Cardiovascular Research

Cardiovascular Research 1997 36(1):60-66; doi:10.1016/S0008-6363(97)00138-7
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Role of endothelin, nitric oxide and L-arginine release in ischaemia/reperfusion injury of rat heart

Friedrich Brunner^a,*, Bernhard Leonhard^b, Walter R Kukovetz^a and Bernd Mayer^a

^aInstitut für Pharmakologie und Toxikologie Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria
^bInstitut für Zoologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria

^* Corresponding author. Tel. +43-316-380-5559; fax: +43-316-380 9890; e-mail: friedrich.brunner@kfunigraz.ac.at

Received 3 February 1997; accepted 22 May 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
Objectives: We tested the hypothesis that endothelin-1 (ET-1) aggravates ischaemia/reperfusion injury by stimulating cellular L-arginine depletion, which would result in reduced synthesis of nitric oxide (NO) and withdrawal of cardioprotection. Methods: Five groups of rat hearts (n = 5 each) were perfused at 9 ml/min per g for 45 min, subjected to 15 min total global ischaemia and reperfused for 30 min; they received, from 5 min pre-ischaemia to end of reperfusion, either vehicle, L-arginine (1 mmol/l), the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 200 µmol/l), the inhibitor of NO formation N^G-nitro-L-arginine (L-NNA; 200 µmol/l), or the ET receptor antagonist PD 142893 (200 nmol/l). Cardiac function and release of L-arginine, cyclic GMP and lactate dehydrogenase (LDH) into coronary effluent were measured. Results: Systolic, diastolic, and coronary reperfusion function were consistently improved by L-arginine, SNAP, or PD 142893, but worsened by L-NNA (P<0.05 in each case). L-arginine release was transiently increased up to 25-fold on reperfusion (vehicle); release was reduced by SNAP (mean: 68%) and entirely prevented by PD 142893. Despite the increased outflow of L-arginine, formation of cyclic GMP was not reduced, but enhanced in reperfusion (11-fold; vehicle), and SNAP further augmented this release, but L-NNA had no significant effect. Release of LDH was decreased by L-arginine, SNAP, and PD 142893 in reperfusion. Finally, release of ET-1 was inhibited by NO in normoxia as well as throughout reperfusion as evident from the stimulatory effect of L-NNA. Conclusion: In ischaemia, ET-1 causes cell necrosis and L-arginine outflow without compromising NO synthesis in this model.

KEYWORDS Nitric oxide; Nitric oxide donor; Ischaemic cell death; Reperfusion injury; Endothelin; Vascular endothelium; Rat

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
The endothelium produces several vasodilator and vasoconstrictor substances that regulate blood vessel tone and exert a number of other cardiovascular effects [1]. Two important factors produced by the endothelium are endothelium-derived relaxing factor [2], generally agreed to be nitric oxide [3, 4], and endothelin-1 (ET-1) [5]. Recent studies focused on the role of the endothelium in cardiac ischaemia-reperfusion injury which is associated with considerable damage to myocytes and coronary arteries. A prominent feature of post-ischaemic endothelial cell dysfunction is a decrease in endothelium-dependent vasodilation in isolated blood vessels and intact vascular beds [6, 7], and studies into its mechanisms have implicated the NO-cyclic GMP pathway. Basal release of NO from rat hearts appeared diminished after ischaemia and reperfusion [8, 9], and the pathophysiological consequences due to impaired NO release were mitigated or abolished by providing exogenous NO donors [8, 10], authentic NO [11], or the NO precursor L-arginine [12–14], suggesting that cellular L-arginine depletion may play a role in these defects. In other studies, however, inhibitors of NO synthase were found to exert protective, rather than injurious effects [15, 16], possibly via indirect mechanisms.

