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Vol. 288, Issue 2, 742-751, February 1999
Departments of Physiology (X.Z., F.A.R., R.B., X.X., T.H.H.) and Pharmacology (A.N.), New York Medical College, Valhalla, New York
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Abstract |
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 Top
 Abstract
 Introduction
Materials and methods
Results
Discussion
References
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Increasing evidence suggests that angiotensin-converting enzyme (ACE) inhibitors can increase vascular nitric oxide (NO) production. Recent studies have found that combined inhibition of ACE and neutral endopeptidase (NEP) may have a greater beneficial effect in the treatment of heart failure than inhibition of ACE alone. Amlodipine, a calcium channel antagonist, has also been reported to have a favorable effect in the treatment of patients with cardiac dysfunction. The purpose of this study was to determine whether and the extent to which all of these agents used in the treatment of heart failure stimulate vascular NO production. Heart failure was induced by rapid ventricular pacing in conscious dogs. Coronary microvessels were isolated from normal and failing dog hearts. Nitrite, the stable metabolite of NO, was measured by the Griess reaction. ACE and NEP inhibitors and amlodipine significantly increased nitrite production from coronary microvessels in both normal and failing dog hearts. However, nitrite release was reduced after heart failure. For instance, the highest concentration of enalaprilat, thiorphan, and amlodipine increased nitrite release from 85 ± 4 to 156 ± 9, 82 ± 7 to 139 ± 8, and 74 ± 4 to 134 ±10 pmol/mg (all *p < .01 versus control), respectively, in normal dog hearts. Nitrite release in response to the highest concentration of these two inhibitors and amlodipine was reduced by 41% and 31% and 32% (all #p < .01 versus normal), respectively, in microvessels after heart failure. The increase in nitrite induced by either ACE or NEP inhibitors or amlodipine was entirely abolished by Nw-nitro-L-arginine methyl ester, HOE 140 (a B2-kinin receptor antagonist), and dichloroisocoumarin (a serine protease inhibitor) in both groups. Our results indicate that: 1) there is an impaired endothelial NO production after pacing-induced heart failure; 2) both ACE and NEP are largely responsible for the metabolism of kinins and modulate canine coronary NO production in normal and failing heart; and 3) amlodipine releases NO even after heart failure and this may be partly responsible for the favorable effect of amlodipine in the treatment of heart failure. Thus, the restoration of reduced coronary vascular NO production may contribute to the beneficial effects of these agents in the treatment of heart failure.
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Introduction |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Nitric
oxide (NO) derived from vascular endothelium plays an important role in
the regulation of many biological functions including vasodilation and
mitochondrial respiration (Moncada et al., 1991
). Increasing evidence
indicates that there is an impaired endothelial NO production during
the development of heart failure in humans and animals (Treasure et
al., 1990; Katz et al., 1993; Drexler et al., 1994; Wang et al., 1994).
Experimental and clinical studies have demonstrated that
angiotensin-converting enzyme (ACE) inhibition has an antihypertensive
and cardioprotective action, partly by preventing kinin degradation and
consequently increasing endothelial NO production (Schwelk et al.,
1993; Linz et al., 1995; Scholkens, 1996). Numerous studies have shown
the existence of the kallikrein-kinin system in cardiac and vascular tissue in animals (Nolly et al., 1993, 1994; Wiemer et al., 1994; Linz
et al., 1995; Scholkens 1996; Zhang et al., 1997). A variety of endo-
and exopeptidases, which are widely distributed in various tissue and
cell types contribute to the degradation of kinins (Erdos and Skidgel,
1989; Skidgel, 1992). ACE is the primary enzyme responsible for this
catabolism. However, growing data from recent studies suggest that
neutral endopeptidase (NEP) is also at least partially responsible for
regulating the metabolism of kinins in the tissue from a variety of
species (Graf et al., 1993; Trippodo et al., 1995a,b;
Dragovic et al., 1996). Yang et al. (1997) recently reported that
inhibition of NEP protects the heart against ischemia/reperfusion injury by a kinin-dependent mechanism. Furthermore, a study from our
laboratory (Zhang et al., 1998a) found that NEP inhibitors can release
nitrite from canine coronary microvessels. All of these data suggest
that NEP might be one of the main peptidases participating in the
metabolism of kinins. Accordingly, we hypothesized that inhibition of
NEP may also have a beneficial effect on the treatment of heart failure
by potentiation of kinins.
