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1 The Hatter Institute, University College London Hospitals and Medical School, London WC1E 6BT, United Kingdom; 2 Institute for Pathophysiology, Universitatsklinikum Essen, Essen 45122, Germany; 3 Department of Biochemistry, University of Szeged, Szeged H-6720, Hungary; and 4 The Royal Veterinary College, London NW1 0TU, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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B-type natriuretic
peptide (BNP) has been reported to be released from the myocardium
during ischemia. We hypothesized that BNP mediates
cardioprotection during ischemia-reperfusion and examined
whether exogenous BNP limits myocardial infarction and the potential
role of ATP-sensitive potassium (KATP) channel opening. Langendorff-perfused rat hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. The control
infarct-to-risk ratio was 44.8 ± 4.4% (means ± SE). BNP
perfused 10 min before ischemia limited infarct size in a
concentration-dependent manner, with maximal protection observed at
10
8 M (infarct-to-risk ratio: 20.1 ± 5.2%,
P < 0.01 vs. control), associated with a 2.5-fold
elevation of myocardial cGMP above the control value. To examine
the role of KATP channel opening, glibenclamide
(106 M), 5-hydroxydecanoate (5-HD; 104 M),
or HMR-1098 (105 M) was coperfused with BNP
(108 M). Protection afforded by BNP was abolished by
glibenclamide or 5-HD but not by HMR-1098, suggesting the involvement
of putative mitochondrial but not sarcolemmal KATP channel
opening. We conclude that natriuretic peptide/cGMP/KATP
channel signaling may constitute an important injury-limiting mechanism
in myocardium.
cGMP; ischemia-reperfusion; infarct size; preconditioning
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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NATRIURETIC PEPTIDES ARE RELEASED from many tissues in response to physiological and pathological stimuli. A-type (atrial) natriuretic peptide (ANP) and B-type (brain or ventricular) natriuretic peptide (BNP) are the predominant natriuretic peptides in mammalian myocardium, stored within secretory granules as propeptides (2, 6, 24). Release of propeptides and the cleaved products in response to dilatation of the cardiac chambers in conditions such as heart failure has been well described. Under such conditions, the classical endocrine actions of ANP and BNP include vasodilatation of some peripheral vascular beds and natriuresis (38, 39). These actions are mediated by elevation of intracellular cGMP after peptide binding to natriuretic peptide receptor type A (NPR-A), a membrane-bound particulate guanylyl cyclase (2, 6, 15, 29).
BNP is the principal natriuretic peptide in ventricular myocardium. Experimental and clinical evidence suggests that brief episodes of ischemia or hypoxia, insufficient to cause alterations in end-diastolic pressure or irreversible tissue injury, can evoke a rapid release of BNP from cardiac tissue. Hypoxic perfusion of isolated hearts led to a rapid increase of BNP immunoreactivity in coronary effluent (35). In patients undergoing percutaneous transluminal coronary angioplasty, coronary sinus BNP concentration increased rapidly after balloon deflation (33), and circulating plasma concentrations of BNP were elevated 4.5-fold in patients after episodes of unstable angina (32). A functional role for the rapid release of BNP in response to brief periods of myocardial ischemia is not known. The recognition in recent years that several neurohormonal mediators are released from myocardium during brief periods of ischemia underpins the current mechanistic model of ischemic preconditioning (1, 8, 28). Autocrine and paracrine mediators acting on G protein-coupled receptors, notably adenosine, bradykinin, opioid peptides, and catecholamines, participate in the activation of a multiple-stage signal transduction pathway. This involves opening of ATP-sensitive potassium (KATP) channels as either a downstream or proximal event essential for conferring resistance to a subsequent episode of ischemia. NPR-A does not couple through G proteins but, through elevation of cGMP, NPR-A activation could modulate KATP channel activity (16). Thus the signal pathway activated by BNP might represent an alternative prosurvival mechanism. To test the hypothesis that BNP is cytoprotective during ischemia-reperfusion through the opening of KATP channels, we examined the ability of exogenous BNP to limit irreversible myocardial injury, defining the involvement of KATP channel opening using pharmacological blockers of KATP channels.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Male Sprague-Dawley rats (300-400 g) were used for these studies. Animals were treated in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. Rat BNP1-32 (hereinafter referred to as BNP), glibenclamide, sodium 5-hydroxydecanoate (5-HD), 8-bromo-cGMP (8-Br-cGMP), and triphenyltetrazolium chloride were from Sigma (Poole, UK). HMR-1098 was a gift of Aventis Pharma. All other reagents were of analytic standard.
