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Servicio de Cardiología, Hospital General Universitario Vall d'Hebron, 08035 Barcelona, Spain
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The objective of this study was to investigate the effect of L-arginine supplementation on myocardial cell death secondary to hypoxia-reoxygenation. Isolated rat hearts (n = 51) subjected to 40 min of hypoxia and 90 min of reoxygenation received 3 mM L-arginine and/or 1 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a selective inhibitor of soluble guanylyl cyclase) throughout the experiment or during the equilibration, hypoxia, or reoxygenation periods. The incorporation of L-[3H]arginine into myocytes during energy deprivation was investigated in isolated adult rat myocytes. The addition of L-arginine to the perfusate throughout the experiment resulted in higher cGMP release (P < 0.05), reduced lactate dehydrogenase release (P < 0.05), and increased pressure-rate product (P < 0.05) during reoxygenation. These effects were reproduced when L-arginine was added only during equilibration, but addition of L-arginine during hypoxia or reoxygenation had no effect. Addition of ODQ either throughout the experiment or only during reoxygenation reversed the beneficial effects of L-arginine. L-[3H]arginine was not significantly incorporated into isolated myocytes subjected to energy deprivation. We conclude that L-arginine supplementation protects the myocardium against reoxygenation injury by cGMP-mediated actions. To be effective during reoxygenation, L-arginine must be added before anoxia.
isolated rat heart; reperfusion; contractile function; nitric oxide; guanylyl cyclase inhibition; myocytes; uptake
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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) is a free radical endogenously produced by a variety of mammalian cells and shown to be a ubiquitous signal transduction molecule. NO plays an important role in blood pressure regulation, vascular tone, neural signaling, and immunological function. It is synthesized by constitutive (I and III) and inducible (II) NO synthases (NOS), enzymes that use L-arginine and molecular oxygen as substrates.
It has been suggested that alterations in NO homeostasis may play a
role in the genesis of myocardial injury secondary to ischemia-reperfusion. NO is a highly reactive molecule and may result in the formation of harmful free radicals when it reacts with
O
2 (14, 26). In some studies,
myocardial NO synthesis has been found to be enhanced during
ischemia-reperfusion (40, 6), and NOS inhibitors have been
found to decrease functional impairment of tissue after
ischemia-reperfusion (7, 23, 37, 38).
However, other studies suggest a decrease in endothelium-derived
relaxing factor-mediated effects in reperfused myocardium (9, 33) and a
protective effect of NO (2, 3, 8, 9, 20, 24, 36). Administration of NO
donors or of the NOS substrate
L-arginine has been shown in
most (although not all) studies to improve functional recovery of
reperfused myocardium (2, 3, 8, 9, 33). This beneficial effect has been explained as a consequence of the inhibiting actions of NO on vascular
constriction and neutrophil and platelet adherence (2, 36) and to
attenuation of the deleterious free radical actions of
O2 (20). On the other hand, NOS itself can be an important site of O2
synthesis when substrate availability is limited (13, 14). A decrease
in NO concentration in the capillary blood has been recently documented in rabbit skeletal muscle at the end of ischemia and during
reperfusion, concomitantly with a symmetrical increase in
O2 concentration (16). Both changes
were attenuated by administration of
L-arginine, suggesting that
insufficient substrate availability during
ischemia-reperfusion may result in a switch of
constitutive NOS enzymes to synthesize
O2 (16).
In addition to reducing the concentration of
O2 and increasing the quench of
oxygen-derived free radicals by NO,
L-arginine supplementation may
increase cGMP synthesis. In contrast to the large number of studies
analyzing the effects of NO on ischemia-reperfusion injury,
data on the role of cGMP in the genesis of these effects are scant,
probably because selective inhibitors of NO-dependent cGMP synthesis
have not been available until very recently. The potentially favorable
cGMP-mediated actions of NO on vascular tone and neutrophil and
platelet adhesion are well recognized (4, 21). However, the effects of
increased cGMP on cardiomyocytes have received much less attention.
cGMP has been found to reduce myofilament responsiveness to
Ca2+ via activation of a
cGMP-dependent protein kinase (25, 29). During reoxygenation,
restoration of cell energy in the presence of elevated cytosolic
Ca2+ concentration may result in
hypercontracture and cell death, and interventions inducing a transient
reduction of myofilament responsiveness to
Ca2+ during the initial phase of
reoxygenation or reperfusion may prevent hypercontracture and limit
myocardial cell death (10, 31). Increasing cytosolic cGMP concentration
by NO-independent stimulation of particulate guanylyl cyclase was shown
recently to prevent reoxygenation-induced hypercontracture (15).
