Cardiovascular Research

Cardiovascular Research 1998 39(3):674-682; doi:10.1016/S0008-6363(98)00167-9
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

The endothelin A receptor antagonist LU 135252 protects the myocardium from neutrophil injury during ischaemia/reperfusion

Adrian T Gonon^*, Qing-Dong Wang and John Pernow

Department of Cardiology, Karolinska Hospital, 171 76 Stockholm, Sweden

^* Corresponding author. Tel.: +46-8-517-73560; Fax: +46-8-311-044.

Received 5 January 1998; accepted 22 May 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Endothelin-1 (ET-1) is not only a potent vasoconstrictor but also a stimulator of polymorphonuclear leukocyte (PMN) aggregation and adhesion. The aim of this study was to investigate whether an ET_A receptor antagonist attenuates the PMN-mediated contractile dysfunction following myocardial ischaemia. Methods: Isolated rat hearts were perfused according to the Langendorff method. The hearts were subjected to global ischaemia and reperfused with buffer solution only, or human PMNs dissolved in rat plasma (HNRP). Results: In an initial study, the ET_A receptor antagonist LU 135252 (1 and 10 µmol/l) or ET-1 (1 and 10 nmol/l) did not significantly affect the recovery of left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), the first derivative of left ventricular pressure (dP/dt) or the rate pressure product (RPP) during reperfusion with buffer solution only compared to a vehicle group. In a second study on hearts reperfused with HNRP, administration of LU 135252 (10 µmol/l) significantly enhanced the recovery of LVDP, dP/dt and RPP in hearts reperfused with HNRP. LVEDP was 20 mmHg lower in hearts given LU 135252 than vehicle in combination with HNRP (P<0.05). The outflow of PMNs in the coronary effluent during reperfusion was 41±8% in hearts given LU 135252 compared to 9±5% in vehicle-treated hearts (P<0.01). There was a significant correlation between the myocardial functional recovery and the outflow of PMNs. Administration of ET-1 (0.1 and 1 nmol/l) in combination with HNRP resulted in complete loss of contractile function and no outflow of PMNs during reperfusion. Conclusion: The ET_A receptor antagonist LU 135252 protects from ischaemia/reperfusion injury in the isolated rat heart in the presence of PMNs. It is suggested that inhibition of PMN-induced injury during reperfusion is an important cardioprotective action of LU 135252.

KEYWORDS Endothelin-1; ET_A receptor; LU 135252; Neutrophils; Ischaemia; Reperfusion; Rat

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The potent endothelium-derived vasoconstrictor peptide, endothelin-1 (ET-1) [1], has been suggested to play an important role in cardiovascular diseases [2]. Several clinical studies have shown that circulating plasma levels of endothelin-like immunoreactivity (ET-LI) are increased in patients with advanced atherosclerosis [3]and myocardial infarction [4]. Exogenous ET-1 causes marked coronary constriction in several species [5, 6], which may result in ischaemic damage [7]. Two different subtypes of ET receptors, ET_A and ET_B, have been characterised and cloned [8, 9]. ET_A and ET_B receptors located on vascular smooth muscle mediate vasoconstriction, whereas ET_B receptors located on the endothelium produce vasodilation by releasing nitric oxide or prostacyclin [10].

Following myocardial ischaemia and reperfusion in experimental animals, there is an enhanced myocardial production and release of ET [11, 12], which seems to be derived from the jeopardised myocardial area [13]. In addition, the vasoconstrictor response to exogenous ET-1 is enhanced [13, 14], and the number of ET-1 binding sites is increased following ischaemia/reperfusion. These findings all indicate a pathophysiological role of ET-1 in the development of ischaemia/reperfusion injury.

A monoclonal antibody against ET-1 (AwETN40) was initially used to elucidate the pathophysiological role of endogenous ET-1. The antibody was shown to reduce the myocardial infarct size in a rat model of ischaemia/reperfusion in vivo [15]. Moreover, the peptide-based, specific ET_A receptor antagonist, BQ 123, and the non-peptide mixed ET_A/ET_B receptor antagonist, bosentan, reduced infarct size in the dog and pig, respectively [16, 17]. The mechanism underlying the proposed cardioprotective effect of ET receptor antagonists remains unclear.

It has been reported that ET-1 produces marked effects on polymorphonuclear leukocytes (PMNs). Thus, ET-1 stimulates aggregation of human PMNs [18], increases PMN intracellular free calcium mobilisation [19, 20]and superoxide anion production by PMNs [21]. ET-1 stimulates PMNs to adhere to endothelial cells and to accumulate in the isolated rabbit heart. These effects are blocked by an antibody against the integrin complex [22].