Isolated Heart Preparation
Rats were anesthetized with pentobarbital sodium (50 mg/kg ip). Heparin (1 IU/g) was administered concomitantly. Excized hearts were perfused retrogradely through the aorta at 11.3 kPa with Krebs-Henseleit buffer [containing (in mmol/l) 118 NaCl, 25 NaHCO3, 11 glucose, 4.7 KCl, 1.2 MgSO4 · 7H2O, 1.2 KH2PO4, and 1.8 CaCl2 · 2H2O; aerated with carbogen, pH 7.3-7.5, at 37°C]. Coronary flow rate (CFR) was determined by timed collection of the coronary effluent. A saline-filled latex balloon connected to a pressure transducer was inserted into the left ventricle (LV), and baseline end-diastolic pressure was set at 5-10 mmHg. Heart rate, LV end-diastolic pressure, and developed pressures were recorded continuously.Infarct Size Evaluation
A 4-0 silk suture was positioned around the left main coronary artery and threaded through a plastic snare to permit reversible occlusion of the coronary artery. Coronary occlusion was induced for 35 min by clamping the snare onto the heart. Reperfusion was achieved by releasing the snare. At the end of 120-min reperfusion, the coronary artery was reoccluded, and the risk zone was delineated with Evans' blue. Hearts were sectioned (2 mm) and incubated in 1% triphenyltetrazolium chloride in phosphate buffer (pH 7.4, 37°C) for 15 min to define white necrotic tissue when fixed in 10% formalin for 24 h. Images of the sections were drawn by an operator blinded to the experimental treatment. Risk zone areas and infarct-to-risk ratios were determined by computerized planimetry (Planimetry+ version 1.0 for Windows).Infarction Protocols
The experimental protocols for the three separate infarction studies are illustrated in Fig. 1.
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Study 1: BNP concentration-response study.
In the control group, hearts were stabilized for 15-20 min and
then subjected to 35 min of regional ischemia, followed by 120 min of reperfusion. BNP (1012-108
mol/l) was added to the Krebs-Henseleit buffer, and perfusion was
started 10 min before ischemia and continued until 30-min reperfusion.
Study 2: KATP channel blockade study.
BNP (108 mol/l) was selected after the experiments
described above. Hearts were randomized to one of the following
experimental groups (Fig. 1): 1) Control group, as described
above. Six control hearts were perfused with 0.016% DMSO (the vehicle
for glibenclamide). Because there was no effect on infarct size of
DMSO, these hearts were combined for statistical evaluation with
non-DMSO-treated control hearts to comprise group 1. 2) BNP (108 mol/l) treatment, as described
above. 3) 5-HD (104 mol/l), a blocker of
mitochondrial KATP channels (9, 36), was
perfused 10 min before ischemia and continued until 30-min reperfusion. 4) 5-HD (104 mol/l) + BNP
(108 mol/l), coperfused as described above. 5)
Glibenclamide (106 mol/l), a nonselective blocker of
sarcolemmal KATP and mitochondrial KATP
channels (7, 17, 23, 37), was perfused 10 min before ischemia and continued until 30-min reperfusion. Glibenclamide was dissolved in DMSO (final concentration not more than 0.016%). 6) Glibenclamide (106 mol/l) + BNP
(108 mol/l), coperfused as described above. 7)
HMR-1098 (105 mol/l), a selective blocker of sarcolemmal
KATP channels (14, 21), was perfused 10 min
before ischemia and continued until 30-min reperfusion.
8) HMR-1098 (105 mol/l) + BNP
(108 mol/l), coperfused as described above.
Study 3: effects of 8-Br-cGMP.
In the third infarct study, 8-Br-cGMP, a cell-permeable analog of cGMP,
was perfused at 108-105 mol/l,
commencing 10 min before ischemia and continued until 30-min
reperfusion. A control group was subjected to coronary artery occlusion
and reperfusion only, as described above.
Myocardial cGMP Concentration
After stabilization, hearts were perfused for 10 min with BNP (1012-108 mol/l) as described above.
Ventricular myocardium was then rapidly freeze-clamped to the
temperature of liquid nitrogen. After extraction with trichloroacetic
acid, the tissue cGMP concentration was determined by radioimmunoassay
as described previously (5).
