Degradation of bradykinin, a cardioprotective substance, during a single passage through isolated rat-heart.

Angiotensin converting enzyme (ACE) inhibitors have cardioprotective effects in different species including human. This cardioprotective effect is mainly due to the inhibition of bradykinin (BK) degradation rather than inhibition of the conversion of angiotensin I to angiotensin II. Bradykinin, a nonapeptide, has been considered to be the potential target for various enzymes including ACE, neutral endopeptidase 24.11, carboxypeptidase M, carboxypeptidase N, proline aminopeptidase, endopeptidase 24.15, and meprin. In the present study, the coronary vascular beds of Sprague Dawley rat isolated hearts were perfused (single passage) with Krebs solution alone or with different concentrations of BK i.e. 2.75x10(-10), 10(-7), 10(-6) and 10(-5) M solution. Percent degradation of BK was determined by radioimmunoassay. The degradation products of BK after passing through the isolated rat-hearts were determined using RP-HPLC and mass spectroscopy. All the four doses of BK significantly decreased the perfusion pressure during their passage through the hearts. The percentage degradation of all four doses was decreased as the concentration of drug was increased, implying saturation of a fixed number of active sites involved in BK degradation. Bradykinin during a single passage through the hearts degraded to give [1-7]-BK as the major metabolite, and [1-8]-BK as a minor metabolite, detected on HPLC. Mass spectroscopy not only confirmed the presence of these two metabolites but also detected traces of [1-5]-BK and arginine. These findings showed that primarily ACE is the major cardiac enzyme involved in the degradation of bradykinin during a single passage through the coronary vascular of bed the healthy rat heart, while carboxypeptidase M may have a minor role.

Atrial natriuretic peptide reduces the severe consequences of coronary artery occlusion in anaesthetized dogs.

The aim of the present study was to examine the effects of atrial natriuretic peptide (ANP) on the responses to coronary artery occlusion. In chloralose-urethane anaesthetised mongrel dogs either saline (controls) or human synthetic ANP was infused intravenously (10 microg kg(-1) + 0.1 microg kg(-1) min(-1)), starting 30 min before and continuing 10 min during a 25 min occlusion of the left anterior descending coronary artery (LAD). ANP infusion resulted in a fall in mean arterial blood pressure (by 17 +/- 2 mmHg, p < 0.05), a transient (max. at 5 min) increase in coronary blood flow (by 24 +/- 5 ml min(-1), p < 0.05), and a reduction in coronary vascular resistance (by 0.27 +/- 0.05 mmHg ml(-1), p < 0.05). When the LAD coronary artery was occluded, there was a less marked elevation in left ventricular end-diastolic pressure (LVEDP) in the ANP-treated dogs than in the controls (9.0 +/- 0.9 versus 12.2 +/- 0.8 mmHg, p < 0.05). Compared to the controls, ANP reduced the number of ventricular premature beats (VPBs, 26 +/- 12 versus 416 +/- 87, p < 0.05), the number of episodes of ventricular tachycardia per dogs (VT, 0.7 +/- 0.3 versus 12.4 +/- 4.2, p < 0.05), and the incidences of VT (45% versus 100%, p < 0.05) and ventricular fibrillation (VF 18% versus 57%, p < 0.05) during occlusion. Reperfusion of the ischaemic myocardium at the end of the occlusion period led to VF in all the control dogs (survival from the combined ischaemia-reperfusion insult was therefore 0%), but VF following reperfusion was much less in the dogs given ANP (survival 64%; p < 0.05). The severity of myocardial ischaemia, as assessed from changes in the epicardial ST-segment and the degree of inhomogeneity, was significantly less marked in dogs given ANP. We conclude that ANP protects the myocardium from the consequences of myocardial ischaemia resulting from acute coronary artery occlusion and reperfusion in anaesthetized dogs.

Isosorbide-2-mononitrate reduces the consequences of myocardial ischaemia, including arrhythmia severity: implications for preconditioning.