Myocardial ischaemia initiates an acute inflammatory response in which PMNs are of importance. PMNs may contribute to myocardial damage by releasing oxygen-derived free radicals, proteases and arachidonic acid metabolites [23]. Since ET-1 has been demonstrated to stimulate PMNs, it is of interest to evaluate the role of ET-1 in PMN-induced myocardial ischaemia/reperfusion injury.

The aim of the present study was therefore to investigate whether or not a selective non-peptide ET_A receptor antagonist protects from ischaemia/reperfusion injury in the isolated rat heart and whether such an effect is related to inhibition of PMN-induced injury.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
All of the investigations were approved by the regional ethics committee for animal research and conform with the Guide for the care and use of laboratory animals published by the US National Institute of Health (NIH publication No 85-23, revised 1985).

2.1 Isolated heart preparation
Male Sprague-Dawley rats (weight, 250–350 g) were heparinised and anaesthetised with a mixture of fluanisonum, fentanylum and midazolam (2.5, 0.08 and 1.25 mg/kg, respectively, i.m.). The hearts were excised, the ascending aorta was cannulated, and immediately retrogradely perfused with non-recirculating modified Krebs-Henseleit solution (in mmol/l: NaCl 118, KCl 4.7, CaCl₂ 1.5, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25.2 and glucose 11.1) at a constant pressure of 90 cm H₂O or at a constant flow rate by means of a roller pump. The flow was set at a rate (16 ml/min) that resulted in a coronary perfusion pressure of 90 cm H₂O prior to ischemia. The perfusate was bubbled with 95% O₂ and 5% CO₂ and kept at 37°C. Two side arms connected to a mixing chamber just proximal to the heart cannula were used for the administration of drugs prior to ischaemia, PMNs and plasma. During early reperfusion, ET-1 or LU 135252 was administered via the perfusion column. To assess contractile function, a latex balloon connected to a pressure transducer (Gould, USA) was inserted into the left ventricular cavity via the left atrium. Left ventricular end-diastolic pressure (LVEDP) was set at 5 mmHg by inflating the balloon with physiological saline. Left ventricular pressure and its electronically differentiated derivative, dP/dt, were continuously recorded on a polygraph (Model 7D, Grass Instruments, USA). Coronary flow was measured by collecting the effluent, and heart rate was determined from the pressure registration every 5 min.

2.2 Neutrophil preparation
Human buffy coat prepared from citrated whole blood of human volunteer donors was supplied by the Karolinska Hospital Blood Centre. A 20-ml volume of the buffy coat was carefully layered on the top of 15 ml of low density Percoll (55%) and 15 ml of high density Percoll (74%) in conical plastic tubes (Sarstedt, Nümbrecht, Germany). The tubes were centrifuged at 1300 rpm for 25 min at room temperature. The neutrophil-rich band between the two density gradients was aspirated and dissolved in 50 ml of Dulbecco's phosphate-buffered saline without CaCl₂ and MgCl₂ (D-PBS) and centrifuged at 1400 rpm for 5 min. The supernatant was discarded and the pellet was washed twice with D-PBS and centrifuged as before. The precipitant was exposed to 2 ml of distilled water for 30 s, to lyse contaminating erythrocytes. The process was stopped by administration of D-PBS to a volume of 50 ml. After another centrifugation at 1400 rpm for 5 min, the pellet was dissolved in 5 ml of Hank's balanced salt solution without CaCl₂ and MgSO₄ (HBSS) [24]and the PMNs were counted. Pellets that had more than 5% activated PMNs, yielded by Trypan blue (0.4%) staining, were discarded.

2.3 Rat plasma
Rats were anaesthetised as above. Whole rat blood was obtained by performing an open-chest intracardiac puncture using a 10-ml plastic syringe with a needle containing 2000 U heparin. To obtain platelet-poor plasma, whole blood was immediately centrifuged in a Sigma 3K15 refrigerated centrifuge at 2500 rpm and 4°C for 25 min. The plasma layer was collected and stored at 4°C until it was used in the isolated perfused heart.

2.4 Experimental protocol
Two experimental protocols were used for the isolated rat hearts perfused at a constant pressure, according to Fig. 1. In both protocols, baseline LVEDP, left ventricular developed pressure (LVDP), dP/dt, heart rate and coronary flow were recorded after a 30-min equilibration period. Global ischaemia was induced by stopping the buffer perfusion. During the reperfusion period LVDP, LVEDP, dP/dt, heart rate and coronary flow were determined every 5 min.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Schematic diagram of the two experimental protocols. (a) In protocol 1, hearts were perfused with Krebs-Henseleit buffer only. Vehicle or LU 135252 was given to the hearts at the onset of ischaemia (arrows) and vehicle, LU 135252 or ET-1 was given during the first 5 min of reperfusion (horizontal lines). (b) In protocol 2, vehicle, LU 135252 or ET-1 was given as in protocol 1. In addition, human neutrophils and rat plasma (HNRP), or rat plasma only, were administered during the first 5 min (horizontal bars).