Endogenous BNP Release After Ischemia
After stabilization, during which baseline samples were collected, hearts were rendered globally ischemic for 2, 5, or 20 min. Flow was reinstituted, and the coronary effluent was sampled during reflow. Control hearts were perfused without ischemia. The ventricular tissue was immediately frozen in liquid N2. Coronary effluent and ventricular tissue samples were analysed for BNP immunoreactivity by radioimmunassay using a commercially available kit [RIK 9085 BNP-32 (rat), Peninsula Laboratories; San Carlos, CA]. Tissue was extracted with trifluoroacetic acid. The peptide was purified on C18 columns, freeze-dried, and redissolved in "RIA buffer." RIA buffer concentrate was added to the samples of coronary effluent, and aliquots were used for the assay. On the basis of the competition of 125I-labeled BNP and unlabeled BNP binding to a limited amount of specific antibodies, a standard curve was constructed from which the concentration of BNP in the samples was determined.Statistical Analysis
Data are expressed as means ± SE. Infarct-to-risk ratios, risk zone volumes, and BNP tissue concentrations were analyzed using one-way ANOVA and Fisher's protected least-significant-difference post hoc test. LV function parameters, CFR, and BNP coronary effluent concentrations were evaluated using repeated-measures ANOVA with Bonferroni's post hoc test. Statistical significance between group means was defined as P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Technical Exclusions
A total of 217 animals was used. For the concentration-response infarct experiments, 65 hearts were used, of which 5 hearts were excluded: one was damaged by instrumentation, one had failure of the tetrazolium stain, and three had persistent bradyarrhythmia in the stabilization phase. In the second series of experiments, 55 hearts were used, of which 5 hearts were excluded: two failed to reperfuse, one had an instrumentation error that prevented precise LV function assessment, one had failure of the tetrazolium stain, and one had persistent bradyarrhythmia throughout reperfusion. In the third infarct series, 46 hearts were successfully perfused without exclusion. Therefore, we report the data for 156 successfully completed infarct experiments. An additional 36 hearts were used to examine tissue cGMP concentration, and 15 hearts were used to study the release of endogenous BNP.Infarct Study 1: BNP Concentration-Response Study
The risk zone volumes were similar among all the groups (Table 1). The control infarct-to-risk zone ratio was 44.8 ± 4.4% without BNP treatment, consistent with previous results (20). Treatment with BNP limited infarct size in a concentration-dependent manner (Fig. 2). Significant limitation of infarction was observed with BNP (1010, 109, and
108 mol/l). The highest BNP concentration studied
(108 mol/l) resulted in the smallest infarct size
(20.1 ± 5.2%, P < 0.01 vs. control).
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Preischemic global coronary flow rate averaged 12.5 ml/min
among the six experimental groups. After coronary artery occlusion, there was a decrease in the global CFR of ~35% and a recovery to
preischemic values immediately after reperfusion with gradual "rundown" during the remaining 120-min of
perfusion (Fig. 3A). The
global CFR measured at intervals throughout the protocol did not differ
substantially among the experimental groups. There were no detectable
differences among the groups in any of the parameters of LV function
measured (spontaneous heart rate, developed pressure, or the
rate-pressure product; data not presented). Developed pressure and
rate-pressure product declined immediately after the onset of coronary
occlusion to the same extent in all experimental groups.
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Infarct Study 2: KATP Channel Blockade Study
In the second series of infarct experiments, BNP was applied at a concentration of 108 mol/l and KATP channel
blockers were coperfused with BNP.
The risk zone volume did not differ among the groups (Table 1). The
control infarct-to-risk ratio was 47.1 ± 2.8% (Fig.
4). BNP (108 mol/l)
treatment resulted in a significant limitation of infarct size
(21.3 ± 2.8%, P < 0.01 vs. control).
Coperfusion of BNP with either 5-HD or glibenclamide resulted in
abolition of the protective effect of BNP (infarct-to-risk ratio:
52.3 ± 5.7% and 41.0 ± 7.1%, respectively,
P = not significant vs. control). However, the infarct size limitation with BNP was not abolished by HMR-1098 (infarct-to-risk ratio: 14.7 ± 2.7%, P < 0.01 vs. control and
P = not significant vs. BNP). None of the
KATP channel blockers per se influenced infarct size.
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As in study 1, the global CFR and LV function were not altered by BNP perfusion or any of the KATP channel blockers (Fig. 3B).
