasure CB, Vita JA, Cox DA, Fish RD, Gordon JB,
Mudge GH, Colucci WS, St. John Sutton MG, Selwyn AP, Alexander RW, Ganz
P. Endothelium-dependent dilation of the
coronary microvasculature is impaired in dilated
cardiomyopathy.
Circulation. 1990;81:772-779.
5. Drexler H, Lu W. Endothelial dysfunction of hindquarter resistance vessels in experimental heart failure. Am J Physiol. 1992;262(Heart Circ Physiol 31):H1640-H1645.
6. Drexler H, Hayoz D, Munzel T, Hornig B, Just H, Brunner HR, Zelis R. Endothelial function in chronic congestive heart failure. Am J Cardiol. 1992;69:1596-1601.[Medline] [Order article via Infotrieve]
7.
Jones CJH, DeFily DV, Patterson JL, Chilian WM.
Endothelium-dependent relaxation competes with
1- and 2-adrenergic constriction in the
canine epicardial coronary microcirculation.
Circulation. 1993;87:1264-1274.
8. Angus JA, Cocks TM, Satoh K. The alpha adrenoceptors on endothelial cells. Fed Proc. 1986;45:2355-2359.[Medline] [Order article via Infotrieve]
9.
Indolfi C, Piscione F, Villari B, Russolillo E,
Rendina V, Golino M, Chiariello M. Role of alpha-2 adrenoceptors
in normal and atherosclerotic human coronary
circulation. Circulation. 1992;86:1116-1123.
10. Angus JA, Cocks TM, Satoh K. Alpha-2 adrenoreceptors and endothelium-dependent relaxation in canine large arteries. Br J Pharmacol. 1986;88:767-777.[Medline] [Order article via Infotrieve]
11. Chilian WM. Adrenergic vasomotion in the coronary microcirculation. Basic Res Cardiol. 1990;85:111-120.
12. Kichuk MR, Seyedi N, Marboe C, Addonizio L, Michler RE, Hintze TH. Reduced EDRF production from coronary microvessels from the failing explanted human heart. FASEB J. 1994;8(part II):4786. Abstract.
13.
Gerritsen M, Printz M. Sites of
prostaglandin synthesis in the bovine heart and isolated
coronary microvessels. Circ Res. 1981;49:1152-1163.
14.
Seyedi N, Xu X, Nasjletti A, Hintze TH.
Coronary kinin generation mediates nitric oxide release after
angiotensin receptor stimulation.
Hypertension. 1995;26:164-170.
15.
Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze
TH. Chronic exercise in dogs increases coronary vascular
nitric oxide production and endothelial cell
nitric oxide synthase gene expression. Circ
Res. 1994;74:349-353.
16. Hintze TH, Wang J, Wolin MS. Myocardial hypertrophy and failure: association between alterations in the production or release of EDRF/NO and myocardial dysfunction—control of the circulation by flow. In: Rubanyi G, Kaley G, Bevan JA, eds. London, UK: Oxford University Press; 1994.
17.
Belloni FL, Wang J, Hintze TH. Adenosine causes
bradycardia in pacing-induced heart failure.
Circulation. 1992;85:1118-1124.
18.
Zhao G, Shen W, Xu X, Ochoa M, Bernstein R, Hintze
TH. Selective impairment of vagally mediated nitric
oxide–dependent coronary vasodilation in conscious dogs
after pacing-induced heart failure.
Circulation. 1995;91:2655-2663.
19. MacDonald PS, Dubbin PN, Dusting GJ. Beta-adrenoreceptors on endothelial cells do not influence release of relaxing factor in dog coronary arteries. Clin Exp Pharmacol Physiol. 1987;12:525-534.
20.
Weimer G, Schölkens BA, Becker RHA, Busse
R. Ramiprilat enhances endothelial
autacoid formation by inhibiting breakdown of
endothelium-derived bradykinin.
Hypertension. 1991;18:558-563.
21. Busse R, Fleming I, Hecker M. Endothelium-derived bradykinin: implications for angiotensin-converting enzyme-inhibitor therapy. J Cardiovasc Pharmacol. 1993;22:S31-S36.
22. Vanhoutte PM, Auch-Schwelk W, Biondi ML, Lorenz RR, Schini VB, Vidal AMJ. Why are converting enzyme inhibitors vasodilators? Br J Clin Pharmacol. 1989;28:95S-104S.
23. Vanhoutte PM, Miller VM. Heterogeneity of endothelium-dependent responses in mammalian blood vessels. J Cardiovasc Pharmacol. 1985;7:S12-S23.
24. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994;298:249-258.
25. Mombouli JV, Vanhoutte PM. Kinins mediate kallikrein-induced endothelium-dependent relaxations in isolated canine coronary arteries. Biochem Biophys Res Commun. 1992;185:693-697.[Medline] [Order article via Infotrieve]
26. Maddedu P, Gherli T, Bacciu PP, Maioli M, Glorioso N. A kallikrein-like enzyme in human vascular tissue. Am J Hypertens. 1993;6:344-348.[Medline] [Order article via Infotrieve]
27. Westaby S. Aprotinin in perspective. Ann Thorac Surg. 1993;55:1033-1041.[Abstract]
28. Vanhoutte PM, Boulanger CM, Illiano SC, Nagao T, Vidal M, Mombouli JV. Endothelium-dependent effects of converting enzyme inhibitors. J Cardiovasc Pharmacol. 1993;27:S10-S16.
29. Müller-Estrl W. Kininogen, kinins and kinships. Thromb Haemost. 1989;61:2-6.[Medline] [Order article via Infotrieve]
30. Imura O, Shimamoto K. Role of kallikrein-kinin system in the hypotensive mechanisms of converting-enzyme inhibitors in essential hypertension. J Cardiovasc Pharmacol. 1989;13:S63-S66.
31.
Shen W, Xu X, Ochoa M, Zhao G, Wolin MS, Hintze
TH. Role of nitric oxide in the regulation of oxygen consumption
in conscious dogs. Circ Res. 1994;75:1086-1095.
32. Pinsky DJ, Cai B, Yang X, Rodriguez C, Sciacca RB, Cannon PJ. Cytokine-inducible NO-dependent autotoxicity of cardiac myocytes. Circulation. 1994;90:II-1030. Abstract.
33.
Smith CJ, Sun D, Hoegler C, Zhao G, Xu X, Kobari Y,
Pritchard K, Sessa WC, Hintze TH. Reduced gene expression of
vascular nitric oxide synthase and cyclooxygenase-1
in heart failure. Circ Res.. 1996;78:58-64.
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|
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|
|
|
|
|
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