J. R. Parratt

Preconditioning of the ischaemic myocardium; Involvement of the L-arginine nitric oxide pathway

1 Short periods of coronary artery occlusion protect the heart against the effects of a subsequent prolonged period of ischaemia. This phenomenon is known as preconditioning of the ischaemic myocardium. 2 In mongrel, chloralose‐urethane anaesthetized open‐chest dogs, within a restricted body weight range, two 5 min periods of occlusion of the anterior descending branch of the left coronary artery markedly reduced the severity of the early ischaemic arrhythmias resulting from a prolonged (25 min) occlusion of the same coronary artery starting 20 min later. Thus, the number of ventricular premature beats (VPBs) was reduced from 528 ± 140 in controls to 78 ± 27 in preconditioned dogs, the incidence of ventricular fibrillation (VF) was reduced from 47% to 0% and the incidence of ventricular tachycardia (VT) from 100% to 20%. ST‐segment elevation recorded from electrodes within the ischaemic area, and the degree of inhomogeneity of conduction within the ischaemic area were markedly reduced in these preconditioned dogs. 3 The incidence of VF following reperfusion of the ischaemic myocardium at the end of the 25 min occlusion period was reduced in the preconditioned dogs from 100% to 60%; there was thus a 40% survival from the combined ischaemia‐reperfusion insult compared with 0% in the controls. 4 NG‐nitro‐l‐arginine methyl ester (l‐NAME) an inhibitor of the l‐arginine nitric oxide pathway, given in a dose of 10 mg kg−1 intravenously on two occasions, both before the initial preconditioning occlusion and then again before the prolonged occlusion, partially attenuated the protective effects of preconditioning. There were more VPBs (220 ± 75), a higher incidence of VT (60%) and more episodes of VT (11.5 ± 6.0 compared to 0.7 ± 0.3 episodes in the preconditioned dogs not given l‐NAME); none of the animals survived reperfusion (incidence of VF 100%). The improvement in the severity of the degree of inhomogeneity which resulted from preconditioning was abolished by l‐NAME administration. 5 l‐NAME itself elevated blood pressure (from 96 ± 5 mmHg diastolic to 119 ± 7 mmHg), reduced heart rate (from 155 ± 7 to 144 ±4 beats min−1) but did not change LVEDP, LVdP/dtmax, coronary blood flow, ST‐segment elevation or the degree of inhomogeneity of conduction. When given 10 min before the prolonged coronary artery occlusion in dogs not subjected to preconditioning, l‐NAME had no significant effect on the severity of arrhythmias except for more periods of VT (a mean of 11.7 ± 4.7 episodes per dog). 6 It is concluded from these studies that the generation of nitric oxide contributes to the marked antiarrhythmic effects of preconditioning in the canine myocardium, probably through elevation of cyclic GMP.

Protection of the heart by ischaemic preconditioning: mechanisms and possibilities for pharmacological exploitation.

Ischaemic preconditioning can be defined as the protective adaptive mechanism produced by short periods of ischaemic stress resulting in a marked, albeit temporary, resistance of the myocardium to a subsequent more prolonged period of that same stress. This protection includes reductions in ischaemic cellular damage and in life-threatening ventricular arrhythmias. The most likely mechanisms for this protection are discussed in this review by James Parratt and involve the release of endogenous substances from the ischaemic myocardium (for example, adenosine, bradykinin, nitric oxide and prostacyclin) with the possible involvement of ATP-dependent K+ channels, Gi proteins and protein kinase C. If we understood more fully the precise mechanisms of this pronounced protection, it should be possible to exploit them pharmacologically to ultimate therapeutic advantage.