In addition, although calcium antagonists have not been shown to be
beneficial in the treatment of patients with heart failure, a recent
clinical trial (Packer et al., 1996) has demonstrated a favorable
effect of amlodipine on the survival of patients with heart failure
resulting from nonischemic dilated cardiomyopathy. A new concept for
the treatment of heart failure has been suggested to combine ACE
inhibitors with calcium-channel antagonists (Lliceto, 1997; Waeber and
Brunner, 1997), because calcium antagonists and ACE inhibitors exhibit
additive antihypertensive efficacy and counterbalance the negative
effects caused by neurohormonal activation when combined, and their
safety profile is, if anything, improved. However, the mechanism of the
favorable action of amlodipine has not been determined. A recent study
by Lyons et al. (1994) found that like enalaprilat, amlodipine
significantly restored forearm arterial vasoconstriction to local
intra-arterial infusions of NG-monomethyl-L-arginine.
We inferred from that study that amlodipine may mimic the effect of ACE
inhibitors and promote NO production. With this in mind, the present
study was designed to directly measure the hydration product of
NO, nitrite, to determine: 1) whether inhibition of NEP can increase
coronary microvascular NO production, and whether the mechanism is
similar to that of ACE inhibition; 2) whether amlodipine increases NO
production and whether this effect can be affected by blockade of
B2-kinin receptor or inhibition of local kinin
formation; and 3) whether ACE or NEP inhibition or amlodipine can also
increase NO production from coronary microvessels after the development
of severe congestive heart failure.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Animal preparation
All of the studies in dogs were approved by the Institutional
Animal Care and Use Committee of New York Medical College and conform
to current National Institutes of Health and American Physiological
Society Guidelines for the Use and Care of Laboratory Animals.
Twenty-one adult mongrel dogs (body weight 21-30 kg) in two groups,
normal (n = 12) and pacing-induced heart failure (n = 9), were used in this study. Heart failure was
induced by rapid left ventricular pacing for 4 weeks in chronically
instrumented conscious dogs, and hemodynamic data from these awake dogs
were obtained with previously implanted catheters and transducers, as
described in detail (Wang et al., 1994). All normal and failing hearts
were obtained immediately from pentobarbital-anesthetized dogs and kept
in ice-cold phosphate-buffered saline (PBS) containing 0.1% bovine
serum albumin at pH = 7.4.
Isolation of Coronary Microvessels.
Isolation of coronary
microvessels from the left ventricle of the dog heart was performed
according to the method originally developed by Gerritsen and Printz
(1981). Coronary microvessels were obtained free of both large arteries
and veins and also of myocytes by a series of steps involving
sequential dissection, homogenization, sieving, and glass bead
purification. These methods have been used in our previous studies
(Wang et al., 1994; Zhang et al., 1997, 1998a,b).
Incubation of Coronary Microvessels.
Microvessels (external
diameter, 20-70 µm; length, 0.2-0.8 mm) were placed in a small
package of 80-µm nylon mesh, transferred into a tissue bath
containing PBS, and oxygenated with 95% O2 and 5%
CO2 for 30 min. About 20 mg (wet weight) of tissue was placed in 5 ml of plastic tubes which contained 500 µl of PBS as
control or 450 µl of PBS and 50 µl of drugs dissolved in PBS used
to stimulate (e.g., amlodipine and ramiprilat) or inhibit (e.g.,
Nw-nitro-L-arginine methyl
ester (L-NAME)) NO formation. All tissues were incubated
with drug for 20 min at 37°C. At the end of the incubation time, the
tubes were removed from the tissue bath, and sulfanilamide (450 µl of
1%) and N-(1-naphthyl)ethylenediamine (50 µl of
0.2%) were added to each tube for diazotization of sulfanilic acid by
NO. After 5 to 10 min incubation at room temperature, the supernatant
was removed from each tube. Formation of NO was measured as nitrite,
which is the major metabolite of NO in aqueous solution. Nitrite was
measured with a spectrophotometer (Uvikon 930 Spectrophotometer,
Kontron Instruments Inc., Boston, MA) as the increase in
absorbance at 540 nm and compared to known concentrations of nitrite.