 
In protocol 1 (Fig. 1a), five groups of hearts (A–E) were subjected to 30 min of ischaemia followed by 30 min of reperfusion. At the start of ischaemia, the hearts in group A received 3 ml of vehicle (n=8), and groups B (n=7) and C (n=8) received 3 ml of LU 135252 (1 and 10 µmol/l, respectively). Groups B and C were also given LU 135252 (1 and 10 µmol/l, respectively) during the first 5 min of reperfusion. Groups D (n=5) and E (n=5) were given ET-1 (1 and 10 nmol/l, respectively) during the first 5 min of reperfusion. During the following 25 min, the hearts were reperfused with Krebs-Henseleit buffer.

In protocol 2 (Fig. 1b), three groups of hearts (groups F–H) were subjected to 20 min of ischaemia followed by 45 min of reperfusion. Hearts in group F received vehicle (n=8), group G received LU 135252 (10 µmol/l; n=8) and group H received ET-1 (0.1 nmol/l; n=5), as in protocol 1. All three groups were perfused with human PMNs (50x10⁶) in 5 ml of rat plasma (HNRP) along with Krebs-Henseleit buffer at the onset of reperfusion and during the following 5 min. The perfusion was then continued with Krebs-Henseleit buffer alone. An additional group of hearts (group I) was given plasma (n=6) without PMNs during the first 5 min of reperfusion. During the last minute of perfusion with PMNs, coronary effluent was collected and the PMN concentration was counted subsequently using a light microscope and a Bürker chamber.

Two groups of hearts were studied using a constant flow rate. One group received vehicle and the other received LU 135252 (10 µmol/l), as had groups F and G. These experiments were performed in order to distinguish the possible effects of LU 135252 on PMN accumulation from its effect on coronary flow. Accordingly, only the recovery of PMNs in the coronary effluent at 5 min of reperfusion was determined in these experiments.

Separate experiments were performed in order to characterise the receptor selectivity of LU 135252 in the isolated rat heart model. These experiments revealed that LU 135252 (10 µmol/l) significantly attenuated the reduction in coronary flow induced by ET-1 (10 nmol/l) (77±4 vs. 29±6% reduction in coronary flow in the absence and presence of LU 135252, respectively; P<0.01, Mann-Whitney U-test). The ET_B receptor agonist, sarafotoxin 6c (1–100 nmol/l), induced an initial increase followed by a decrease in coronary flow. These effects were not significantly affected by LU 135252. Thus, the maximal increase in coronary flow was 19±6 vs. 22±5% and the decrease in coronary flow was 35±5 vs. 23±10% in the absence and presence of LU 135252 (10 µmol/l), respectively (n=5).

The method of perfusing rat hearts with human PMNs and rat plasma has been validated previously [25]. A mixture of human PMNs and rat plasma does not cause PMN activation or any measurable complement activation. Furthermore, reperfusion with PMNs alone does not alter postischaemic myocardial function, whereas PMNs together with plasma causes marked myocardial injury. Therefore, PMNs were only given together with plasma in the present protocol. The postischaemic mean recovery of PMNs in untreated hearts using the present experimental model is 7.4% (95% CI, 3.5–11.4; n=25).

2.5 Materials
Sodium heparin was obtained from Lövens (Ballerup, Denmark), Dormicum (midazolam) was from Hoffmann-LaRoche (Basel, Switzerland), Hyponorm (fluanisonum+fentanylum) was from Janssen (Beerse, Belgium) and ET-1 was from Alexis Corporation (Läufelfingen, Switzerland). Percoll, modified Hanks' balanced salts and modified Dulbecco's phosphate-buffered saline were purchased from Sigma (St Louis, MO, USA). LU 135252 (2-[(4,6-dimethoxypyrimidin-2-yl)oxy]-3-methoxy-3,3-diphenylpropionic acid) is the active enantiomer of LU 127043 with high affinity to the human ET_A receptors (K_i=2 nmol/l for ET_A and K_i=184 nmol/l for ET_B) [26]and was kindly supplied by Dr. Manfred Raschack, Knoll (Ludwigshafen, Germany). LU 135252 was dissolved in 1 mol/l NaOH and saline and adjusted with 0.1 mol/l HCl to obtain a pH of 7.5.