The effects of the intracoronary administration of isosorbide-2-mononitrate (ISMN; 3 microg kg(-1) min(-1)), a major metabolite of isosorbide dinitrate, were examined in chloralose-urethane anaesthetized dogs before and during a 25 min, acute occlusion of the left anterior descending coronary artery. The only significant haemodynamic effects of ISMN administration were a slight (-11 +/- 2 mmHg) decrease in arterial blood pressure and a decrease (< 12%) in diastolic coronary vascular resistance. Coronary occlusion in the presence of ISMN led to a markedly reduced incidence and severity of ventricular arrhythmias compared to those in control, saline-infused dogs. There were fewer ectopic beats (62 +/- 35 versus 202 +/- 72; p < 0.05), a lower incidence (25% versus 75%; p < 0.05) and number of episodes (0.7 +/- 0.4 versus 4.3 +/- 2.1; p < 0.05) of ventricular tachycardia and fewer dogs fibrillated during the ischaemic period (17% versus 82%; p < 0.05). More dogs given ISMN survived the combined ischaemia-reperfusion insult (50% versus 0%; p < 0.05). Changes in ST-segment elevation (recorded by epicardial electrodes) and in the degree of inhomogeneity of electrical activation within the ischaemic area were much less pronounced throughout the occlusion period in dogs given ISMN. These results add weight to the hypothesis that the previously reported antiarrhythmic effects of ischaemic preconditioning, and of the intracoronary administration of nicorandil, involve nitric oxide.

The effects of Z13752A, a combined ACE/NEP inhibitor, on responses to coronary artery occlusion; a primary protective role for bradykinin.

The effects on the responses to coronary artery occlusion of a combined ACE/NEP inhibitor (Z13752A) were examined in anaesthetized dogs. A 1 h infusion of Z13752A (128 microgram kg(-1) min(-1) intravenously) decreased arterial blood pressure (by 11+/-3%; P<0. 05) and increased coronary blood flow (by 12+/-4%, P<0.05). There were no other significant haemodynamic changes. Z13752A inhibited both NEP and ACE enzymes both in dog plasma and in tissue (lung ACE; kidney NEP). Pressor responses to angiotensin I in vivo were inhibited and systemic vasodilator responses to bradykinin were potentiated. When the left anterior descending coronary artery was occluded for 25 min, Z13752A markedly reduced the severity of the resultant ventricular arrhythmias. No ventricular fibrillation (VF) occurred (compared to 7/16 in the controls; P<0.05), and ventricular tachycardia (VT) was reduced (VT in 2/9 dogs treated with Z13752A cp. 16/16 of controls; episodes of VT 0.2+/-0.1 c.p. 10.7+/-3.3; P<0. 05). Reperfusion of the ischaemic myocardium led to VF in all control dogs but occurred less frequently in dogs given Z13752A (survival from the combined ischaemia-reperfusion insult 67% c.p. 0% in controls; P<0.05). Z13752A reduced two other indices of ischaemia severity; epicardial ST-segment elevation and inhomogeneity of electrical activation. These protective effects of Z13752A during ischaemia and reperfusion were abolished by the administration of icatibant (0.3 mg kg(-1), i.v.) a selective antagonist of bradykinin at B(2) receptors; the ischaemic changes in dogs given both icatibant and Z13752A were similar to those in the controls. We conclude that this ACE/NEP inhibitor is effective at reducing the consequences of coronary artery occlusion in this canine model and that this protection is primarily due to potentiation of released bradykinin. British Journal of Pharmacology (2000) 129, 671 - 680

Delayed protection against ventricular arrhythmias by monophosphoryl lipid-A in a canine model of ischaemia and reperfusion.