L-NAME was used to block NO synthase. HOE-140 (Icatibant) was used to block the kinin B2-receptor and
dichloroisocoumarin (DCIC) was used to block the action of
kinin-forming enzymes. We have described these methods recently (Wang
et al., 1994; Zhang et al., 1997, 1998a,b).
Effects of Ramiprilat and Enalaprilat on NO Production from
Coronary Microvessels in Normal and Failing Dog Hearts.
The
effects of two ACE inhibitors on nitrite production from isolated
coronary microvessels were compared from normal and failing hearts.
Increasing concentration of ramiprilat (10
10 to
107 mol/liter) and enalaprilat (1010 to
107 mol/liter) were incubated with tissue for 20 min, and
nitrite was measured. To determine the role of kinin and kinin-forming enzyme in ACE inhibitor-induced NO production, 50 µl of
105 mol/liter HOE 140 or DCIC was preincubated with
tissue before addition of the highest concentration of ramiprilat or
enalaprilat. To confirm that nitrite release reflects NO production,
the effect of the highest concentration of ramiprilat and enalaprilat
was also assessed after preincubation of the microvessels with
L-NAME. A standard dose-response curve for bradykinin
(108 to 105 mol/liter) was also performed,
and the effects of L-NAME and HOE 140 on NO production
induced by the highest concentration of bradykinin were also examined.
Effects of Phosphoramidon, Thiorphan, and Kininogen on NO
Production from Coronary Microvessels in Normal and Failing Dog
Hearts.
We compared the ability of neutral endopeptidase inhibitor
to release NO with that of ACE inhibitor. In coronary microvessels from
both normal and failing hearts, the effects of increasing concentration
of phosphoramidon and thiorphan (108 to 105
mol/liter) on NO production were assessed. A comparison of the effects
of kininogen (0.5-10 µg/ml) on NO production from coronary microvessel was also performed. The effects of the highest
concentration of each of these agents were also analyzed after
preincubation of the microvessels with L-NAME, HOE-140, or DCIC.
Effects of Amlodipine on NO Production from Coronary Microvessels
in Normal and Failing Dog Hearts.
We investigated the ability of
amlodipine to release NO in this study. In coronary microvessels, the
effects of increasing concentration of amlodipine (1010
to 104 mol/liter) on NO production were assessed in both
normal and failing hearts. The effect of the highest concentration of
amlodipine was also studied after preincubation of the microvessels
with L-NAME, HOE-140, or DCIC.
Drugs and Chemicals. The PBS used in these studies was made of: NaCl, 139 mM; KCl, 2.7 mM; NaHPO4, 8.1 mM; KH2PO4, 1.5 mM; CaCl2, 0.68 mM; MgCl2, 0.49 mM; and bovine serum albumin, 0.1%. L-NAME is an inhibitor of NO synthase, HOE-140 (Icatibant) is a bradykinin 2 receptor antagonist, and DCIC is a serine protease inhibitor which blocks the activity of kinin-forming enzymes (serine proteases). Drugs (bradykinin, enalaprilat, phosphoramidon and thiorphan) and chemicals (L-NAME, DCIC and nitrite) were purchased from Sigma Chemical Co. (St. Louis, MO). Amlodipine was generously supplied by Pfizer Pharmaceutical Inc. (Groton, CT). Ramiprilat and HOE 140 were generously supplied by Hoechst-Roussel Inc. (Somerville, NJ). Bovine kininogen was purchased from Seikagaku Kogyo Co Ltd. (Tokyo, Japan).
Statistical Analysis and Calculation.
To construct a
standard curve for nitrite, a stock solution of NaNO2
(105mol/liter) was prepared and diluted each day.