2.6 Calculation and statistical analysis
The recovery of myocardial performance is expressed in percent of the pre-ischaemic value. Rate pressure product (RPP) was calculated as the heart rate multiplied by the LVDP. Outflow of PMNs was calculated as the PMN concentration in the coronary effluent multiplied by coronary flow. The resulting outflow rate of PMNs (cells/min) is expressed in percent of the number of PMNs (10⁷ cells/min) added to the perfusate during the same time period.

All values are presented as the mean±SEM. Comparison between the groups were made by one-way analysis of variance (ANOVA) followed by Tukey's test. The significance level, , was set at 0.05. Correlation coefficients were calculated using Pearson's linear correlation.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Reperfusion with buffer alone (protocol 1)
There was a tendency towards improved recoveries of coronary flow, LVDP and RPP in hearts given LU 1352525 compared to vehicle-treated hearts. However, there were no significant differences between the groups (Table 1). Furthermore, LU 135252 did not affect LVEDP in comparison with the vehicle-treated hearts (Table 1). Administration of ET-1 (10 nmol/l) resulted in a significantly lower coronary flow (29±5% recovery) at 5 min of reperfusion in comparison with the vehicle group (74±8% recovery; P<0.01). At 30 min of reperfusion, the percentage recovery in coronary flow and myocardial performance was only slightly and non-significantly lower in the groups receiving ET-1 (1 and 10 nmol/l) compared to the vehicle-treated group (Table 1). However, the recovery of coronary flow was significantly higher in the group given LU 135252 (10 µmol/l) than in the group given ET-1 (10 nmol/l) (Table 1).

View this table: [in this window] [in a new window]   Table 1 223Haemodynamic parameters at 30 min reperfusion in protocol 1

 
3.2 Reperfusion with HNRP or plasma (protocol 2)
The stability of the model in the presence of PMNs was evaluated in hearts that were not subjected to ischaemia. This revealed that coronary flow, LVDP, RPP and dP/dt 45 min following administration of PMNs were 80% of basal values (n=5).

Pre-ischaemic values of coronary flow, LVDP, LVEDP, dP/dt, heart rate and RPP of the groups (F–H) receiving HNRP during the first 5 min of reperfusion are shown in Table 2. There were no significant differences between the groups.

View this table: [in this window] [in a new window]   Table 2 Pre-ischaemic haemodynamic parameters for hearts reperfused with human neutrophils+rat plasma (HNRP) during early reperfusion, in combination with vehicle (group D; n=8), LU 135252 (group E; n=8) or ET-1 (group F; n=5)

 
The functional recovery of the HNRP vehicle group (F) was generally poor. During the late part of reperfusion, the recovery of LVDP, dP/dt and RPP was 30–35% of the pre-ischaemic values (Fig. 2). LVEDP in this group increased continuously, to 93±4 mmHg, during ischaemia and remained high during reperfusion (Fig. 3).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Recovery of (a) left ventricular developed pressure (LVDP), (b) the first derivative of left ventricular pressure (dP/dt) and (c) the rate pressure product (heart rate multiplied by LVDP) during 45 min of reperfusion following 20 min of ischaemia. The hearts were given human neutrophils in rat plasma (HNRP) plus vehicle, HNRP plus LU 135252, or plasma only. The recovery is expressed in percent of the pre-ischaemic values. Results are presented as the mean and SEM (n=6–8). Significant differences from the HNRP vehicle group are shown; *P<0.05, **P<0.01 and ***P<0.001.

 

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Left ventricular end-diastolic pressure (LVEDP) before ischaemia and during reperfusion in hearts given human neutrophils in rat plasma (HNRP) plus vehicle, HNRP plus LU 135252, or plasma alone. Results are presented as the mean and SEM. Significant differences from the HNRP vehicle group are shown; *P<0.05, **P<0.01 and ***P<0.001.

 
The recovery of LVDP of the HNRP plus LU 135252 group (G) increased during reperfusion and was 60±3% at 45 min of reperfusion, compared to 34±9% in the HNRP vehicle group (Fig. 2a, P<0.05). The recovery of dP/dt of the HNRP plus LU 135252 group increased continuously during reperfusion and was significantly higher than that of the HNRP vehicle group after 30 min of reperfusion (Fig. 2b).

Heart rate was not significantly different between the two groups reperfused with HNRP plus vehicle or LU 135252. The recovery of RPP in the HNRP plus LU 135252 group was higher at 30 and 40 min of reperfusion compared with the HNRP vehicle group (Fig. 2c). At 45 min of reperfusion, the RPP of the HNRP vehicle group increased by 5%, due to three hearts with tachycardia.