Bacterial endotoxin reduces the severity of ventricular arrhythmias which occur when a coronary artery is occluded several hours later. We have now examined in anaesthetised dogs the effects on ischaemia and reperfusion-induced arrhythmias, of a non-toxic derivative component of the endotoxin molecule of the lipid-A (monophosphoryl lipid-A). This was given intravenously, in doses of 10 and 100 microg kg(-1), 24 h prior to coronary artery occlusion. Arrhythmia severity was markedly reduced by monophosphoryl lipid-A. During ischaemia, ventricular premature beats were reduced from 315+/-84 in the vehicle controls to 89+/-60 (with the lower dose of monophosphoryl lipid-A) and 53+/-23 (P<0.05) with the higher dose. The incidence of ventricular tachycardia was reduced from 75% to 25% (P<0.05) and 31% (P<0.05), and the number of episodes of ventricular tachycardia from 13.4+/-4.9 per dog to 1.1+/-1.1 (P<0.05) and 1. 2+/-0.9 (P<0.05) after doses of 10 and 100 microg kg(-1), respectively. The incidence of ventricular fibrillation during occlusion and reperfusion in the control group was 96% (15/16), i.e., only 6% (1/16) dogs survived the combined ischaemia-reperfusion insult. Monophosphoryl lipid-A (100 microg kg(-1)) significantly reduced the incidence of occlusion-induced ventricular fibrillation (from 50% to 7%; P<0.05), and increased survival following reperfusion to 54% (P<0.05). Monophosphoryl lipid-A also significantly reduced ischaemia severity as assessed from ST-segment elevation recorded from epicardial electrodes as well as the degree of inhomogeneity of electrical activation within the ischaemia area. There were no haemodynamic differences prior to coronary occlusion between vehicle controls and monophosphoryl lipid-A-treated dogs. These results demonstrate that monophosphoryl lipid-A reduces arrhythmia severity 24 h after administration. Although the precise mechanisms are still unclear, there is some evidence that nitric oxide and prostanoids (most likely prostacyclin) may be involved because the dual inhibition of nitric oxide synthase and cyclooxygenase enzymes by administration of aminoguanidine and meclofenamate abolished the marked antiarrhythmic protection resulted from monophosphoryl lipid-A treatment 24 h previously.

Repeated cardiac pacing extends the time during which canine hearts are protected against ischaemia-induced arrhythmias: role of nitric oxide.

Right ventricular pacing in lightly anaesthetized dogs (4x5 min periods at a pacing rate of 220 beats/min) protects against the consequences of coronary artery occlusion when this is initiated 24 h after the pacing stimulus. The main purpose of the present experiments was to determine whether repeating the pacing stimulus, at a time when protection from the initial stimulus had faded (48 h), prolonged the protection afforded against ischaemia-induced ventricular arrhythmias and other ischaemic changes (epicardial ST-segment mapping; changes in the degree of electrical inhomogeneity in the ischaemic region). Dogs were paced on two occasions, with a 48 h period between and, at different times (48, 72 and 96 h) after the second pacing stimulus, were re-anaesthetized and subjected to occlusion of the left anterior descending coronary artery. There was a marked reduction in the severity of ischaemia-induced arrhythmias 48 and 72 h after the second pacing stimulus (reduction in occlusion-induced and reperfusion-induced ventricular fibrillation, e.g. at 72 h 0/11 during occlusion and only 3/11 following reperfusion, compared to 7/21 and 10/21 respectively in the controls P<0.05). The protection had disappeared 96 h following the second pacing stimulus. Changes in ST-segment elevation and in the degree of inhomogeneity largely followed these changes in the severity of ventricular arrhythmias. The results suggest the possibility of maintaining protection against life-threatening arrhythmias following coronary occlusion by repeating a preconditioning pacing stimulus. We also demonstrate that this prolonged protection afforded by repeated cardiac pacing is mediated by nitric oxide, since the marked antiarrhythmic effect observed, e.g. 72 h after the second pacing stimulus, was abolished when S-(2-aminoethyl)-isothiourea (AEST), a particularly selective inhibitor of iNOS, had been administered before coronary artery occlusion.

The influence of maturation and gender on the anti-arrhythmic effect of ischaemic preconditioning in rats.