The objective of this study was to test the hypothesis that L-arginine supplementation limits cell death caused by hypoxia-reoxygenation through cGMP-mediated actions of NO. For this purpose, a novel and specific inhibitor of the soluble guanylyl cyclase was used.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolated, Perfused Heart
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and experimental procedures were approved by the Research Commission on Ethics of the Hospital Vall d'Hebron, Barcelona, Spain.Adult male Sprague-Dawley rats, weighing 300-350 g, were anesthetized with an intraperitoneal injection of thiopental sodium (200 mg/kg). The hearts were excised and immediately arrested in ice-cold saline solution. The aorta was quickly cannulated, and the heart was perfused with a Krebs-Henseleit bicarbonate buffer (KHB) at 37°C using a nonrecirculating Langendorff apparatus at a constant perfusion pressure of 60 mmHg. The composition of KHB was as follows (in mM): 140 NaCl, 24 NaHCO3, 2.7 KCl, 0.4 KH2PO4, 1 MgSO4, 1.8 CaCl2, and 5 glucose. KHB was filtered through a 0.45-µm cellulose filter to remove any particulate matter and continuously gassed with 95% O2-5% CO2.
Left ventricular pressure was monitored by means of a water-filled latex balloon inserted through the left atrium and into the left ventricle. The balloon was fixed in the tip of a Cordis 5-F catheter (Cordis, Miami, FL) and connected to a pressure transducer (43600 F, Baxter). At the beginning of the experiment the left ventricular end-diastolic pressure (LVEDP) was set between 8 and 12 mmHg by adjusting the filling of the balloon. The signal obtained was digitized and recorded continuously on hard disk with the aid of ad hoc developed software. Left ventricular developed pressure (LVDP) was calculated as the difference between left ventricular peak systolic pressure and LVEDP.
Experimental Protocol and Groups of Treatment
Five hearts were perfused for 160 min under normoxic conditions (normoxic controls). Hearts to be submitted to hypoxia-reoxygenation (n = 51) were previously stabilized for 30 min, followed by 40 min of hypoxic perfusion and 90 min of reoxygenation. Hypoxic perfusion was performed with the same buffer without glucose and gassed with 95% Ar-5% CO2. In a first series of experiments hearts were allocated to one of four groups receiving the following treatments: none (control, n = 12); 3 mM L-arginine (Sigma Chemical, St. Louis, MO) (n = 12); 3 mM L-arginine plus 1 µM 1H-[1,2,4]oxadiazolo4,3,-a]quinoxalin-1-one (ODQ; Alexis, San Diego, CA), a selective inhibitor of soluble guanylyl cyclase (12) (n = 5); and 1 µM ODQ (n = 5). In all groups, drugs were added to the KHB 10 min before hypoxia and maintained throughout the rest of the experiment. Before dilution in buffer L-arginine was neutralized with hydrochloric acid and ODQ was dissolved in dimethyl sulfoxide. Final concentrations of dimethyl sulfoxide in the KHB (0.025%) had no effect on any of the parameters measured. To determine at what moment of the hypoxia-reoxygenation protocol L-arginine exerts its protective actions, a second series of experiments were performed in which hearts received L-arginine only during the equilibration period (n = 7), the hypoxic period (n = 4), or the reoxygenation period (n = 2). Finally, another group of hearts received 3 mM L-arginine throughout the experiment plus ODQ during the last 5 min of hypoxia and all through the reoxygenation period (n = 4).Lactate Dehydrogenase and cGMP Release