Sulfanilamide (450 µl of 1%) and
N-(1-naphthyl)ethylenediamine (50 µl of 0.2%) were added to each tube and mixed well. The tubes were allowed to stand at
room temperature for 5 to 10 min for full color (pink) development and
absorbance of nitrite was measured at 540 nm. Absorbance was computed
and converted to a straight line with a regression analysis (Y = ax + b, R > 0.99). Nitrite production was calculated with the
linear regression formula and values computed. Data were expressed as
mean ± S.E.M. in picomoles/mg wet weight/20 min. Differences of
nitrite production from two groups were determined by an analysis of
variance. The differences between individual data points were determined with Tukey's test. p < .05 was
considered to be statistically significant. Statistical analysis and
graphs were produced on a 486 computer (Everex, Freemont, CA) with
commercially available software (Lotus 123; Lotus Dev. Corp.,
Emeryville, CA; GBSTAT; Dynamic Microsystems; Silver Spring, MD; Slide
Write; Advanced Graphics Software, Inc., Carlsbad, CA).
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Results |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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The average of body weight, the average weight of the heart, and the average weight of the left ventricular wall in these dogs were 24 ± 0.83 and 24 ± 0.78 kgs, 189 ± 9 and 259 ± 16 g (p < .05 compared with normal), and 71 ± 3 and 85 ± 6 g in normal and heart failure groups, respectively. The average amount of microvessels collected from both groups was 1.7 ± 0.3 g/heart. The data in the figures are the actual values of nitrite production in pmol/mg wet weight/20 min incubation, whereas the data in the text are the actual values and percent changes. Hemodynamic data are shown in Table 1. There were marked changes in all of the indices measured, indicative of severe decompensated heart failure.
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Effects of Ramiprilat and Enalaprilat on NO Production from
Coronary Microvessels in Normal and Failing Dog Hearts.
The
effects of increasing concentration of ramiprilat (1010
to 107 mol/liter) and enalaprilat (1010 to
107 mol/liter) on NO production in coronary microvessels
are shown in Fig. 1. Both agents
substantially increased nitrite production in a concentration-related
manner in normal and failing hearts. However, nitrite production was
significantly reduced after heart failure. The highest concentration of
ramiprilat and enalaprilat increased nitrite production from 79 ± 4 to 122 ± 14 pmol/mg and 85 ± 4 to 156 ± 9 pmol/mg,
respectively, in normal hearts; but increased nitrite production from
50 ± 3 to 91 ± 5 pmol/mg and 54 ± 5 to 92 ± 4 pmol/mg (all *p < .01), respectively, in failing hearts. Compared with the normal group, nitrite production in response
to the highest concentration of these two inhibitors was reduced by
44% by ramiprilat and 41% by enalaprilat (all *p < .01), respectively, after heart failure. The effects of the highest
concentration of these two inhibitors on NO production were entirely
blocked by L-NAME, HOE 140, or DCIC in both groups (Fig.
2).
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8 to 105
mol/liter) caused a significant, comparable, and
concentration-dependent increase in nitrite production in coronary
microvessels of normal and failing dog hearts. The highest
concentration of bradykinin increased nitrite release from 78 ± 8 to 153 ± 9 pmol/mg in normal hearts, and from 46 ± 4 to
92 ± 6 pmol/mg (all *p < .01) in failing hearts.
Compared with the normal group, nitrite release in response to the
highest concentration of bradykinin was reduced by 46%
(*p < .01) after heart failure. The effects of the
highest concentration of bradykinin on NO production were entirely
abolished by L-NAME and HOE 140 in both
groups (Fig. 2).
Effects of Phosphoramidon, Thiorphan, and Kininogen on NO
Production from Coronary Microvessels in Normal and Failing Dog
Hearts.
Phosphoramidon, thiorphan (108 to
105 mol/liter), and kininogen (0.5-10 µg/ml), all
significantly increased nitrite production in a concentration-dependent
manner in both groups (Fig. 3). However, nitrite release was significantly reduced after heart failure. The
highest concentration of phosphoramidon, thiorphan, and kininogen increased nitrite release from 80 ± 6 to 133 ± 7 pmol/mg,
82 ± 7 to 139 ± 8 pmol/mg, and 81 ± 5 to 181 ± 15 pmol/mg, respectively, in normal hearts, but increased nitrite
production from 52 ± 4 to 100 ± 7 pmol/mg, 53 ± 4 to
96 ± 6 pmol/mg, and 59 ± 3 to 110 ± 8 pmol/mg (all
*p < .01), respectively, in failing hearts.