LVEDP was 20–30 mmHg higher in the HNRP vehicle group than in the HNRP plus LU 135252 group during the reperfusion period (Fig. 3).

The initial recovery of coronary flow was significantly higher in the HNRP plus LU 135252 group than in the HNRP vehicle group (Fig. 4). Following this initial improvement, there were no significant differences between the groups, however.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Recovery of coronary flow during 45 min of reperfusion following 20 min of ischaemia. The hearts were given human neutrophils in rat plasma (HNRP) plus vehicle, HNRP plus LU 135252, or plasma alone. The recovery is expressed in percent of the pre-ischaemic values. Results are presented as the mean and SEM (n=6–8). Significant differences from the HNRP vehicle group are shown; *P<0.05, **P<0.01 and ***P<0.001.

 
Myocardial performance during reperfusion of the hearts given ET-1 (0.1 nmol/l) in combination with HNRP (group H) was extremely poor, and all hearts ceased beating within 14 min of reperfusion and did not recover. Two hearts were given 1 nmol/l ET-1 in combination with HNRP during reperfusion. In these hearts, coronary flow was reduced to zero within 1 min and there were signs of PMN adhesion to the wall of the plastic mixing chamber.

In the group reperfused with rat plasma without PMNs (group I), the functional recovery was significantly better than in the HNRP vehicle group (Figs. 2–4). On the other hand, myocardial performance at the end of reperfusion did not differ significantly between the plasma group and the HNRP plus LU 135252 group.

3.3 Outflow of PMNs
The percentage recovery of PMNs in the coronary effluent was four-times higher in the HNRP plus LU 135252 group than in the HNRP vehicle group and was 20-times higher than in the group given ET-1 together with HNRP (Fig. 5). When data from the HNRP vehicle and HNRP plus LU 135252 groups were compiled, significant correlations were found between the percentage recovery in cardiac function and the outflow of PMNs in the coronary effluent (Fig. 6). There was also a significant inverse correlation between the LVEDP during reperfusion and the outflow of PMNs (Fig. 6).

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Recovery of polymorphonuclear leukocytes (PMN) in the coronary effluent collected during the last minute of PMN administration in hearts given either vehicle, LU 135252 or ET-1. The recovery is expressed in percent of the number of PMNs given during 1 min. Results are presented as the mean and SEM. **P<0.01, ***P<0.001 vs. LU 135252 group.

 

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Correlation between the percentage recovery of polymorphonuclear leukocytes (PMN) in the coronary effluent and the percentage recovery of (a) the rate pressure product (RPP), (b) the left ventricular developed pressure (LVDP) and (c) the left ventricular end diastolic pressure (LVEDP) at 45 min of reperfusion. The correlation coefficients were calculated using Pearson's linear correlation.

 
To explore the possibility that the differences on outflow of PMNs were due to changes in coronary flow, the recovery of PMNs was also investigated in hearts perfused at a constant flow rate. The percentage recovery of PMNs in this model was 26±3% in the HNRP vehicle group and 55±8% in the HNRP plus LU 135252 group (P<0.01; n=5, Mann-Whitney U-test), indicating that the higher recovery in hearts given LU 135252 was unrelated to coronary flow.

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The pathophysiological role of ET in the development of ischaemia/reperfusion injury has been investigated in several studies in vivo as well as in vitro, but is still far from clarified. It has been reported that ET receptor antagonists protect against myocardial ischaemia and reperfusion injury in vivo [17]. Thus, the non-peptide mixed ET_A/ET_B receptor antagonist, bosentan, reduced infarct size in a pig model of regional ischaemia/reperfusion [17]. In addition, the peptide ET_A-specific antagonist, BQ 123, given directly into the left circumflex coronary artery of dogs resulted in a 40% reduction in infarct size [16]. Moreover, another peptide-based ET_A receptor antagonist, FR 139317, reduced infarct size in the rabbit when given before, but not after, coronary artery occlusion [27]. Other studies in vivo have resulted in no or inconsistent protection by ET_A receptor antagonists [28, 29].