The coronary effluent was collected at different times to measure lactate dehydrogenase (LDH) activity and cGMP. LDH was determined in samples collected during the reoxygenation at 1, 2, 3, 4, 6, 8, 10, 12, 15, and 20 min and then every 10 min and spectrophotometrically assayed as described previously (17). Results of LDH release were expressed as units of LDH activity released during the first 30 min of reoxygenation per gram of dry weight. To measure cGMP, samples (9 ml) of the coronary effluent were collected at 25 min of the equilibration period, at 5 and 35 min of the hypoxic period, and at 2, 15, 30, and 90 min of the reoxygenation period and rapidly frozen in liquid N2. In normoxic controls the coronary effluent was collected at 30, 60, 120, and 160 min of perfusion. Samples were boiled for 10 min and centrifuged (1,250g × 10 min), and the supernatant was lyophilized. cGMP was determined in concentrated samples by radioimmunoassay using acetylated [3H]cGMP as previously described (1).Uptake of L-Arginine in Isolated Cardiomyocytes
Ventricular cardiomyocytes were isolated from adult Sprague-Dawley rat hearts as previously described (27). Whole hearts were retrogradely perfused for 20 min with a buffer containing (in mM) 110 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11 glucose (pH 7.4) and 0.03% collagenase. Dissociated tissue was filtered and centrifuged, and the pellet was subjected to a progressive normalization of Ca2+ levels to 1 mM. Rod-shaped cells were selected by gravity sedimentation and plated in medium 199 (Sigma Chemical) with 4% fetal calf serum in preincubated Falcon dishes.Cultures were washed twice with HEPES-buffered saline (in mM: 140 NaCl, 3.6 KCl, 1.2 MgSO4, 1 CaCl2, and 20 HEPES, pH 7.4) and incubated in the same buffer plus 5 mM glucose (control dishes) or plus 4 mM sodium cyanide (dishes with energy deprivation) for 1 h. L-[3H]arginine (50 µM) then was added to the cell cultures. The reaction of L-arginine incorporation was terminated at different times by rapid washing with cold HEPES-buffered saline and addition of 0.3 M perchloric acid. L-[3H]arginine in the intracellular extracts was quantified by liquid scintillation spectrometry. Uptake of L-[3H]arginine was linear for up to 5 min and was expressed as picomoles of total L-arginine incorporated per minute and per milligram of protein in this period of time.
Data Analysis and Statistics
Statistical analysis was performed by using commercially available software (Instat, GraphPad Software). Differences between groups were assessed by means of one-way analysis of variance. Individual comparisons between groups were performed by using the Student-Newman-Keuls test. A critical P value of 0.05 was used. Values are expressed as means ± SE.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Normoxic Hearts
Hemodynamic parameters were measured in hearts submitted to 160 min of perfusion with a normoxic solution. LVEDP, LVDP, heart rate, and pressure-rate product did not change significantly throughout the perfusion, whereas coronary flow showed a significant decrease (from 9.3 to 7.1 ml/min) (Table 1). cGMP release to the perfusion medium showed a nonsignificant trend toward decrease during perfusion (at 160 min of perfusion cGMP release was 82 ± 11% of that observed at the end of the equilibration period). There was no measurable LDH release during continuous normoxic perfusion.
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Administration of L-Arginine to Hearts Subjected to Hypoxia-Reoxygenation
Contractile function.
In a first series of experiments hearts received
L-arginine, ODQ, both, or
neither during the whole experimental protocol (equilibration, hypoxia,
and reoxygenation periods). At the end of the equilibration period,
LVEDP, LVDP, and heart rate were 9.0 ± 0.8 mmHg, 83 ± 5 mmHg,
and 279 ± 14 beats/min, respectively, without differences between
groups. During hypoxia, LVEDP increased steeply, with a peak of 184 mmHg 3.4 min after the onset of hypoxic perfusion. Reoxygenation
induced a second increase in LVEDP, with a peak of 140 mmHg 2.2 min
after its onset (Fig. 1). Changes in LVEDP
were virtually identical in the four groups. Pressure-rate product
(LVDP × heart rate) fell to zero in all four groups because of
total abolition of LVDP at the beginning of the hypoxic period, with
virtually no recovery during reoxygenation in the control group (Fig.