Compared with the normal group, nitrite production in response to the
highest concentration of these two inhibitors or kininogen was reduced by 25% by phosphramidon, 31% by thiorphan, and 39% by kininogen (all
*p < .01), respectively, after heart failure. The
effect of the highest concentration of phosphoramidon, thiorphan, or kininogen on nitrite production was entirely blocked by
L-NAME, HOE 140, or DCIC in both groups (Fig.
4).
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Effects of Amlodipine on NO Production from Coronary Microvessels
in Normal and Failing Dog Hearts.
Amlodipine (1010
to 104 mol/liter) also caused a significant and
concentration-related increase in nitrite production from coronary microvessels in both normal and failing hearts (Fig.
5). The highest concentration of
amlodipine increased nitrite production from 74 ± 5 to 134 ± 10 pmol/mg in normal hearts, and increased nitrite production from
53 ± 5 to 91 ± 7 pmol/mg (all *p < .01) in failing hearts. Compared with the normal group, nitrite release
in response to the highest concentration of amlodipine was reduced by
31% (*p < .01) after heart failure. The effect of
the highest concentration of amlodipine on nitrite production was
markedly blocked not only by L-NAME, but also by HOE 140 or
DCIC in both groups (Fig. 5).
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Discussion |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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The most significant findings of the current study were that NEP
and ACE inhibitors can significantly increase NO production in isolated
canine coronary microvessels from both normal and failing hearts. These
effects were completely blocked by NO synthase inhibitor
L-NAME, specific B2-kinin receptor
antagonist HOE 140, and kinin-forming enzyme inhibitor DCIC.
These results indicated that both ACE and NEP are important
participants in the modulation of coronary vascular NO production by
blocking bradykinin degradatory enzymes. Similar to inhibition of
ACE, NEP inhibitor-induced NO production is related to the stimulation
of B2-kinin receptor and is dependent on the
activation of NO synthase and local kinin-forming enzymes. Amlodipine
also significantly increased NO production in either normal coronary
microvessels or those from the failing heart, whereas other calcium
channel blockers, nifedipine and diltiazem, did not increase NO release
under similar condition to this study (Zhang et al., 1998b). It is,
therefore, very likely that at least part of the difference between
amlodipine and other calcium channel blockers stems from the ability of
amlodipine to release NO. This effect could be blocked by not only
L-NAME, but also HOE 140 and DCIC, indicating a
kinin-related NO production induced by amlodipine. All of these data
suggest that kinin-mediated coronary NO production may contribute to
the therapeutic actions of these agents that are currently used in the
clinical treatment of heart failure.
The biological effect of neutral endopeptidase (EC 3.4.24.11) has been
recognized for some time. NEP was originally discovered in the kidney
as a brush border enzyme in rabbit (Kerr and Kenny, 1974) and
was later found to be identical with an "enkephalinase" in the
brain (Grafford et al., 1983). NEP is widely distributed among various
organs, fluids, and cells. However, important sources of this enzyme
seem to be in brush border structures, the lungs, and the brain (Erdos
1990, Skidgel 1992). NEP cleaves a variety of peptides in vivo,
including bradykinin, Substance P, and atrial natriuretic factor (ANF)
(Erdos and Skidgel 1989, Skidgel 1992). Because there was an interest
in ANF in the past, a number of studies have focused on the effect of
NEP on the clearance of ANF in the kidney (Erdos and Skidgel, 1989). In
1987, Ura et al. first demonstrated the effect of NEP on the metabolism
of kinins in vitro and in vivo. Until that time, not much attention was paid to the effect of NEP on the regulation of kinin metabolism, although the Km of enkephalin hydrolyzed by
human NEP is only slightly higher than that of bradykinin (Erdos and
Skidgel, 1989). Importantly, Llorens-Cortest et al. (1992) and
Soleilhac et al. (1992) recently identified and characterized
NEP in bovine, porcine, rabbit, and human vascular endothelial cells.
Llorens-Cortest et al. found that 50% of bradykinin hydrolysis in
vascular tissue was due to NEP activity. They concluded that the
endothelium is an important site for the metabolism of circulating
vascular peptides. Graf et al. (1993) found that phosphoramidon, a
potent NEP inhibitor, significantly diminished the breakdown of
bradykinin even without ACE inhibition in the cultured human
endothelial cells. Gafe et al. (1995) also found that NEP is
constitutively expressed in human endothelial cells. The concentration
of NEP in endothelium from coronary microvessels was from 40% to 200 to 300% higher than the endothelium from other organs. In our study,
inhibition of NEP alone with phosphoramidon or thiorphan significantly
increased NO production from isolated coronary microvessels from both
normal and failing hearts, suggesting a distinct effect of NEP on the modulation of coronary microvascular NO production. NO production increased by NEP inhibitors was completely blocked by a
B2-kinin receptor antagonist and a serine
protease inhibitor, clearly indicating an enhanced effect of local kinins.