The aim of this study was to investigate the influence of maturation and gender on the anti-arrhythmic effect of myocardial ischaemic preconditioning in rats. Coronary artery occlusion was carried out in either rats anaesthetised with sodium pentobarbitone or in rat isolated hearts. Cardiac arrhythmias occurring in the 30 min post-occlusion period were assessed. In anaesthetised 3 month (m) old male rats ischaemic preconditioning, with a 3 min temporary coronary artery occlusion, significantly reduced the total number of ventricular ectopic beats (VEBs) from 2074 +/- 206 to 490 +/- 139 and the incidence of ventricular fibrillation (VF) from 40 to 0% during a subsequent 30 min occlusion (P < 0.05). In middle-aged male rats (16 m) the anti-arrhythmic effect of preconditioning was unaltered (VEBs were reduced from 1958 +/- 121 to 245 +/- 66 and VF from 70 to 0%). In 3 m old anaesthetised female rats the effect of ischaemic preconditioning was also evident (VEBs reduced from 961 +/- 170 to 154 +/- 48; P < 0.05). In non-preconditioned age-matched female animals the total number of VEBs (961 +/- 170), VF (0%) and mortality (0%) were significantly (P < 0.05) lower than in respective male animals. In female rats, attenuation of ischaemia-induced arrhythmic severity was most pronounced in the oestrus state. In hearts isolated from weight-matched male and female rats the incidence of ventricular tachycardia (81 vs 25%) and the total number of VEBs (351 +/- 73 vs 81 +/- 50) were significantly (P < 0.05) different. It is concluded that in rats neither maturation nor gender influence the anti-arrhythmic effect of ischaemic preconditioning. However, female rats exhibit a lower level of arrhythmic activity during sustained coronary artery occlusion than male rats both in vivo and in vitro.

Coronary vascular endothelium-myocyte interactions in protection of the heart by ischaemic preconditioning.

Because ischaemia preconditioning is a general phenomenon - it is present in almost all tissues and organs - we must look for a mechanism which involves cell types common to all organs. The hypothesis outlined below is that endothelial cells, in response to a brief ischaemic stimulus, release protective mediators which (in the heart) diffuse to cardiac myocytes to increase resistance to a subsequent ischaemic stress. These substances include nitric oxide, generated through initial bradykinin release, and prostacyclin. The evidence includes measurement of mediator release and the pharmacological attenuation of the antiarrythmic effects of preconditioning by blockade of bradykinin receptors and inhibition of nitric oxide production.

Coronary endothelial dysfunction increases the severity of ischaemia-induced ventricular arrhythmias in rat isolated perfused hearts.

In order to determine the role of coronary vascular endothelial cells in generating cardioprotective substances during myocardial ischaemia, rat isolated hearts, perfused at constant flow by the Langendorff technique, were subjected to treatment with the detergent Triton X100 and the responses of these hearts to a 30 or 60 min period of coronary artery occlusion was determined. Endothelial damage or denudation was shown both by histological examination and by the altered vasodilator response to the endothelium-dependent vasodilator bradykinin (100 nM), which was reversed to vasoconstriction in hearts treated with Triton X-100. In contrast, the responses to sodium nitroprusside (100 microM) were unimpaired in these hearts and were not different from control responses. Ventricular ectopic activity was much more pronounced in hearts with endothelial dysfunction (e.g., 3329 +/- 361 ventricular premature beats over a 30 min occlusion period compared to 243 +/- 34 in controls; P < 0.01), and the duration of ventricular tachycardia was greatly increased (1162 +/- 391 s v 9 +/- 12 s in the controls; P < 0.01). Ventricular ectopic activity was still marked when the occlusion was prolonged to 1 h and was still apparent at the end of this 1 h occlusion period. Reperfusion arrhythmias (ventricular tachycardia and ventricular fibrillation) were marked in endothelium-damaged hearts (50%); whereas there were no such arrhythmias after a 30 or 60 min occlusion period in control hearts. Hearts were also preconditioned by a 3 min coronary artery occlusion period 10 min prior to a 30 min coronary artery occlusion. This reduced ventricular ectopic activity in both control and endothelium-damaged hearts to about the same extent (between 80 and 90% suppression). The results suggest that under normal conditions substances generated from endothelial cells protect the myocardium against ventricular arrhythmias both during ischaemia and reperfusion. However, in this species, preconditioning is still possible in hearts from which the coronary vascular endothelium has been removed. If these results can be extrapolated to the clinical situation, it suggests that in patients with endothelial dysfunction ventricular arrhythmias may be more pronounced following a period of ischaemia and especially of reperfusion.