There is now substantial evidence that the formation of ET-1 is enhanced during myocardial ischaemia [17]and that pretreatment of hearts with receptor antagonists reduces myocardial infarct size/injury in several [18–20]albeit not all models [21, 22]. These discrepancies may in part relate to the balance, in a given experimental setting, of pro-ischaemic actions of ET-1, which include coronary constriction and impairment of systolic and diastolic function, and anti-ischaemic effects such as increased release of vasodilator prostanoids and endothelium-derived NO. In addition, reciprocal interactions between the ET and L-arginine/NO systems in conjunction with increased cytosolic free Ca²⁺ concentrations observed in ischaemic hearts [23]might be of importance. Data from cultured endothelial cells suggest that the formation of ET-1 and NO is increased in this condition [24, 25]and that feed-back inhibition of ET-1 formation by NO may also play a role. However, the importance of these mechanisms in intact hearts subjected to ischaemia and reperfusion is not known.

Based on this evidence we hypothesized that increased release of ET-1 might promote ischaemia/reperfusion injury resulting in myocardial and endothelial cell death which would secondarily lead to depletion of cellular L-arginine, withdrawal of cardioprotection due to reduced synthesis of NO, and further ET-1 release. Therefore, the purposes of this study were (1) to determine the role of L-arginine/NO and ET-1 after myocardial ischaemia with reperfusion and (2) to relate the recovery of coronary and myocardial function after reperfusion to alterations in release of L-arginine and cyclic GMP as well as lactate dehydrogenase (LDH), an established marker of cell viability.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
2.1 Materials
L-arginine and L-NNA were purchased from Sigma (Vienna, Austria); SNAP was from Tocris Cookson (Bristol, UK); 2,2-diethyl-1-nitrosooxyhydrazinex1 Na (DEA/NO) was from Research Biochemicals (Natick, MA, USA); PD 142893 (Ac-D-diphenylalanine-Leu-Asp-Ile-Ile-Trpx2 Na) was the kind gift of Dr. Annette Doherty from Parke-Davis Pharmaceutical Research (Ann Arbor, MI, USA). All other chemicals were of standard purity.

2.2 Heart perfusion
Sprague–Dawley rats (260–320 g) were anaesthetized with diethyl ether, the hearts removed, arrested in cold solution and immediately mounted in a Langendorff perfusion system. Heart beats resumed within 85 s of thoracotomy (within 2.5–3 min after start of anaesthesia). Hearts were perfused in the non-recirculating mode at a flow rate of 9 ml/min per g heart wet weight with a modified Krebs–Henseleit bicarbonate buffer (composition in mmol/l: NaCl 118, NaHCO₃ 25, KH₂PO₄ 1.2, KCl 4.8, MgSO₄ 1.2, CaCl₂ 1.25, glucose 11) using the ISOHEART perfusion system (Hugo Sachs Elektronik, March–Hugstetten, Germany) as described previously [26]. Cardiac parameters were monitored continuously and included heart rate, coronary perfusion pressure (CPP), left ventricular developed pressure (LVDevP; difference between left ventricular peak systolic pressure and end-diastolic pressure), and left ventricular end-diastolic pressure (LVEDP). All animals received care in accordance with the "Austrian Law on Experimentation with Laboratory Animals" (last amendment, 1989) which is based on the principles of laboratory animal care as adopted by the American Heart Association and the Declaration of Helsinki.

2.3 Experimental protocol
Hearts were perfused for 45 min (baseline), followed by 15 min of global ischaemia, and finally reperfused for 30 min at normal flow. The hearts were thermostated at 37 (ischaemia: 35°C). Some hearts were perfused for 90 min under normoxic conditions. The following drugs were added to the perfusion medium 5 min before start of ischaemia to the end of reperfusion (final concentrations): L-arginine (1 mmol/l), the physiological precursor for the formation of NO; S-nitroso-N-acetyl-DL-penicillamine (SNAP; 200 µmol/l); N^G-nitro-L-arginine (L-NNA; 200 µmol/l), a specific inhibitor of NO formation; and PD 142893 (200 nmol/l), an inhibitor of ET_A and ET_B receptors [27].