Recently, considerable interest has been devoted to the development of
dual-acting inhibitors of NEP and ACE (Fink et al., 1995), because
combining inhibition of both enzymes could produce synergistic effects
on improving systemic hemodynamics and maintaining cardiac and renal
function in experimental studies of hypertension and heart failure.
Although the design of dual inhibitors of ACE and NEP was expected to
block the formation of angiotensin II by inhibiting ACE and protect ANF
by inhibiting NEP (Coric et al., 1996), many studies have clearly shown
the enhanced effect of kinins after inhibition of ACE (Schwelk et al.,
1993; Linz et al., 1995; Scholkens, 1996; Zhang et al., 1997) and NEP
(Trippodo et al., 1995a,b; Yang et al., 1997; Zhang et al., 1998a).
Yang et al. (1997) have shown that inhibition of NEP protects the heart against ischemia/reperfusion injury as evidenced by significant reduction of myocardial infarct size. These effects were blocked by the
kinin receptor antagonist HOE 140, but were only slightly attenuated by
ANF antagonist Hs-142-1. They also demonstrated that the
cardioprotective effect of inhibition of NEP is mediated primarily
through a kinin-dependent mechanism rather than by potentiation of
endogenous ANF. Trippodo et al. (1995a,b) also showed that combined
inhibition of ACE and NEP significantly deceased left ventricular end
diastolic pressure and total peripheral resistance and increased
cardiac output compared with the selective inhibition of ACE or NEP
alone in hamsters with heart failure. They suggested that inhibition of
both enzymes could result in a significant potentiation of endogenous
bradykinin and that the bradykinin-mediated enhanced effect of combined
inhibition of ACE and NEP may improve the beneficial effects of ACE
inhibition in the treatment of heart failure. In our study, inhibition
of either of these enzymes significantly increased NO production even
after heart failure. A B2-kinin receptor antagonist and local kinin-forming enzyme inhibitor completely blocked
these effects. Our data provide direct evidence for the possible
contribution of enhanced-kinin activity to at least part of the
beneficial effect of NEP and ACE inhibition in the treatment of heart
failure (Trippodo et al., 1995 a,b).
Amlodipine, a promising second-generation dihydropyridine long
half-life calcium channel antagonist, has been widely used in
antihypertensive and cardioprotective therapy in experimental and
clinical studies. However, the use of this class of drugs is
controversial because calcium antagonists have important additional effects, such as depressing cardiac contractility and activating hormonal systems, in addition to their potent vasodilatory effect (Lliceto, 1997). Treatment with calcium channel blockers may worsen heart failure and increase the risk of death of patients with advanced left ventricular dysfunction ( Messerli, 1996; Packer et al.,
1996; Lliceto 1997). However, a recent clinical trial (PRAISE) (Packer
et al., 1996) demonstrated a favorable effect of amlodipine on the
survival of patients with heart failure resulting from nonischemic
dilated cardiomyopathy. Data from our laboratory and others (Treasure
et al., 1990; Katz et al., 1993; Drexler et al., 1994; Wang et al.,
1994) have shown that NO production substantially decreased after the
development of heart failure, and suggested that endothelial cell
dysfunction may contribute to cardiac deterioration. Our data in this
study also show an impaired NO production in coronary microvessels from
pacing-induced failing heart. Therefore, restoring the ability of
endothelial cells to produce NO production in heart failure may be most
important for improving the hemodynamics and protecting the heart. In
the present study, amlodipine (10-8
M, which is a concentration 15 times lower than the lowest
dose used in the clinical treatment) significantly increased NO
production, clearly showing the ability of amlodipine to release NO in
coronary microvessels from failing heart. This strongly suggests that
at least a portion of the beneficial effect of amlodipine on
cardiovascular diseases could be due to the production of NO. Actually,
a study by Lyons et al. (1994) has also found a NO-related beneficial effect of amlodipine which could normalize endothelial NO production in
humans. They found that chronic treatment of patients with enalaprilat
or amlodipine significantly reduced systolic/diastolic blood pressure.