Incubation with endotoxin activates the L-arginine pathway in vascular tissue

Rat aortic rings incubated with a low dose of endotoxin (100 ng ml-1) for 5 h exhibited depressed reactivity to norepinephrine (NE) which was independent of the presence of endothelium. An inhibitor of nitric oxide synthesis from L-arginine NGmonomethyl-L-arginine (300 microM), but not the inactive D-enantiomer, restored the contractile response of endotoxin-treated rings to control. The effect of NGmonomethyl-L-arginine was reversed by L-arginine (1 mM). In the absence of NGmonomethyl-L-arginine, L- but not D-arginine relaxed endotoxin-treated rings but was without effect on control tissues. This response was reversed following inhibition of guanylate cyclase by methylene blue (3 microM). In addition, tissue cyclic GMP content was 10 times greater in endotoxin-treated compared to control tissue. These data indicate that endotoxin can act directly on vascular tissue to induce a hyporeactivity to NE which is secondary to the activation of the L-arginine pathway and subsequent activation of soluble guanylate cyclase.

The effect of inhibitors of the L-arginine/nitric oxide pathway on endotoxin-induced loss of vascular responsiveness in anaesthetized rats.

1 The effects on blood pressure and on pressor responses to noradrenaline (NA), of NG‐monomethyl‐l‐arginine (l‐NMMA) and NG‐nitro‐l‐arginine methyl ester (l‐NAME), inhibitors of the l‐arginine/nitric oxide pathway, were investigated in anaesthetized rats receiving an infusion of bacterial endotoxin (E. coli lipopolysaccharide, LPS). 2 Infusion of LPS (10 mg kg−1 h−1) for 50min had no effect on mean arterial blood pressure (MABP) but induced a reduction in responsiveness to noradrenaline (100 ng–1 μg kg−1). l‐NMMA (30 mg kg−1), but not d‐NMMA, caused an increase in MABP of approximately 30 mmHg and restored responses to NA. This effect was reversed by l‐ but not d‐arginine (100 mg kg−1). 3 In LPS‐treated rats, blood pressure responses to NA were only marginally increased by the cyclooxygenase inhibitor, indomethacin (5 mg kg−1). l‐NAME (1 mg kg−1) caused a similar increase in MABP and restored pressor responses to NA both in the presence and absence of indomethacin. 4 Co‐infusion of vasopressin (100 ng kg−1, for 10 min) with LPS (10 mg kg−1 h−1) in order to reproduce the hypertensive effect of l‐NMMA and l‐NAME increased pressor responsiveness to 100 and 300 ng kg−1 NA but not to 1 μg kg−1 NA. 5 Infusion of sodium nitroprusside (30 μg kg−1 min−1) decreased responsiveness to NA even when the hypotension was corrected by co‐infusion of vasopressin (50 ng kg−1 min−1). 6 These results demonstrate that the restoration of vascular responsiveness to NA in LPS‐treated anaesthetized rats by inhibitors of the l‐arginine/nitric oxide pathway is stereospecific and reversible. Furthermore, the experiments involving indomethacin suggest that although cyclo‐oxygenase products of arachidonic acid may contribute to the development of LPS‐induced hyporeactivity, the effect of l‐NAME is unlikely to involve inhibition of the cyclo‐oxygenase pathway. Comparison of NA responsiveness during vasopressin and l‐NMMA/l‐NAME‐induced hypertension shows that increasing the blood pressure may modify LPS‐induced hyporeactivity, but cannot account for the complete restoration of responses to NA by l‐NMMA and l‐NAME. These observations suggest that activation of nitric oxide formation from l‐arginine makes a direct contribution to the production of vascular hyporeactivity by LPS in vivo.

Evidence that an L-arginine/nitric oxide dependent elevation of tissue cyclic GMP content is involved in depression of vascular reactivity by endotoxin