Monophosphoryl lipid A reduces both arrhythmia severity and infarct size in a rat model of ischaemia.

A non-toxic derivative of the active lipid A component of the endotoxin molecule (monophosphoryl lipid A) when given to rats in a dose of 5 mg kg(-1) by intraperitoneal injection 24 h prior to anaesthesia and coronary artery occlusion, markedly decreased the severity of ischaemia-reduced ventricular arrhythmias (ventricular fibrillation reduced from 60 to 21%; P < 0.05) and reduced myocardial infarct size (from 35.8 +/- 1.6% of the area at risk to 22.7 +/- 2.0%; P < 0.05). It did not modify blood pressure or heart rate either before or during the period of ischaemia.

The dopamine receptor agonist Z1046 reduces ischaemia severity in a canine model of coronary artery occlusion.

Z1046, (S)-6[[6-[[2-(2-methoxyphenoxy)ethyl]amino]propyl]amino]-5,6,7,8-tetra-h ydro-1,2-naphtalenediol dihydrochloride, is an agonist at both dopamine D1 and D2 receptors. Since stimulation of dopamine D2 receptors inhibits noradrenaline release, and because cardiac noradrenaline release has been implicated in the genesis of early ischaemia-induced, life-threatening ventricular arrhythmias, the effect of Z1046 has been examined for its effects on coronary artery occlusion in chloralose urethane anaesthetised mongrel dogs. Z1046 (10 microg kg(-1) intravenously or 1 microg kg(-1) by local intracoronary injection) decreased heart rate and reduced arterial blood pressure and coronary blood flow, effects prevented by the prior administration of domperidone (40 microg kg(-1) i.v.). The ischaemic changes induced by a 25-min occlusion of the left anterior descending coronary artery (including ST-segment elevation and ventricular ectopic activity) were much less marked in those dogs administered Z1046 and survival from the combined ischaemia reperfusion insult was increased from 7% to 36% (P < 0.05). These effects of Z1046 were partly attenuated by domperidone. We conclude that the anti-ischaemic effects of Z1046 are due to inhibition of cardiac sympathetic responses. Studies using rat isolated perfused mesenteric vascular bed preparations subjected to sympathetic nerve stimulation confirmed that Z1046 inhibits synaptic transmission without modifying vascular responses to noradrenaline.

Tolerance to ischaemia and ischaemic preconditioning in neonatal rat heart.

Although there is much information on ischaemic preconditioning in the adult myocardium, this phenomenon has not yet been investigated in neonatal hearts. To examine the early development of cardiac tolerance to ischaemia and the possible protective effects of preconditioning, rat hearts isolated on days 1, 4 and 7 of postnatal life were perfused (Langendorff) with Krebs-Henseleit solution at constant pressure, temperature (37 degrees C) and rate (200 beats/min). Developed force (DF) of contraction was measured by an isometric force transducer, and analysed using an on-line computer. Hearts were exposed to 40 or 60 min of global ischaemia followed by 30 min of reperfusion. Preconditioning was induced by three 3-min periods of global ischaemia, each separated by 5-min periods of reperfusion. Developmental changes in expression of protein kinase C (PKC) isoforms, and their activation following preconditioning, were estimated using Western blot analysis. Recovery of contractile function during reperfusion decreased from day 1 (48 +/- 2%) to day 4 (42 +/- 1%) and day 7 (33 +/- 2%). Preconditioning failed to improve ischaemic tolerance on day 1 (46 +/- 2%) and on day 4 (43 +/- 3%), but pronounced effect was observed on day 7 (40 +/- 2%). Prolonging the period of sustained ischaemia from 40 to 60 min on day 1 did not lead to a better recovery of contractile function in preconditioned hearts. PKC isoforms alpha, delta, epsilon and zeta were expressed in the ventricular myocardium during the first week of life, but there was no evidence of translocation following preconditioning on day 7. It may be assumed that the decreasing tolerance of the heart to ischaemia during early postnatal life is counteracted by the development of an endogenous protection.