2.4 Measurement of L-arginine and LDH
The HPLC method used for quantitative analysis of L-arginine was adapted from procedures described previously [28]. Coronary effluent samples (0.1 ml) were treated with acetonitrile (0.1 ml) at ambient temperature for 10 min, followed by centrifugation at 12000xg for 3 min. The supernatant was dried and the resulting sediment dissolved in 20 µl Na-bicarbonate buffer (50 mmol/l, pH 8.1). This solution was combined with 40 µl of the derivatization solution [4 mmol/l 4-(dimethylamino)-azobenzenesulfonyl chloride in acetonitrile] and heated for 12 min at 70°C in a water bath. After derivatization, samples were diluted with citrate buffer (10 mmol/l, pH 4.6, containing 30% acetonitrile and 4% dimethyl formamide) to a final volume of 0.5 ml (dilution of effluent=1:5). Aliquots (10 µl) of the diluted samples were injected onto an Ultrasphere XL-ODS 3 µm (4.6x70 mm) column equipped with an integrated guard column (Beckman; Vienna, Austria). Separation was achieved at ambient temperature with a binary gradient at a flow rate of 1.2 ml/min. Solvent A was Na-citrate (13 mmol/l, pH 6.4) containing 4% dimethyl formamide; solvent B was a mixture of 30% solvent A and 70% acetonitrile containing 4% dimethyl formamide. The gradient was 30% B to 56% B in 10.6 min, 56% B to 86% B in 1.7 min, then kept at 86% B for 1.38 min, 86% B to 100% B in 0.5 min, kept at 100% B for 3 min, returned to 30% B in 0.5 min and equilibrated for 2 min at starting conditions. The cycle time from injection to injection was 22 min. The detector wavelength was 436 nm. Calibration of the method with authentic L-arginine yielded linear responses of peak areas vs concentration in the range of 1 to 20 pmol/injection. The effluent samples contained 1–10 pmol L-arginine per injection.

LDH was measured as described previously [25].

2.5 Determination of ET-1 and cyclic GMP
ET-1 was concentrated by solid-phase extraction followed by quantitative radioimmunoassay as described previously [26]. Briefly, coronary effluents were chromatographed on C2 Ethyl Spe-ed^TM cartridges (Inovec; Vienna, Austria), and ET-1 was eluted with acetonitrile (70%), the eluate freeze-dried, and ET-1 contained in the sediment was dissolved in buffer and determined by RIA using an ET-1-specific antibody (Peninsula; Belmont, CA, USA). Cyclic GMP in coronary effluents was determined directly by radioimmunoassay essentially as described previously [29]. Standard curves were established in Krebs–Henseleit buffer (pH 8.0) containing 1 mmol/l 3-isobutyl-1-methylxanthine to inhibit cyclic nucleotide-dependent phosphodiesterases.

2.6 Statistical analysis
Group data are presented as arithmetic mean values±SEM. Hemodynamic parameters were evaluated by two-way analysis of variance (ANOVA) for repeated measurements to account for different treatments (control, ischaemia, reperfusion) and additions (vehicle and drugs). When a significant overall effect was detected, the Scheffé test was used to compare single mean values. A probability of less than 5% was considered significant. P values less than 0.01 were not indicated separately.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
3.1 Control perfusions in normoxic hearts
First, control perfusions in normoxic perfused hearts not subjected to ischaemia were done in hearts not treated (vehicle) or treated with drugs. For vehicle, heart rate was 311±6 beats/min, LVDevP was 90±2 mm Hg, and CPP was 54±2 mm Hg; LVEDP was set at 0 mm Hg at the end of equilibration (45 min). All parameters were stable throughout the duration of experiment. The NO synthase inhibitor L-NNA (200 µmol/l) had no effect on heart rate (300±10 beats/min), but reduced LVDevP to 67±3 mm Hg (74% of vehicle), and increased CPP to 143±5 mm Hg (2.7-fold) after 90 min perfusion (P<0.05 in each case). The NO donor SNAP (200 µmol/l) and L-arginine (1 mmol/l) affected neither heart rate (303±5 and 311±4 beats/min) nor LVDevP (86±3 and 86±4 mm Hg) or CPP (53±2 and 53±2 mm Hg). In the presence of the ET receptor antagonist PD 142893, functional parameters were likewise indistinguishable from time-matched vehicle values (heart rate: 306±4 beats/min; LVDevP: 88±1 mm Hg; CPP: 52±2 mm Hg) (n = 5 in each case). Whereas in the absence of PD 142893 exogenous ET-1 (40 pmol/l) increased CPP to 110±3 mm Hg in untreated hearts, CPP was unchanged in the presence of the antagonist (55±2 mm Hg, n = 3 in each case), lending support to the specificity of antagonist action.