1 The aim of this investigation was to study the relationship between contractile responsiveness, activation of the l‐arginine pathway and tissue levels of guanosine 3′:5′‐cyclic monophosphate (cylic GMP) in aortic rings removed from rats 4 h after intraperitoneal administration of bacterial endotoxin (E.coli. lipopolysaccharide, LPS, 20 mg kg−1). 2 LPS‐treatment resulted in a reduction of the sensitivity and maximal contractile response to noradrenaline (NA). 3 Depression of the maximal contractile response was restored to control by 6‐anilo‐5,8‐quinolinedione (LY 83583, 10 μm), which prevents activation of soluble guanylate cyclase. 4 Cyclic GMP levels in tissue from LPS‐treated rats were 2 fold greater than cyclic GMP levels detected in tissue from control (saline‐treated) rats. The LPS‐induced increase in cyclic GMP content was observed both in the presence and absence of functional endothelium. 5 Addition of l‐arginine (1 mm) to maximally contracted aortic rings produced significant relaxation of rings from LPS‐treated rats but not rings from control animals. In the LPS‐treated group, addition of l‐arginine was also associated with a significant increase in cyclic GMP content. l‐Arginine had no effect on the cyclic GMP content of control rings. d‐Arginine (1 mm) was without effect. 6 In rings from LPS‐treated rats, NG‐nitro‐l‐arginine methyl ester (l‐NAME, 300 μm) an inhibitor of nitric oxide (NO) production, increased the contractile response to NA and prevented the LPS‐induced increase in cyclic GMP content. In control rings, l‐NAME increased the NA sensitivity only when the endothelium remained intact and reduced the cyclic GMP content of these rings to that of control endothelium‐denuded rings. 7 These results demonstrate that LPS‐induced hyporeactivity to NA occurs secondarily to activation of the l‐arginine pathway and subsequent activation of soluble guanylate cyclase in vascular tissue. In addition they suggest that LPS induces the production of an NO‐like relaxing factor in non‐endothelial cells.

Release of 5-hydroxytryptamine and histamine from tissues of the rat

5-Hydroxytryptamine (5-HT) occurs in many tissues of the rat, but its association with histamine in tissue mast cells remains in doubt. For example, 5-HT has been found to be concentrated in areas of the skin where histamine levels are low and mast cells are few (Parratt & West, 1957 a). Experiments were therefore designed to study the changes in the 5-HT and histamine content of the tissues of the rat following treatment with drugs. The results reported in this paper show that the release of histamine and disruption of mast cells can occur without the simultaneous release of 5-HT, and vice versa, and it is most unlikely that the mast cells contain any considerable. quantity of 5-HT.

Myocardial and circulatory effects of E. coli endotoxin: modification of responses to catecholamines

1 The predominant acute effect of E. coli endotoxin in anaesthetized, ventilated cats was pulmonary hypertension resulting from a 8–12 fold increase in pulmonary vascular resistance. This was followed by decreases in left ventricular (LV) and systemic arterial pressures and in LV dP/dt max. Recovery occurred within 2–4 min and was dependent upon increased sympathetic drive; recovery did not occur in cats treated with the β‐adrenoceptor blocking drug alprenolol. 2 The pulmonary vasoconstriction was reduced in cats given compound 48/80 and evidence is presented that it results primarily from histamine release. 3 Over the 2–3 h period following endotoxin injection, systemic arterial pressure tended to decrease and heart rate and myocardial metabolic heat production to increase. Myocardial blood flow and LV dP/dt remained fairly stable until the terminal stages of shock. 4 The predominant delayed effects of E. coli endotoxin in cats were a markedly reduced stroke volume, an increase in peripheral vascular resistance and a severe metabolic acidosis (arterial base excess—20 mEq/litre). Arterial pO2 and pCO2 were not significantly affected. It is concluded that myocardial contractility is maintained at this time through the release of catecholamines and that endotoxin itself depresses contractility. 5 The effects of adrenaline and noradrenaline infusions on systolic and diastolic blood pressures, heart rate, cardiac output, myocardial blood flow and LV dP/dt max were markedly reduced in the period 2–3 h after endotoxin. In a few animals some recovery of the response to noradrenaline occurred and was associated with a general circulatory improvement and a reduced metabolic acidosis.