Infarct Study 3: 8-Br-cGMP Study
In the third series of experiments, 8-Br-cGMP was applied across a range of concentrations to examine the role of receptor-independent elevation of intracellular cGMP in myocardial responses to ischemia-reperfusion. Risk zone volume was similar among the experimental groups, and the control infarct-to-risk ratio was 38.7 ± 3.6%. We observed a paradoxical inverse concentration response with increasing concentrations of 8-Br-cGMP (Fig. 5). Statistically significant limitation of infarction was observed with the lowest concentrations examined (107 mol/l: 23.1 ± 6.3%, P < 0.05 vs. control; 108 mol/l: 20.8 ± 4.2%,
P < 0.05 vs. control). However, treatment with higher
concentrations did not result in a significant limitation of
infarction.
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Perfusion with 8-Br-cGMP at 105 and 106
mol/l resulted in a nonsignificant increase in the preischemic
CFR (Fig. 3C; 16.0 ± 1.0 ml/min in the control group
vs. 19.5 ± 1.1 and 20.3 ± 1.1 ml/min in the
105 and 106 mol/l 8-Br-cGMP-treated groups,
respectively, P = not significant). These
influences of 8-Br-cGMP were not observed with the lower, infarct-limiting concentrations of the agent. No consistent patterns of
8-Br-cGMP treatment on LV contractility were observed during the
perfusion protocol.
Effect of BNP on Myocardial cGMP Concentration
Perfusion with BNP for 10 min caused a concentration-dependent increase of the cGMP concentration in ventricular myocardium (Fig. 6). In control tissue, at the time point corresponding to the onset of myocardial ischemia, the cGMP concentration was 11.6 ± 0.5 pmol/g wet wt (n = 9). Significant increases in the tissue cGMP concentration were observed after perfusion with 109 mol/l BNP (cGMP:
20.3 ± 5.2 pmol/g wet wt, n = 5, P < 0.05) and 108 mol/l BNP (cGMP:
28.6 ± 1.5 pmol/g wet wt, n = 8, P < 0.01). Lower concentrations of BNP did not cause
statistically significant elevation of the myocardial cGMP
concentration.
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BNP Release and Ventricular Myocardial BNP Concentrations
We observed a low basal release of BNP into the coronary effluent in the range of 1.1-9.7 pmol/l (see Fig. 7). In the control group (no ischemia), this efflux of BNP remained stable throughout 35 min of perfusion. Peak postischemic BNP concentrations in the coronary effluent were related to the duration of preceding ischemia: 2-min ischemia, 11.0 ± 2.4 pmol/l; 5-min ischemia, 20.1 ± 2.2 pmol/l; and 20-min ischemia, 41.5 ± 3.3 pmol/l (all P < 0.05 vs. control values; Fig. 7). Global ischemia for 2 and 5 min was not associated with changes in end-diastolic pressure above baseline. Hearts subjected to 20-min ischemia displayed an increase in end-diastolic pressure of ~10 mmHg above baseline values. Myocardial BNP concentration was 1.58 ± 0.16 pmol/g wet wt in control hearts and 1.77 ± 0.16, 3.69 ± 0.65 (P < 0.05 vs. control), and 1.82 ± 0.14 pmol/g wet wt, respectively, in hearts subjected to 2-, 5-, or 20-min global ischemia with 5-min reperfusion (n = 3-4 hearts/group).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present investigation provides two principal new findings. First, acute infusion of exogenous BNP is markedly protective against myocardial ischemia-reperfusion injury, leading to concentration-dependent infarct size limitation. Second, the mechanism of protection afforded by BNP is associated with elevation of cGMP and appears to involve KATP channel opening. The pharmacological selectivities of the widely used KATP channel blockers, applied at conventional inhibitory concentrations, may indicate involvement of the mitochondrial KATP channel rather than the sarcolemmal KATP channel. Although recent studies have reported that ANP (30) and the related noncardiac peptide urodilatin (27) limit infarct size in vivo, this is the first study to show the cardioprotective effect of BNP and to provide evidence of a primary mechanism of action of a natriuretic peptide on myocardium mediated by KATP channel activation.
The cell surface receptor mediating the biological actions of BNP is a
particulate guanylyl cyclase A receptor, NPR-A, abundantly expressed in
cardiac tissue (2, 29). Unlike receptors for adenosine,
bradykinin, and opioids, the NPR-A receptor is not G protein coupled.
This receptor contains an intracellular guanylyl cyclase catalytic
domain that mediates most of the biological action of the natriuretic
peptide through the conversion of GTP to cGMP (4, 13, 20).