3.2 Effect on ischaemic and reperfusion function
As expected, no-flow ischaemia resulted in cessation of left ventricular contractile activity (Fig. 1A), an increase in LVEDP (Fig. 1B) and a collapse of CPP (Fig. 1C); heart beat slowly ceased. After 30 min of reperfusion, heart rate returned to baseline (=pre-ischaemic) level, but LVDevP remained depressed to 74%, LVEDP was elevated to 24±1 mm Hg, and CPP was increased 2.1-fold (vehicle; each P<0.05 vs. baseline). In the presence of L-arginine or SNAP (200 µmol/l), recovery of reperfusion function was considerably improved, so that parameters approached baseline values (LVDevP: 90% and 96% of baseline; LVEDP: 4±2 and 1±1 mm Hg; CPP: 1.2-fold and 1.1-fold of baseline, respectively) (P>0.05 vs. baseline). NO synthase inhibition by L-NNA had opposite effects: LVEDP was elevated to 34±4 mm Hg, and CPP was increased 2.5-fold; LVDevP was unchanged (57%). Addition of the ET receptor antagonist PD 142893 (200 nmol/l) to the perfusion medium consistently improved cardiac and vascular function to a similar extent as SNAP. A statistical comparison between effects of test drugs and effects of vehicle during reperfusion was also done and is indicated in Fig. 1.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Cardiac functional parameters at baseline, during 15 min of ischaemia (Isch.), and during reperfusion (Rep.) after addition of vehicle, L-NNA (200 µmol/l), L-arginine (1 mmol/l), SNAP (200 µmol/l), or PD 142893 (200 nmol/l). Effects on left ventricular developed pressure (LVDevP) (A), left ventricular end-diastolic pressure (LVEDP) (B), and coronary perfusion pressure (CPP) (C) are shown. Drugs were added to perfusion fluid 5 min before onset of ischaemia to end of reperfusion. Results are means ±SEM, n = 5. *P<0.05 vs. vehicle.

 
3.3 Effect on L-arginine and cyclic GMP release
The concentration of endogenous L-arginine in coronary effluent was 0.06±0.01 µmol/l at baseline (Fig. 2A). Restoration of coronary flow resulted in a considerable increase (25-fold) within 5 s, followed by a quick decline to baseline within 5 min. The L-arginine concentration was not significantly altered by L-NNA, but reduced by SNAP (mean: 68%); the ET receptor antagonist PD 142893 effectively suppressed L-arginine release so that effluent levels were at or below baseline throughout reperfusion. (P<0.05 in each case).

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Concentration of L-arginine (A) and cyclic GMP (B) in coronary effluent during reperfusion. Sampling was performed at baseline (marked with "B" at abscissa; i.e. 45 min post mounting of heart), and 5, 10, 20 and 300 s after initiation of reperfusion. Because of total no-flow ischaemia (45–60 min post mounting), no samples were obtained during the ischaemic period. Drugs were added to perfusion fluid 5 min before onset of ischaemia to the end of reperfusion. For concentration of drugs, see Fig. 1. Results are means ±SEM, n = 5. *P<0.05 vs. vehicle.

 
To complement these measurements, the effector of NO, cyclic GMP, was also determined (Fig. 2B). After infusion of vehicle, the concentration of cyclic GMP was elevated 11.1-fold in the first effluent sample collected after initiation of reperfusion (P<0.05 vs. baseline); baseline level was reached again after 5 min. As expected, cyclic GMP was greatly increased by SNAP (52-fold vs. baseline). Neither PD 142893 nor L-NNA affected cyclic GMP in reperfusion (P>0.05 vs. vehicle). Cyclic GMP levels were never lower than baseline throughout reperfusion.

3.4 Effect on LDH release
The LDH concentration in coronary effluent increased 9-fold within 5 s of reperfusion as compared to baseline and declined to baseline level within 5 min (Fig. 3, vehicle). The LDH concentration was decreased after infusion of L-arginine (50–80%) or SNAP (60–90%) (P<0.05 in each case); L-NNA had no effect (P>0.05). Antagonism of ET receptors with PD 142893 reduced LDH release to the same level as observed with SNAP. LDH release was indistinguishable from baseline between 5 and 30 min of reperfusion (data not shown).