Attenuation of the antiarrhythmic effects of ischaemic preconditioning by blockade of bradykinin B2 receptors

1 The possibility that bradykinin is involved in the pronounced antiarrhythmic effects of ischaemic preconditioning in anaesthetized mongrel dogs was examined with the use of the selective B2 antagonist, icatibant (Hoe‐140). 2 Preconditioning, achieved by two 5 min occlusions of the left anterior descending coronary artery, followed 20 min later by a 25 min occlusion of the same artery resulted, during this prolonged occlusion, in less severe arrhythmias (VF 0% versus 47% in control non‐preconditioned dogs), reductions in the incidence and number of episodes of ventricular tachycardia (VT) and in the number of ventricular premature beats and increased survival following reperfusion (50% versus 0% in the controls). 3 Hoe‐140 was given in a dose of 300μg kg−1 either before the preconditioning procedure or after preconditioning but before the prolonged occlusion. This dose of Hoe‐140 had insignificant haemodynamic effects and failed to modify the increase in coronary blood flow that occurred in the circumflex coronary artery when the anterior descending branch was occluded. 4 It was difficult to precondition dogs in the presence of Hoe–140. There were more ventricular arrhythmias during the initial 5 min occlusion (44 ± 12 versus 10 ± 3) and a higher incidence of ventricular fibrillation (50% versus 21%) during the preconditioning procedure. There was also a more pronounced ST‐elevation (recorded from epicardial electrodes) during the preconditioning occlusions in those dogs given Hoe‐140. 5 In those dogs that survived to the long (25 min) occlusion, Hoe‐140 prevented the antiarrhythmic effects of preconditioning (reduction in ventricular premature beats and in VT) although all the dogs survived the occlusion period. However on reperfusion, survival in the preconditioned dogs given Hoe‐140 was less than in those dogs preconditioned without the B2 antagonist. 6 Changes in the degree of inhomogeneity of conduction within the ischaemic area, which were markedly suppressed by preconditioning, were attenuated in those dogs preconditioned in the presence of Hoe–140. 7 These results suggest that bradykinin acts as both a ‘trigger’ for preconditioning and as one of the mediator protective (antiarrhythmic) substances generated by the myocardium under these conditions. Since the protection afforded both by preconditioning and by local intracoronary infusions of bradykinin is markedly attenuated by an inhibitor of the 1‐arginine nitric oxide pathway, we suggest that much of the protection afforded by ischaemic preconditioning results from the generation of nitric oxide, and that bradykinin, released early during ischaemia, acts as a stimulant for this generation.

Local intracoronary infusions of bradykinin profoundly reduce the severity of ischaemia-induced arrhythmias in anaesthetized dogs.

Bradykinin in a dose (25 ng kg−1min−1) which did not alter coronary flow, or saline, were infused into a small branch of the left anterior descending coronary artery in dogs anaesthetized with chloralose and urethane, for 10 min prior to coronary artery occlusion and throughout the 25 min occlusion period. The degree of inhomogeneity of conduction and epicardial ST‐segment changes were measured in the ischaemic zone with a composite electrode. In control dogs, coronary artery occlusion led to severe arrhythmias with an incidence of ventricular fibrillation of 47% and tachycardia of 80% and with a mean of 528 ± 140 ventricular premature beats. In marked contrast, those dogs administered bradykinin had no ventricular fibrillation or tachycardia and the number of premature beats was significantly less (53 ± 19). ST‐segment changes were also much less in these dogs. These results raise the possibility that bradykinin might contribute to the protective effects of preconditioning and acts as an ‘endogenous myocardial protective substance’.

Prevention by dexamethasone of the marked antiarrhythmic effects of preconditioning induced 20 h after rapid cardiac pacing

Dogs were paced, via a pacing electrode in the right ventricle, for four 5 min periods at a rate of 220 beats min−1. On the following day they were reanaesthetized, thoracotomized and the left anterior descending coronary artery occluded for 25 min. Pacing markedly reduced the severity of ischaemia‐induced arrhythmias (e.g. reduction in VF from 45% in unpaced dogs to 10% in paced dogs; P < 0.05), an effect reversed by dexamethasone (4 mg kg−1 i.v., 45 min prior to pacing). This protection may be due to the induction of nitric oxide synthase or cyclo‐oxygenase.