2). However, in hearts receiving
L-arginine pressure-rate product
showed a significant recovery (Fig. 2) that was abolished when ODQ was
included together with
L-arginine. Pressure recordings from hearts receiving ODQ alone were not different from those in the
control group.
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Coronary flow.
Coronary flow increased during the first minute of hypoxic perfusion
and then decreased to values below those obtained before hypoxia (Fig.
3). Reoxygenation did not induce any
significant change in coronary flow. No intergroup differences were
observed.
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LDH release.
There was no measurable LDH release during equilibration and hypoxic
perfusion. During reoxygenation LDH activity in the effluent followed a
characteristic pattern with an early peak 2 min after the onset of
reoxygenation, followed by a rapid decay (Fig.
4). LDH release during the first 30 min of
reoxygenation was reduced in hearts receiving
L-arginine compared with
controls, and this effect was reversed by ODQ treatment (Fig. 4,
inset). ODQ alone had no effect.
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cGMP release.
In control hearts cGMP release was 350 ± 17 fmol/min at the end of
the equilibration period. Hypoxia induced a marked and progressive
decrease in cGMP release into the perfusion medium, and at the end of
the hypoxic period cGMP release was ~30% of the value measured
before hypoxia (Fig. 5). The decrease in
cGMP was not modified by reoxygenation and persisted until the end of
the experiment.
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Critical Timing of L-Arginine Supplementation
In a second series of experiments L-arginine was added to the perfusate during only one of the three phases of the perfusion protocol. When added during hypoxia or reoxygenation L-arginine had no effect on functional recovery (Fig. 6A) or LDH release (Fig. 6B) during reoxygenation. However, addition of L-arginine during the 10 min before hypoxia afforded the same protective effect on recovery of pressure-rate product and LDH release than when it was added all throughout the experiment (Fig. 6, A and B). This protective effect was associated with a significant increase of cGMP release at the end of hypoxia (Fig. 6C). Addition of L-arginine to the perfusate only during hypoxia had no effect on cGMP release.
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Finally, in a series of experiments, hearts were supplemented with L-arginine throughout the experiment and treated with ODQ only for the last 5 min of hypoxia and during reoxygenation. ODQ added during this period was able to inhibit 80 ± 12% (P < 0.05) of the functional recovery and 82 ± 8% (P < 0.05) of the reduction in LDH release evoked by L-arginine (in comparison with 91 ± 9 and 63 ± 5% of inhibition by ODQ treatment throughout the experiment of functional recovery and LDH reduction, respectively).
Uptake of L-Arginine in Isolated Cardiomyocytes
During normoxic conditions, uptake of L-arginine by isolated cardiomyocytes was 43.9 ± 6.3 pmol · min1 · mg
protein1.
L-Arginine uptake was markedly
reduced in isolated myocytes submitted to energy deprivation (7.8 ± 6.9 pmol · min1 · mg
protein1,
P < 0.05 with respect to normoxic controls).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study demonstrates that L-arginine supplementation reduces myocardial cell death and improves functional recovery in myocardium submitted to hypoxia-reoxygenation. These beneficial effects are associated with attenuation of the reduction of cGMP synthesis during hypoxia-reoxygenation, as assessed by cGMP release to the coronary circulation, and are abolished when soluble guanylyl cyclase is inhibited by ODQ. L-Arginine supplementation during the equilibration period before hypoxia is both necessary and sufficient to attenuate the reduction of cGMP release and limit myocardial cell death, whereas addition of ODQ during the reoxygenation period is sufficient to abolish the beneficial effects of L-arginine. Supplementation of L-arginine during hypoxia and reperfusion has no effect on cGMP release or cell death. The beneficial effects of L-arginine are independent of any change in coronary flow. These results indicate that the beneficial effects of L-arginine are cGMP mediated, and they are in agreement with previous observations showing that cGMP has a protective effect against reoxygenation-induced hypercontracture.