We observed significant increases in the ventricular cGMP concentration
after 10-min perfusion with BNP (109 or 108
mol/l). Although this observation is consistent with NPR-A activation, we are unable to conclude at present that the infarct-limiting action
of BNP in ischemic myocardium is a receptor-mediated action. Unfortunately, reliable NPR-A inhibitors such as HS-142-1 and A71915
are not available in sufficient quantity to undertake perfused heart or
in vivo studies. Approaches using cultured cardiac myocytes may be
feasible but may not accurately model endogenous protective mechanisms
in intact tissue where there are interactions among several cell types
and humoral mediators. Alternative approaches using mutant mouse
strains with targeted deletion of either pro-BNP or NPR-A genes may
also be possible in future studies. However, interpretation of findings
from such animals, in the absence of complementary pharmacological
data, may be clouded by the uncertainties of altered expression of
other gene products, high levels of redundancy in signaling pathways,
and the spontaneous development of cardiac pathology in these animals.
For example, Izumi et al. (12 ) recently reported that
mice with targeted deletion of the NPR-A gene sustained infarcts that
were 20% smaller than wild-type control animals. However, the mutant
animals had a substantial degree of LV hypertrophy. Moreover,
interpretation of this study is predicated on the observation that
natriuretic peptides upregulated vascular adhesion molecule expression
in vitro, an effect that has not been demonstrated in humans or in
intact animal models.
Our observation that the synthetic cGMP analog 8-Br-cGMP evoked, at low
concentrations, an infarct-limiting effect similar to that observed
with BNP is consistent with the notion that elevation of intracellular
cGMP may indeed be a mechanism that is central to the cardioprotective
action of BNP. cGMP elevation has been proposed to be a mechanism of
injury limitation in ischemic myocardium (26), but
the distal molecular mechanisms resulting in enhanced tolerance to
ischemic injury associated with cGMP elevation are unclear.
Proposed mechanisms include inhibition of L-type calcium channel
opening (18), decreased intracellular concentrations of
cAMP through a feedback mechanism and stimulation of cAMP
phosphodiesterase (11, 18, 22), inhibition of the
mitochondrial permeability transition pore (31), and
opening of KATP channels (16). However, the
unexpected finding that higher concentrations of 8-Br-cGMP (similar to
those frequently applied in isolated cell and tissue pharmacology) were
not protective is intriguing and unexplained at present. Previous work
has reported that high concentrations of cGMP are associated with cell
injury. For example, Nakamura et al. (25) reported that
the cytotoxic effects of a NO donor in a phaeocromocytoma line were
augmented by a cell-permeable cGMP analog. A role of cGMP and
cGMP-dependent protein kinase (cGK) in mediating apoptosis of
pancreatic 
-cells has been reported (19). Tepperman et
al. (34) extended these observations to rat intestinal
epithelial cells, showing that dibutyryl cCMP at millimolar
concentrations reduced cell viability in culture. This cytotoxic effect
of high intracellular concentrations of cGMP may be related to the
generation of reactive oxygen species, because superoxide dismutase
attenuated the injurious effects of the cGMP analog in intestinal
epithelial cells. Thus our apparently paradoxical observation that
increasing concentrations of 8-Br-cGMP were not associated with infarct
limitation, whereas low concentrations were protective, may reflect an
ambivalent effect of cGMP, both prosurvival and proinjury effects being
mediated by this second messenger depending on concentration and
pathophysiological context.
In myocardium, sarcolemmal KATP channels were originally postulated to participate in salvage from irreversible ischemia-reperfusion injury, because their opening would produce an increase in the outward potassium current leading to shortening of action potential duration, which would in turn reduce the Ca2+ influx through voltage-dependent Ca2+ channels and increase the time during which the Na+/Ca2+ exchanger would operate to extrude Ca2+ from the cell. Since 1998, attention has focused on mitochondrial KATP channels in both ischemic preconditioning and pharmacological preconditioning studies (28). Much of the evidence implicating a role of mitochondrial KATP channels is reliant on the reputed selectivity of pharmacological agents such as 5-HD (a blocker of mitochondrial KATP channels) and HMR-1098 (a blocker of sarcolemmal KATP channels). With the caution that pharmacological specificity and selectivity may be subject to revision, our study provides pharmacological evidence for involvement of a KATP channel subtype, possibly a mitochondrial KATP channel, in the infarct-limiting action of BNP. Further studies in appropriate isolated cell and mitochondrial preparations using biophysical approaches are now being planned to probe the specific involvement of mitochondrial KATP channel opening.
