Endothelin in the healthy and ischaemic myocardium

The effects of intravenous infusion of endothelin-1 (ET-1) on the incidence and severity of arrhythmias induced by a period of myocardial ischaemia were assessed in anaesthetized rats. To determine the role of ETA-receptor-mediated events during ischaemia the effects of the ET^ antagonist, BQ 123, on the pro-arrhythmic actions of both exogenous and endogenous endothelin were examined. Infusion of ET-1 (0.05 and 0.1 nmol.kg"Vmin" 1) resulted in a significant increase in the total number of ventricular ectopic beats (VEBs), and in beats occurring as ventricular tachycardia (VT). There was a marked increase in the incidence of ventricular fibrillation (VF). Over a range of doses of BQ123 (5-100 pg.kg" 1.min" 1), only one dose (10 pg.kg" 1.min" 1) significantly attenuated the total number of VEBs by reducing all types of ischaemic arrhythmias. With the highest dose of BQ 123, there was an increased incidence of irreversible VF. This may reflect the U shaped dose-response curve which has been reported for the antagonism of the pressor effect of ET 1 by BQ123 (Bird et al., 1993). BQ123 (10 pg.kg" 1.min" 1) also significantly reduced the pro arrhythmic effects of infused ET-1. These findings suggest that exogenous ET-1 can intensify ischaemia-induced arrhythmias through the activation of ET^ receptors. However, while endogenous ET-1 may make some contribution to the genesis of arrhythmias resulting from ischaemia by an action at ET^ receptors, the observed pro-arrhythmic effect of high doses of BQ 123 suggests that this may unmask an effect of ET-1 at other receptors. This study also examined the plasma and tissue concentrations of endothelin during coronary artery occlusion and reperfusion. Following 30 min of coronary artery occlusion and 30 min reperfusion the mean immunoreactive ET-1 (IR-ET-1) concentration in right atrial plasma was elevated 2.5-fold and 2.9-fold respectively (from 3.2±0.5 pg.mH in sham-operated animals to 8.2±0.9 and 9.1±1.7 pg.mH). The myocardial tissue mean level of endothelin, unchanged during the ischaemia, increased from 11.2±1.5 pg.g'^ tissue to 17.8±0.7 pg.g‘1 tissue only after 30 min reperfusion. Thus, it is unlikely that the ischaemic heart is the source of the endothelin released into the plasma. To evaluate the involvement of the myocardium in the conversion of big ET-1 to ET-1, the degradation of exogenous big ET-1 (the precursor of ET-1) in rat ventricular homogenates, was studied by using reversed phase HPLC and electrospray mass spectroscopy. Big ET-1 was degraded optimally at pH 7.2 and produced two detectable metabolites. Neither was mature ET-1. One of the fragments was ET-1 sequence 1 17, which was produced in only trace amounts by incubating ET-1 itself with the ventricular extract at pH 7.2. Thus, even if the myocardium produced endothelin, the level of myocardial ET-1 formation was so slow and small as to be unlikely to have a direct influence on circulating levels. The present study also investigated the enzymic activity involved in ET-1 degradation by homogenized rat ventricle and compared it with that in the lung extract. ET-1 was degraded by both ventricular and lung extracts optimally at acid pH. Degradation of ET-1 by the lung and ventricular extracts at pH 5.2 produced respectively 4 and 2 detectable metabolite peaks on HPLC. The two ET-1 metabolites produced by ventricular extracts were identified as tryptophan and des-Trp-ET-1. The degrading activity was not inhibited by EDTA, aprotinin, pepstatin A or captopril. However, phosphoramidon and PMSF (methyl sulfonyl fluoride) produced significant inhibitions. These results indicate that myocardial degradation of ET-1 at pH 5.2 involves cleavage of the C-terminal tryptophan and that more than one enzyme may be involved.

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