View larger version (43K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 LDH concentration in coronary effluent during reperfusion. Sampling was performed as described in Fig. 2 and concentrations of drugs are given in Fig. 1. Drugs were added to perfusion fluid 5 min before onset of ischaemia to the end of reperfusion. Results are means ±SEM, n = 3. *P<0.05 vs. vehicle.

 
3.5 Effect of the L-arginine/NO pathway on ET-1 secretion
In normoxic hearts, basal ET-1 release was constant (0.028±0.002 pg/ml effluent; Fig. 4A). Addition of L-NNA to the perfusate increased the ET-1 concentration within 30 min to 0.098±0.011 pg/ml (3.5-fold; P<0.05). As expected, ET-1 secretion was increased several fold after ischaemia (Fig. 4B) (P<0.05 vs. baseline). SNAP and a second NO donor, DEA/NO [30]slightly reduced ET-1 secretion throughout reperfusion, whereas L-NNA significantly increased it (P<0.05 vs. vehicle in each case).

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 ET-1 concentration in coronary effluent of normoxic (A) and reperfused hearts (B). Normoxic hearts were equilibrated (45 min; baseline) and vehicle or L-NNA was then added to perfusion fluid for 45 min. In (B), hearts were equilibrated for 45 min, made ischaemic for 15 min, and reperfused for 30 min. Sampling was performed at baseline (marked with "B" at abscissa) and 3, 6, 20 and 30 min after initiation of reperfusion. Because of total no-flow ischaemia (45–60 min post mounting), no samples were obtained during the ischaemic period. The effects of L-NNA (200 µmol/l), SNAP (200 µmol/l) and DEA/NO (10 µmol/l), added from 5 min before onset of ischaemia to the end of reperfusion, are shown. Results are means ±SEM, n = 5. *P<0.05 vs. vehicle (A) or P<0.05 vs. baseline (B).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
The present study tested the hypothesis that ET-1 released in ischaemia/reperfusion is associated with a loss of cellular L-arginine, reduced synthesis of NO, and withdrawal of cardioprotection by NO/cyclic GMP. We found, during reperfusion following ischaemia, (1) a protective effect of L-arginine, SNAP, and the ET receptor antagonist, and a deleterious effect of NO synthase inhibition on haemodynamic function, L-arginine release, and LDH release, and (2) that the synthesis of NO (measured as cyclic GMP) was never lower than during normoxic perfusion despite augmented efflux of L-arginine from the heart. These results indicate for the first time that ET-1 damages myocytes and endothelial cells in the setting of ischaemia/reperfusion, which results in cell necrosis, L-arginine outflow, and impaired reperfusion function but not in reduced formation of NO.

Neither PD 142893 nor SNAP had any effect on functional parameters under normoxic conditions, indicating that ET-1 production is substantially down-regulated by NO and that ET-1 exerts no measurable deleterious effects in normoxic-perfused hearts. In view of the protective effects of PD 142893 in ischaemia (see below), the specificity of action of PD 142893 is of particular importance. In support, no non-specific vasodilator effect was ever observed with PD 142893 in normoxic perfusions, rather CPP was indistinguishable from vehicle values in all measurements throughout the protocol. Furthermore, PD 142893 completely blocked the vasoconstrictor effect of a test concentration of exogenous ET-1. Together, these data indicate that non-specific actions of the ET receptor antagonist can be excluded.

After ischaemia, impairment of vascular relaxation was evident from a doubling of CPP after 30 min of reperfusion. The additional reduction of vasodilator tone observed in the presence of L-NNA, as well as the substantial protective effect of L-arginine and SNAP, clearly indicated a lack of NO or NO activity in reperfusion vascular dysfunction. Endogenous ET-1 is likely to be an important factor in this impaired vascular relaxation because the post-ischaemic increase in CPP observed with vehicle was effectively antagonized by the ET receptor antagonist (Fig. 1C). Besides these coronary effects, the antagonist also improved myocardial function as evident from a higher recovery of post-ischaemic LVDevP and a better post-ischaemic diastolic relaxation (lower LVEDP). Because these effects were matched by a greatly reduced release of LDH after reperfusion, these data indicate that endogenous ET-1, in amounts generated by the ischaemic insult, plays a causal role in reperfusion injury in this model. A similar vascular protective effect was observed in the isolated rabbit heart [31]and in hearts of anaesthetized pigs [19].