While not constituting proof of mechanism, the association between concentration-dependent elevation of cGMP by BNP and infarct limitation that we observed leads us to hypothesize a role for cGK-I (protein kinase G). The cGMP/cGK-I pathway could promote KATP channel opening, representing an alternative signal cascade to the widely studied G protein receptor-coupled-protein kinase C pathway. Indeed, recent studies support the concept that KATP channel activation may be promoted by cGK in a variety of cell types, including ventricular myocytes (10). The contribution of cGK signaling to mitochondrial KATP channel activation and infarct limitation after BNP treatment will be the subject of further studies, using mice with targeted deletion of cardiac cGK-I.
BNP is a vasodilator in several vascular beds including coronary
epicardial conductance arteries and coronary microvessels (3,
40) A surprising finding in our studies was the absence of any
gross alterations in the global CFR secondary to BNP-mediated coronary
vasodilatation. It likely that a more consistent and marked effect
would be observed at higher concentrations than those used in our
studies. The highest concentration of BNP we used (108
mol/l) is at the threshold for vasorelaxation in rat aortic rings (13). At present, we must conclude that the protective
effect of BNP on ischemic myocardium is apparently independent
of coronary vasodilatation or collateral vessel recruitment, because
the rat heart is devoid of native coronary collateral vessels. It is of interest that in a previous study examining the coronary vasodilator mechanisms of ANP in a constant flow preparation, ANP reduced coronary
perfusion pressure, an effect sensitive to inhibition by
N
-nitro-L-arginine methyl ester
(40). The possibility that BNP-associated cardioprotection may be related, at least in part, to NO generation and
activation of soluble guanylyl cyclase is currently being investigated
in our laboratory.
We observed that postischemic release of endogenous BNP increased in a graded fashion with ischemia severity. Moreover, the increase of tissue BNP after 2- and 5-min ischemia likely reflects cleavage of the stored propeptide in response to ischemia; after a 20-min ischemic stimulus, tissue levels of BNP were reduced as a consequence of massive release of the peptide. The immediate stimulus to BNP release could be either ischemia per se or local tissue deformation as a result of ischemia. At present, we are unable to comment on this except to say that we observed graded release of BNP after ischemia that was not associated with substantial changes in end-diastolic pressure. It is impossible to directly relate the concentrations of BNP in coronary effluent to the concentrations required to protect against infarction. Although the coronary effluent concentrations were two to three orders of magnitude less than the protective concentrations infused, local interstitial concentration during ischemia, when there is no flow and thus no washout, would be considerably higher than that detected in the coronary effluent during reflow. Thus changes in the coronary effluent concentrations reflect changes in the interstitial concentrations but cannot predict the interstitial concentrations.
In conclusion, this study is the first to demonstrate a cardioprotective effect of exogenous BNP against ischemia-reperfusion injury. The abrogation of this protective effect by glibenclamide and 5-HD, but not by HMR-1098, is consistent with, but does not constitute proof of, a mechanism involving opening of the putative mitochondrial KATP channel. Although we postulate that elevation of cGMP with activation of cGK-I is a plausible mechanism of KATP channel opening, the signaling pathway underlying this newly defined action of BNP requires further elucidation, as does the involvement of NPR-A activation. Further studies in vivo and in other species are indicated to elucidate fully the cytoprotective potential of BNP and cGMP signaling in myocardial ischemia-reperfusion, especially the therapeutic application of recombinant BNP and inhibitors of neutral endopeptidase, the major enzymatic route for BNP degradation.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the support of British Heart Foundation Programme Grant RG/99002 and a cooperative award (to P. Ferdinandy and G. F. Baxter) from the Hungarian Ministry of Science and Technology and the British Council. S. P. D'Souza was supported by a grant from the Wellington Hospital Trust.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. F. Baxter, Dept. of Basic Sciences, The Royal Veterinary College, Univ. of London, Royal College St., London NW1 0TU, UK (E-mail: gfbaxter{at}rvc.ac.uk).
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. Section 1734 solely to indicate this fact.
First published January 9, 2003;10.1152/ajpheart.00902.2002
Received 17 October 2002; accepted in final form 6 January 2003.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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P < 0.01 vs. HMR-1098 control (one-way ANOVA).
; n = 3), after 2-min ischemia
(
; n = 4), 5-min ischemia
(
; n = 4), or 20-min ischemia
(
; n = 4). * P < 0.05 vs.
the corresponding control value (repeated-measures ANOVA).