Nitric oxide in sepsis and endotoxaemia.

Nitric oxide (NO) is one of many vasoactive substances released, from a variety of cells, under conditions of endotoxaemia and sepsis. Under physiological conditions it is produced by two constitutive calcium-dependent enzymes (nitric oxide synthase; NOS) in neurones (nNOS) and endothelial cells (eNOS) and has functions ranging from neurotransmission and vasodilatation to inhibition of platelet adhesion and aggregation. Following bacterial infection, especially with Gram-negative organisms, its formation from L-arginine is enhanced due to the cytokine-mediated induction of a NOS enzyme (iNOS) in cells (e.g. cardiac myocytes, vascular smooth muscle) that do not normally have the ability to synthesize NO. The result of this excessive NO production is enhanced bacterial lysis by activated macrophages, vasoplegia and myocardial depression. These cardiovascular effects can be alleviated by inhibitors of the L-arginine NO pathway, which results in elevated perfusion pressure, restored responsiveness to sympathetic nerve stimulation and to exogenous catecholamines, and to enhanced (endothelin-dependent) myocardial contractility. In patients in shock this approach also leads to detrimental effects (increased systemic vascular resistance, elevated pulmonary artery pressure, reduced cardiac output and oxygen delivery, increased platelet accumulation) and survival is not improved. Because some of these detrimental effects are due to inhibition of eNOS, attempts have been made to examine the effects of substances with a higher selectivity for the induced form of the enzyme. In experimental animals, one of these (L-canavanine) protects endothelial cells from damage, increases survival time and restores vascular responsiveness without increasing blood pressure or peripheral vascular resistance. However, whether even this approach will be of benefit to patients with sepsis remains in doubt since studies in iNOS knock-out mice do not support the concept that eliminating this particular source of NO improves ultimate survival.

Lack of involvement of mast cell degranulation in the antiarrhythmic effect of preconditioning in rats.

It has been proposed that the cardioprotective effects of myocardial ischaemic preconditioning may involve the release of mast cell mediators. The aim of the study was to determine whether mast cells are involved in the antiarrhythmic effect of ischaemic preconditioning in rat hearts. Preconditioning was achieved, both in anaesthetised rats and in rat isolated hearts, by a 3-min temporary occlusion of the left main coronary artery followed by 10 min of reperfusion before a 30-min permanent occlusion. Preconditioning had a marked antiarrhythmic effect, reducing the number of ventricular ectopic beats from 1,176 +/- 69 to 490 +/- 139 and the incidence of ventricular fibrillation from 40% to 0. Administration of the mast cell-stabilising drugs lodoxamide tromethamine and sodium cromoglycate (20 mg/kg/h i.v. 30 min before and throughout experimental protocol) did not modify the antiarrhythmic effect of preconditioning. Sodium cromoglycate, but not lodoxamide tromethamine, itself significantly reduced the number of ectopic beats that occurred over a 30-min period of ischaemia (from 760 +/- 181 to 153 +/- 33 in nonpreconditioned animals). Both drugs abolished the decrease in arterial blood pressure that occurred on coronary artery occlusion. The decrease in arterial blood pressure produced by the mast cell-degranulating compound 48/80 (50 microg/kg; i.v.) was attenuated to a similar degree by both drugs (decreases in pressure of 53 +/- 7, 31 +/- 1, and 25 +/- 3 mm Hg in control, sodium cromoglycate-treated, and lodoxamide tromethamine-treated animals, respectively). In rat isolated hearts, degranulation of mast cells with three consecutive doses of 50 microg of compound 48/80 had no antiarrhythmic effects and did not modify the antiarrhythmic effect of preconditioning. It is concluded that cardiac mast cells do not play a major role in the protection offered by ischaemic preconditioning on arrhythmogenesis in rat hearts.