In the present study, the non-peptide ET_A receptor antagonist, LU 135252, did not produce any significant cardioprotective effects in the isolated rat heart reperfused with buffer solution. This is in contrast to our observations that LU 135252 reduced infarct size by 60% in a porcine model of regional ischaemia and reperfusion in vivo [30]. The apparent discrepancy may relate to species differences, with variations in ET receptor subtypes between pigs and rats [13, 31]. It may also depend on differences between in vivo and in vitro conditions. One important difference is the small number or absence of PMNs in the buffer-perfused isolated heart. Under in vivo conditions, on the other hand, PMNs are likely to play an important role in the development of tissue damage during ischaemia and reperfusion [23]. It was therefore of importance to further investigate the effect of LU 135252 on ischaemia/reperfusion injury in the presence of PMNs during reperfusion.

The administration of LU 135252 to hearts reperfused with HNRP significantly enhanced the recovery of LVDP, dP/dt and RPP in comparison with the HNRP vehicle group. Furthermore, the LVEDP was lower in the HNRP plus LU 135252 group than in the HNRP vehicle group. Interestingly, the outflow of PMNs in the coronary effluent was significantly greater in hearts given LU 135252. Administration of a low concentration (0.1 nmol/l) of ET-1 in combination with HNRP, on the other hand, impaired the recovery of myocardial function and outflow of PMNs. A ten-fold higher dose of ET-1, in combination with HNRP, completely stopped coronary perfusion. By contrast, ET-1 (up to 10 nmol/l) did not significantly impair post-ischaemic myocardial function in the absence of PMNs. Taken together, these results indicate that ET-1 is involved in the accumulation of PMNs in the reperfused myocardium and in the development of myocardial injury in the presence of PMNs. Inhibition of PMN accumulation and PMN-induced injury in the post-ischaemic myocardium seems to be an important mechanism of action of LU 135252. This assumption is supported by the close correlation between myocardial functional recovery and the outflow of PMNs during reperfusion. Since LU 135252 also enhanced the outflow of PMNs in hearts perfused at a constant flow rate, the inhibition of PMN accumulation does not seem to be secondary to changes in coronary flow, but rather to a direct effect on PMN adhesion.

The duration of the ischaemic periods differed in protocols 1 and 2 due to the aggravated injury in the presence of PMNs [25]. It can not be entirely excluded that this may have influenced the results. However, pilot experiments revealed that 20 min of ischaemia is too short to induce marked post-ischaemic dysfunction in buffer-perfused hearts, while 30 min of ischaemia will cause damage that is too severe, in the presence of PMNs. Accordingly, the post-ischaemic dysfunction in group I (20 min ischaemia, plasma) was less severe than in group A (30 min ischaemia, buffer). In agreement with the study by Shandelya et al. [25], administration of HNRP (group D) caused impairment of post-ischaemic recovery in comparison with the group given plasma only (group I), clearly indicating a PMN-induced injury. This impairment was almost completely inhibited by LU 135252, suggesting that the effect of LU 135252 was related to inhibition of PMN-induced injury.

It has previously been demonstrated that PMNs both generate and degrade ET-1 [32–34]. ET-1 is also known to be a chemoattractant for human PMNs [35]and to stimulate PMN accumulation in the rabbit heart [22]. The present observation, that exogenous ET-1 abolished PMN outflow in the post-ischaemic heart, supports this view. In agreement with the report by López Farré et al. [22], there were clear signs of PMN adhesion to the plastic walls of the perfusion system in the presence of the high dose of ET-1. The mechanism by which ET-1 may stimulate PMN accumulation in the post-ischaemic heart remains to be established. ET-1 has been suggested to cause aggregation of human PMNs by the production of platelet activating factor via a Ca²⁺-dependent mechanism [18]. Furthermore, ET-1 increases the expression of the adhesion molecule CD11b/CD18 on the PMN surface and an anti-CD18 antibody blocks the ET-1-mediated PMN adhesion [22]. It has also been shown that ET-1 induces neutrophil adhesion to cardiac myocytes and aortic endothelial cells by increasing the expression of the intercellular adhesion molecule, ICAM-1 [36]. At least on myocytes, this effect is mediated via the ET_A receptor and is dependent on activation of protein kinase C.

Another possible mechanism by which LU 135252 may protect against PMN-induced injury is that endogenous ET-1 will more selectively activate ET_B receptors on the endothelium when ET_A receptors are blocked. This may increase the release of nitric oxide [10], which is known to limit PMN-induced cardiac dysfunction after myocardial ischaemia and reperfusion in the isolated rat heart [37]. Moreover, nitric oxide is known to inhibit adhesion of PMNs [38], partially by inhibition of ICAM-1 expression [39].

In conclusion, the selective ET_A receptor antagonist LU 135252 protects the myocardium from ischaemia/reperfusion injury in the presence of PMNs and inhibits the accumulation of PMNs in the post-ischaemic heart. The results suggest that an important cardioprotective effect is mediated via inhibition of PMN-mediated injury during reperfusion.