Previous studies demonstrated that
L-arginine supplementation may
attenuate endothelial dysfunction (36), reduce edema (16) and necrosis
(36), and improve functional recovery in reperfused skeletal (16) and
heart (2, 9, 21) muscle. However, the mechanism of these beneficial
effects has not been completely elucidated.
L-Arginine can increase NO
synthesis by NOS (19), may prevent switch of NOS activity to
O2 production (13, 14), and has been
described to release NO by a NOS-independent mechanism (22). All these
actions of L-arginine
supplementation could contribute to reduce
O2 concentration and to limit
O2-dependent tissue damage thanks to
the strong scavenging action of NO toward peroxyl radicals, OH, and
oxoferryl hemoproteins (20) overproduced during myocardial reperfusion
and reoxygenation (11, 35). In fact, previous studies using methylene
blue (a less specific inhibitor of soluble guanylyl cyclase) suggested
that the beneficial effects of
L-arginine were mainly mediated
by the antioxidant effects of NO and only to a little extent by
cGMP-mediated actions (20). The results of the present study do not
support this hypothesis, and they clearly indicate that the protective
effects of L-arginine
supplementation on reoxygenation injury as assessed by enzyme release
and contractile recovery are mainly mediated by increased cGMP
availability during reperfusion. However, the present results do not
exclude the possible existence of other beneficial effects of
L-arginine related to cGMP-independent actions.
Maintenance of cGMP concentration during ischemia-reperfusion by L-arginine supplementation may reduce necrosis by decreasing neutrophil accumulation in reperfused myocardium (36) and/or by improving coronary flow (2, 16). Reduced neutrophil accumulation can be excluded in our crystalloid-perfused model, and the lack of any effect of L-arginine on coronary flow indicates that its beneficial effect is not mediated by flow preservation. The results of the present study are consistent with the beneficial effect of L-arginine being caused by a cGMP-mediated protection against reoxygenation-induced hypercontracture. cGMP inhibits contraction in cardiomyocytes by mechanisms not completely understood and probably involving modulation of both Ca2+ availability (34) and myofilament sensitivity to Ca2+ (25, 29). Increased cGMP concentration at the time of reoxygenation by stimulation of NO-independent guanylyl cyclase has been shown to prevent hypercontracture in isolated cardiomyocytes (15), and exposure to the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate has been found to improve relaxation in posthypoxic cardiomyocytes (28). This effect was similar to that produced by transient contractile blockade with 2,3-butanedione monoxime, a drug that directly inhibits actin-myosin interaction (10, 31). Because recovery of cell energy and hypercontracture occurs during the first seconds of restoration of oxygen supply (32), contractile blockade must be fully established at the onset of reoxygenation. Stimulation of particulate guanylyl cyclase (15), or administration of 2,3-butanedione monoxime (10, 31), thus had to be started during the last few minutes of hypoxia to be effective. In the present study, the failure of L-arginine added only at the time of reoxygenation could be explained by the time required for incorporation of L-arginine into the pool of L-arginine acting as NOS substrate within the cells. On the other hand, uptake of L-arginine by the cells involves an electrogenic transport system that seems to be impaired during hypoxia (Ref. 39 and present study). In fact, addition of L-arginine to the hypoxic buffer had no effect on cGMP release. Thus, to exert its beneficial effect during reoxygenation, L-arginine must be administered before hypoxia.
It could be expected that a protective effect of
L-arginine against
hypercontracture would result in a detectable reduction of the
reoxygenation-induced rise in LVEDP. However, in the present study
LVEDP was not modified by
L-arginine. This apparent
discrepancy can be explained by a nonlinear relationship between the
number of cells undergoing hypercontracture and the magnitude of the increase in LVEDP. This nonlinear relationship is suggested by previous
unpublished observations in the very same model used in this study
(Fig. 7). Regression analysis shows that
hypercontracture of a relatively small proportion of cardiomyocytes, as
indicated by release of a small fraction of total LDH content during
reoxygenation, suffices to produce a maximal rise in LVEDP.