Does the opening of ATP-sensitive K+ channels modify ischaemia-induced ventricular arrhythmias in anaesthetised dogs?

These experiments were designed to determine whether there is any change in the severity of ventricular arrhythmias resulting from coronary artery occlusion in anaesthetised mongrel dogs if ATP-sensitive potassium channels were already open at the time of coronary occlusion. To achieve this we locally infused the K(ATP) channel opener levcromakalim, in a total dose of 3 microg/kg, and given by slow infusion over a 30 min period directly into a side branch of the left anterior descending coronary artery. This dose increased blood flow in that main artery by 30% (and by 7% in the adjacent left circumflex artery). The degree of inhomogeneity of electrical activation, measured from the left ventricular wall distal to the occlusion site, was unaffected by levcromakalim administration but there was significant epicardial ST-elevation, perhaps indicating K+ egression from cells. Following coronary artery occlusion there was no marked difference in the severity of arrhythmias between control and levcromakalim-treated dogs, except for an increased number of episodes of ventricular tachycardia due entirely to effects in two of the nine treated dogs. We conclude that opening cardiac K(ATP) channels with levcromakalim, at this one dose level, and administered directly to the left ventricular wall, does not significantly modify arrhythmia severity during ischaemia. These results cannot be extrapolated to studies in which such drugs markedly reduce coronary perfusion pressure.

Bradykinin and endothelial-cardiac myocyte interactions in ischemic preconditioning.

Myocardial ischemia results in the release of a variety of vasoactive substances from coronary vascular endothelial cells and/or from cardiac myocytes. Some of these substances appear to be protective and include nitric oxide and bradykinin. One hypothesis for the pronounced antiarrhythmic effects of preconditioning involves the early generation of bradykinin and, subsequently, nitric oxide. Evidence for early bradykinin release has come from clinical studies involving patients undergoing coronary angioplasty where, in 4 of 5 patients, there was evidence for elevated kinin levels in coronary sinus blood either during balloon inflation (i.e., ischemia) or deflation (reperfusion). The levels reached are sometimes considerable (increases 10-20 fold). The second piece of evidence comes from dogs subjected to a preconditioning stimulus (2 x 5 min periods of ischemia), followed 20 min later by occlusion of the same artery for a 25-min period. This preconditioning procedure markedly reduces ischemia-induced ventricular arrhythmias and, although under resting conditions there was little difference between arterial and coronary sinus bradykinin levels (125 +/- 22 and 157 +/- 41 pg/mL, respectively), there was a marked increase in coronary sinus levels in preconditioned dogs before the prolonged occlusion (637 +/- 293 pg/mL compared with 114 +/- 18 pg/mL in nonpreconditioned dogs); levels at the end of the prolonged occlusion in the preconditioned dogs were also higher (577 +/- 305 pg/mL compared with 162 +/- 34 pg/mL in control dogs). Other evidence for the involvement of bradykinin and nitric oxide comes from studies in which the generation, or effects, of these mediators have been suppressed (e.g., with the bradykinin B2 receptor blocking agent icatibant, with inhibitors of the L-arginine-nitric oxide pathway, and by methylene blue). The conclusion is that early bradykinin release is protective under conditions of ischemia, is presumably enhanced during therapy with angiotensin-converting enzyme (ACE) inhibitors and is suppressed under conditions of endothelial dysfunction.

Modification by bradykinin B2 receptor blockade of protection by pacing against ischaemia-induced arrhythmias.