NG-monomethyl-L-arginine, a potent NO
synthase inhibitor, significantly reduced forearm blood flow in both
enalaprilat- and amlodipine-treated patients, but there was no effect
on the placebo-treated group, clearly implicating that part of the
vasodilatory effect of enalaprilat and amlodipine is NOdependent.
This is puzzling, because endothelial constitutive NO synthase is
calcium/calmodulin dependent (Moncada et al., 1991). If anything,
calcium channel antagonists may impair the activity of NO synthase and
reduce endothelial NO release by affecting endothelial intracellular
calcium. However, Mugge et al. (1991) demonstrated that production of
NO from endothelium was not affected by diltiazem or nifedipine in
cultured bovine aortic endothelial cells. Furthermore, Hashimoto et al.
(1997) and Drummond and Cocks (1996) reported that L-type
calcium channel blockers nifedipine and diltiazem did not affect
bradykinin-induced endothelial increase of intracellular calcium, but
clearly decreased intracellular calcium in vascular smooth muscle. It
seems that there are no L-type calcium channels in vascular
endothelium. Indeed, amlodipine markedly increases coronary blood flow,
decreases myocardial oxygen consumption, and reduces myocardial oxygen
demand with a minimal cardiac depressant effect and minimal activity on
the neurohormonal system (Murdoch and Heel, 1991; Lliceto, 1997).
Perhaps amlodipine protects cardiac function by acting not only as a
calcium channel blocker in vascular smooth muscle, but also an
important NO-releasing agent. This speculation may explain why
amlodipine has a unique beneficial effect when compared with other
drugs of this class in the treatment of heart failure (Packer et
al.,1996).
It is very interesting that the same mechanism that is responsible for ACE and NEP inhibitor-induced NO production, that is, a kinin-dependent mechanism, appears also to be responsible for the ability of amlodipine to release NO because nitrite release induced by amlodipine was significantly reduced by not only L-NAME, but also HOE 140 and DCIC. This supports our hypothesis that amlodipine increases NO production, probably by promoting the effect of kinins. In the present study, both exogenous bradykinin and the precursor of kinins, kininogen, markedly increased coronary NO production in normal and failing heart, indicating that both endogenous and exogenous kinins release NO in coronary microvessels.
In summary, inhibition of ACE or NEP, or amlodipine, all significantly increased nitrite production from isolated canine coronary microvessels. These effects were blocked by the NO synthase inhibitor, B2-kinin receptor antagonist and kinin-forming enzyme inhibitor. NEP inhibitors and amlodipine stimulate NO production from endothelium, most likely by diminishing degradation of local kinins or promoting the effect of local kinin, respectively. The ACE and NEP inhibitors and amlodipine also significantly increased NO release from coronary microvessels after heart failure. However, NO release was markedly decreased, supporting our previous studies indicating endothelium dysfunction. This may partly contribute to the beneficial therapeutic effects of these agents in the treatment of heart failure.
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Footnotes |
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Accepted for publication September 2, 1998.
Received for publication March 16, 1998.
1 These studies were supported by PO-1-HL 43023, HL 50142, HL 18579, and HL 53053 from the National Heart, Lung and Blood Institute and by Fellowship 96-103 from the New York Affiliate of the American Heart Association (to X.Z.).
Send reprint requests to: Thomas H. Hintze, Ph.D., Department of Physiology, New York Medical College, Valhalla, NY 10595. E-mail: Thomas_Hintze-{at}NYMC.edu
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Abbreviations |
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ACE, angiotensin-converting enzyme; NO, nitric oxide; NEP, neutral endopeptidase; PBS, phosphate-buffered saline; L-NAME, Nw-nitro-L-arginine methyl ester; DCIC, dichloroisocoumarin; ANF, atrial natriuretic factor.
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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-thiol dipeptide dual inhibitors of angiotensin-I converting enzyme and neutral endopeptidase EC 3.4.24.11.
J Med Chem
38:
5023-5030[Medline].This article has been cited by other articles:
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