Time for primary review 31 days.

	Acknowledgements

 
This study was supported by the Swedish Medical Research Council (10857), the Swedish Heart and Lung Foundation, King Gustav and Queen Victoria Foundation, Magnus Bergvalls Foundation and Laerdal Foundation. We are grateful to Professor Lars Rydén for valuable criticism of the manuscript.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Yanagisawa M, Kurihara H, Kimura S, et al. Endothelin: a novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (1988) 322:411–415.[CrossRef]
2. Rubanyi G.M, Polokoff M.A. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev (1994) 46:325–415.[Web of Science][Medline]
3. Lerman A, Edwards B.S, Hallet J.W, et al. Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. New Engl J Med (1991) 325:997–1001.[Abstract]
4. Miyauchi T, Yanagisawa M, Tomizawa T, et al. Increased plasma concentrations of endothelin-1 and big endothelin-1 in acute myocardial infarction. Lancet (1989) 2:53–54.[CrossRef][Web of Science][Medline]
5. Pernow J, Boutier J.F, Franco-Cereceda A, et al. Potent selective vasoconstrictor effects of endothelin in the pig kidney in vivo. Acta Physiol Scand (1988) 134:573–574.[Web of Science][Medline]
6. Nichols A.J, Koster P.F, Ohlstein E.H. The effect of diltiazem on the coronary haemodynamic and cardiac functional effect produced by intracoronary administration of endothelin-1 in the anaesthetized dog. Br J Pharmacol (1990) 99:597–601.[Web of Science][Medline]
7. Ezra D, Goldstein R.E, Czaja J.F, Feuerstein G.Z. Lethal ischemia due to intracoronary endothelin in pigs. Am J Physiol (1989) 257:H339–H343.[Web of Science][Medline]
8. Arai H, Hori S, Aramori I, et al. Cloning and expression of a cDNA encoding an endothelin receptor. Nature (1990) 348:730–732.[CrossRef][Medline]
9. Sakurai T, Yanagisawa M, Takuwa Y, et al. Cloning of a cDNA encoding a non isopeptide selective subtype of the endothelin receptor. Nature (1990) 348:732–735.[CrossRef][Medline]
10. DeNucci G, Thomas R, D'Orleans-Juste P, et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci USA (1988) 85:9797–9800.[Abstract/Free Full Text]
11. Tønnessen T, Giaid A, Saleh D, et al. Increased in vivo expression and production of endothelin-1 by porcine cardiomyocytes subjected to ischemia. Circ Res (1995) 76:767–772.[Abstract/Free Full Text]
12. Watanabe T, Suzuki N, Shimamoto N, Fujino M, Imada A. Contribution of endogenous endothelin to the extension of myocardial infarct size in rats. Cardiovasc Res (1991) 69:370–377.[CrossRef]
13. Wang Q.-D, Uriuda Y, Pernow J, et al. Myocardial release of endothelin (ET) and enhanced ET_A receptor-mediated coronary vasoconstriction after coronary thrombosis and thrombolysis in pigs. J Cardiovasc Pharmacol (1995) 26:770–776.[Web of Science][Medline]
14. Saito T, Furshimi E, Abe T. Augmented contractile response to endothelin and blunted endothelium-dependent relaxation in post-ischemic reperfused coronary arteries. Jpn Circ J (1992) 56:657–670.[Medline]
15. Watanabe T, Suzuki N, Shimamoto N, Fujino M, Imada A. Contribution of endogenous endothelin to the extension of myocardial infarct size in rats. Circ Res (1991) 69:370–377.[Abstract/Free Full Text]
16. Grover G.J, Dzwonczyk S, Parham C.S. The endothelin-1 receptor antagonist BQ 123 reduces infarct size in a canine model of coronary occlusion and reperfusion. Cardiovasc Res (1993) 27:1613–1618.[Abstract/Free Full Text]
17. Wang Q.-D, Li X.-S, Lundberg J.M, Pernow J. Protective effects of non-peptide endothelin receptor antagonist bosentan on myocardial ischaemic and reperfusion injury in the pig. Cardiovasc Res (1995) 29:805–812.[Abstract/Free Full Text]
18. Gómez Garre D, Guerra M, González E, et al. Aggregation of human polymorphonuclear leukocytes by endothelin: role of platelet activating factor. Eur J Pharmacol (1992) 224:167–172.[CrossRef][Web of Science][Medline]
19. López Farré A, Riesco A, Moliz M, et al. Inhibition by L-arginine of the endothelin-mediated increase in cytosolic calcium in human neutrophils. Biochem Biophys Res Commun (1991) 173:496–500.[Web of Science]