In dogs, rapid cardiac pacing, by way of a pacing electrode in the right ventricle, protects against ventricular arrhythmias when a coronary artery is occluded immediately after cessation of the pacing period. This represents a form of ischaemic preconditioning. The role of bradykinin in mediating the protective effects of rapid cardiac pacing in this model was investigated using a selective antagonist of bradykinin at B2 receptors (icatibant; HOE 140). In the presence of icatibant cardiac pacing (220 beats min(-1)) resulted in more severe ischaemia (as assessed by ST-segment elevation from the pacing electrode at the end of the stimulus) and to a higher incidence of ventricular arrhythmias during the pacing protocol. When the coronary artery was occluded under such conditions the antiarrhythmic protection afforded by cardiac pacing was not seen although other indices of reduced ischaemia severity (epicardial ST-segment mapping; changes in the degree of inhomogeneity of electrical activation within the ischaemic area) were not affected by icatibant treatment. These results suggest that bradykinin is an important trigger mediator involved in the protective effects of cardiac pacing. Whether this is due to the generation of endothelium-derived protective substances (such as nitric oxide and prostacyclin) or whether it results from a direct effect on B2 receptors in cardiac myocytes is unclear.

The case of low-dose aspirin for the prevention of myocardial infarction: but how low is low?

Although there is firm consensus that aspirin reduces the incidences (and severity) of reinfarction if given to patients after a myocardial infarction or in patients with unstable angina, there is disagreement about the optimum dose that should be used. We make the case that it could be considerably lower than the "medium dose" (75-320 mg daily) in current usage and put forward the view that a dose of 30 mg daily is sufficient. The arguments are based on the presystemic, exclusively pharmacokinetic inhibition of thromboxane synthesis in platelets by very low-dose aspirin and the importance of maintaining prostacyclin production by endothelial cells. Little attention has been in the past to the endogenous myocardial protection that results from prostacyclin generation. Such low doses have the additional advantage of not inducing gastrointestinal ulceration. We conclude that more extensive clinical trials with such "low-dose" aspirin preparations are warranted.

Nitric oxide. A key mediator in sepsis and endotoxaemia?

A very large number of biologically active substances are released into the circulation under conditions of endotoxaemia and sepsis. One of the most important of these is nitric oxide. Under these conditions nitric oxide is produced through an induced enzyme (nitric oxide synthase) in a variety of tissues and the nitric oxide so generated is largely responsible for the loss of vascular reactivity, which occurs under these conditions, for the resulting unrelenting hypotension associated with the hypodynamic phase of septic shock. Nitric oxide also contributes to the myocardial depression in this condition. The question as to whether it is a worthwhile therapeutic approach to inhibit nitric oxide synthase is discussed with particular reference to the generation of inhibitors selective for the induced form of the enzyme. This approach has certain benefits but may also be detrimental. The fact that nitric oxide is not the key mediator involved in ultimate mortality in this condition is suggested by the failure to improve mortality in iNOS knockout mice given endotoxin.

Genistein, an inhibitor of tyrosine kinase, prevents the antiarrhythmic effects of preconditioning.

The possible involvement of tyrosine kinase in the signal transduction processes associated with the antiarrhythmic effects of ischaemic preconditioning was assessed by the administration, prior to the preconditioning stimulus, of the tyrosine kinase inhibitor genistein (5 mg/kg i.v.) to pentobarbitone anaesthetised male rats. The hearts were then removed, perfused by the Langendorff technique and preconditioned by a single brief (3 min) coronary artery occlusion prior to the 30 min test ischaemic insult. Preconditioning reduced ventricular arrhythmias during occlusion (e.g., premature ventricular beats from 333 +/- 60 in controls to 52 +/- 5 in preconditioned hearts; P < 0.05) but this protective effect was not observed in the hearts of rats given genistein (272 +/- 37 premature beats). A similar abolition of the protective effects of preconditioning could be demonstrated in hearts perfused in vitro with genistein (100 microM). This suggests the involvement of tyrosine kinase activation in this powerful form of endogenous cardioprotection.