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The present results are in agreement with previous studies investigating the effect of NO donors on myocardial injury secondary to experimental ischemia. With a few exceptions (5), NO donors, including S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and 3-morpholinosydnonimine, have been consistently found to exert a beneficial effect on cell death (30) and functional recovery (3, 8). Our data help to explain these beneficial effects through cGMP-mediated actions.
In other studies, inhibition of NO synthesis before ischemia
has been found to improve functional recovery during reperfusion in the
isolated, perfused heart (7, 23, 37). The mechanism of this protective
effect has not been established, and stimulation of adenosine-mediated
preconditioning (37), reduction of hydroxyl free radical (23) or
peroxynitrite (38) generation, and preservation of glucose transport
and glycolysis (7), with delayed onset of rigor- contracture, have been
proposed to play a role. It seems, however, that the protective effect
of NOS inhibitors were independent of a reduction in cGMP concentration
(7). The beneficial effects of NOS inhibitors on
ischemia-reperfused myocardium are not in conflict with our
working hypothesis of a cGMP-mediated beneficial effect of
L-arginine. Inhibition of NOS
during ischemia-reperfusion can result in a beneficial
reduction of O2 and ONOO production (13) that
could outweigh the potentially harmful effects of reduced NO synthesis.
In fact, these unfavorable effects should be small if, as suggested by
this study, cGMP synthesis is already markedly reduced during
ischemia-reperfusion.
Study Limitations
In this study, a model of hypoxia-reoxygenation was used. Although this model may be less relevant than ischemia-reperfusion to most clinical situations, it allows better control of drug delivery during energy deprivation and continuous measurement of cGMP release. The actual cellular cGMP concentrations were not measured in this study. However, changes in cGMP release have been shown to be a reliable index of changes in myocardial cGMP synthesis in previous studies (3, 18) and can be continuously monitored in the same heart. The lack of effect of L-arginine on coronary flow in the present study could be caused by the existence of a maximal vasodilatation in the crystalloid-perfused heart. Our results thus do not exclude the possibility of additional vascular related effects of L-arginine during myocardial reperfusion in other models.Implications
The demonstration of a protective effect of cGMP on ischemia-reperfusion injury opens new possibilities of pharmacological prevention of reperfusion injury. In fact, L-arginine concentrations required to observe beneficial effects are similar to those obtained in plasma after intravenous infusion (16). Although the potential value of L-arginine in the prevention of reoxygenation/reperfusion injury is limited by the need to administer it before energy deprivation, this limitation could be circumvented by substances directly stimulating cGMP synthesis. Currently available substances are able to effectively increase cGMP concentration by NO-independent stimulation of particulate guanylyl cyclase without the side effects of increased NO concentration. The potential therapeutic value of these substances on ischemia-reperfusion injury needs to be investigated.| |
ACKNOWLEDGEMENTS |
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The authors acknowledge the excellent technical work of Yolanda Puigfel and the contribution of Dr. Marisol Ruiz-Meana and Dr. Juan Cinca in reviewing the manuscript.
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FOOTNOTES |
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This work was partially supported by the BIOMED Concerted Action BMH1-PL95/1254 from the European Union and by a grant from the Fondo de Investigación de la Seguridad Social (FIS 97/0948).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. García-Dorado, Servicio de Cardiología, Hospital General Universitario Vall d'Hebron, Paseo Vall d'Hebron 119-129, 08035 Barcelona, Spain (E-mail: dgdorado{at}hg.vhebron.es).
Received 25 September 1998; accepted in final form 22 January 1999.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). Relation between the 2 variables is clearly nonlinear
but followed a hyperbolic model (r = 0.882, P < 0.0001), and LDH release
of one-third of maximal values is associated with maximal values of
peak LVEDP. 
and 
, mean values in control and
L-arginine groups, respectively,
in present study (n = 12 in each
group). Vertical and horizontal bars indicate SE for peak LVEDP and LDH
release. Although reoxygenation-induced LDH release, indicating
sarcolemmal disruption and cell death secondary to hypercontracture, is
clearly reduced by L-arginine,
this change is not associated with a reduction in peak LVEDP because it
occurs along horizontal segment of regression curve.