20. Elferink J.G.R, DeKoster B.M. Endothelin-induced activation of neutrophil migration. Biochem Pharmacol (1994) 48:865–871.[CrossRef][Web of Science][Medline]
21. Ishida K, Takshige K, Minakami S. Endothelin-1 enhances superoxide generation of human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. Biochem Biophys Res Commun (1990) 178:884–891.[CrossRef][Web of Science]
22. López Farré A, Riesco A, Espinosa G, et al. Effect of endothelin-1 on neutrophil adhesion to endothelial cells and perfused heart. Circulation (1993) 88:1166–1171.[Abstract/Free Full Text]
23. Hansen P.R. Role of neutrophils in myocardial ischemia and reperfusion. Circulation (1995) 91:1872–1885.[Abstract/Free Full Text]
24. Wright D.G. Human neutrophil degranulation. Methods Enzymol (1988) 162:538–551.[Web of Science][Medline]
25. Shandelya S.M.L, Kuppusamy P, Weisfeldt M.L, Zweier J.L. Evaluation of the role of polymorphonuclear leukocytes on contractile function in myocardial reperfusion injury. Evidence for plasma-mediated leukocyte activation. Circulation (1993) 87:536–546.[Abstract/Free Full Text]
26. Münter K, Hergenröder L, Unger L, Kirchengast M. Oral treatment with a selective ETA-receptor antagonist inhibits neointima formation induced by endothelial injury. Pharm Pharmacol Lett (1996) 6:90–92.
27. Burke S.E, Nelson R.A. Endothelin-receptor antagonist FR 139317 reduces infarct size in a rabbit model when given before, but not after, coronary artery occlusion. J Cardiovasc Pharmacol (1997) 29:87–92.[CrossRef][Web of Science][Medline]
28. Krause S.M, Lynch J.J jr, Stabilito I.I, Woltmann R.F. Intravenous administration of the endothelin-1 antagonist BQ 123 does not ameliorate myocardial ischaemic injury following acute coronary occlusion in the dog. Cardiovasc Res (1994) 28:1672–1678.[Abstract/Free Full Text]
29. McMurdo L, Thiemermann C, Vane J.R. The effects of the ETA receptor antagonist, FR 139317, on infarct size in a rabbit model of acute myocardial ischaemia and reperfusion. Br J Pharmacol (1994) 112:75–80.[Web of Science][Medline]
30. Gonon AT, Wang Q-D, Shimizu M, Pernow J. The novel non-peptide selective endothelin A receptor antagonist LU 135252 protects against myocardial ischaemic and reperfusion injury in the pig. Acta Physiol Scand 1998, in press.
31. Wang Q.-D, Li X.-S, Pernow J. Characterization of endothelin-1-induced vascular effects in the rat heart by using endothelin receptor antagonists. Eur J Pharmacol (1994) 271:25–30.[CrossRef][Web of Science][Medline]
32. Sessa W.C, Kaw S, Hecker M, Vane J.R. The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes. Biochem Biophys Res Commun (1991) 174:613–618.[CrossRef][Web of Science][Medline]
33. Sessa W.C, Kaw S, Zembowicz A, et al. Human polymorphonuclear leukocytes generate and degrade endothelin-1 by two distinct neutral proteases. J Cardiovasc Pharmacol (1991) 17:S34–S38.
34. Patrignani P, Del Maschio A, Bazzoni G, et al. Inactivation of endothelin by polymorphonuclear leukocyte-derived lytic enzymes. Blood (1991) 78:2715–2720.[Abstract/Free Full Text]
35. Wright C.D, Cody W.L, Dunbar J.B jr, et al. Characterization of endothelins as chemoattractants for human neutrophils. Life Sci (1994) 55:1633–1641.[CrossRef][Web of Science][Medline]
36. Hayasaki Y, Nakajima M, Kitano Y, et al. ICAM-1 expression on cardiac myocytes and aortic endothelial cells via their specific endothelin receptor subtype. Biochem Biophys Res Commun (1996) 229:817–824.[CrossRef][Web of Science][Medline]
37. Pabla R, Buda A.J, Flynn D.M, et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Circ Res (1996) 78:65–72.[Abstract/Free Full Text]
38. Kubes P, Suzuki M, Granger D.N. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA (1991) 88:4651–4655.[Abstract/Free Full Text]
39. DeCaterina R, Libby P, Peng H, et al. Nitric oxide decreases cytokine-induced endothelial activation: NO selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest (1995) 96:60–68.[Web of Science][Medline]

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