Examining the cellular mechanism of action of ET-1 was beyond the scope of our present objectives. With respect to myocyte function, it may involve an increase in intracellular Ca²⁺ concentration (Ca²⁺ overload) [32], depletion of energy stores due to the positive inotropic effect of ET-1 [33], or increased Ca²⁺ sensitivity of myofibrils [34].

Despite suggestions that L-arginine deficiency may account for reduced NO synthesis and impaired vasorelaxation [8, 13, 35], so far L-arginine levels have apparently not been determined during post-ischaemic reperfusion. The present HPLC measurements (Fig. 2A) show clearly that L-arginine release into coronary effluent is increased some 25-fold above pre-ischaemic level after restoration of coronary flow, which amounted to a total L-arginine efflux of 3 nmol within 5 min of reperfusion (vehicle). Judgment of the significance of this efflux is hampered by the ignorance of its cellular origin. Clearly, the amount released is a substantial fraction of the endothelial cell content (5–10 nmol based on published endothelial cell dimensions and cytosolic L-arginine concentrations [36]), but would not represent a significant fraction of total cardiac L-arginine. The question was resolved by determining the time-course of cardiac cyclic GMP release into the same coronary effluents in which L-arginine was determined. They clearly showed an increased, rather than decreased level, indicating that the L-arginine release occurring at the same time does not result in reduced NO synthesis. This may be due to relatively high cellular L-arginine levels [36]being well above the concentration required for substrate saturation of NO synthases, which exhibit K_m values in the low micromolar range [37, 38]. The cyclic GMP recovered in reperfusion may have accumulated during preceding low-flow ischaemia and been washed out during reperfusion, because a model of low-flow ischaemia (0.2 ml/min) showed that the cyclic GMP appeared during the ischaemic phase rather than during reperfusion (unpublished observations).

The lack of effect of L-NNA on cyclic GMP release was somewhat surprising and is presently being investigated. A preliminary explanation is that the steps leading to activation of guanylyl cyclase by NO are more complex than currently known, possibly involving storage forms of NO.

In cultured cells, NO attenuates the synthesis of ET-1 [39], but since NO formation may be impaired in ischaemia-reperfusion (see Introduction), we expected the formation of ET-1 to be enhanced. Under normoxic conditions, ET-1 release into coronary effluent was tonically inhibited by NO as shown by the several-fold increase in ET-1 release in the presence of L-NNA (Fig. 4A). Interestingly, even after ischaemia (Fig. 4B), ET-1 formation was still subject to modulation by NO, as evident from its inhibition by donor-derived NO and the higher rate of ET-1 secretion in the presence of L-NNA. These novel observations indicate that the negative feed-back inhibition of ET-1 formation by NO remains functional, yet insufficient after ischaemia as evident from the suppression of all deleterious effects by the ET receptor antagonist (Figs. 1–3). Examining the mechanism of this counter-regulation was not intended here; it might result from stimulation of endothelial NO synthase [24]induced by a rise in cytosolic Ca²⁺ during ischaemia. Thus, our results suggest that the negative feed-back component of ET-1 formation remains operational in ischaemia, but is overridden by the increased net release and action of ET-1 [17].

	5 Conclusion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
Our results indicate that under normoxic conditions, basal NO release results in tonic inhibition of ET-1 production, while the ischaemic insult triggers the synthesis and release of ET-1 which leads to receptor-mediated cell necrosis with concomitant LDH and L-arginine efflux. However, the latter does not cause reduced NO synthesis and does not appear to account for impairment of reperfusion coronary function in this buffer-perfused rat heart model.

Time for primary review 22 days.

	Acknowledgements

 
We thank Dr. Annette Doherty (Parke-Davis Pharmaceutical Research, Ann Arbor, MI) for the generous supply of PD 142893 and Mr. G. Wölkart for excellent technical assistance. This study was supported by the Austrian Research Council (Österreichischer Forschungs-Fonds) Projects 11040, 10655 and 11478.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 

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