Protection from AMP 579 can be added to that from either cariporide or ischemic preconditioning in ischemic rabbit heart

AMP 579, an adenosine A /A receptor agonist, is cardioprotective when administered at reperfusion. Pretreatment with the Na /H exchanger inhibitor cariporide or ischemic preconditioning (PC) also limits infarct size. To gain insight into the mechanism of AMP 579 we investigated whether its protection could be added to that from either cariporide or PC. rabbit hearts were subjected to 45 min of regional ischemia followed by 3 h of reperfusion. Infarct size in the control group was 55.8 +/- 3.9% of the risk zone. PC significantly reduced infarct size to 26.0 +/- 6.7% (p<0.05). AMP 579 (30 micro g/kg) given just before reperfusion followed by 3 micro g/kg/min infusion for 70 min also limited infarct size (32.1 +/- 1.8%,) but the combination of AMP 579 and PC showed a significantly greater limitation of infarct size (5.5 +/- 2.7%, p < 0.05). Because cariporide pretreatment was so protective (8.5 +/- 3.7% infarction), we had to increase the ischemic insult to 60 min to test for any additive effect of the combination of AMP 579 + cariporide. Infarct size in the untreated group was 66.0 +/- 4.9% of the risk zone. Cariporide (0.5 mg/kg) 5 min prior to ischemia significantly reduced infarct size to 41.5 +/- 7.7%. When cariporide pre-treatment was combined with AMP 579 at reperfusion, infarction was further limited (14.2 +/- 4.5%). Because AMP 579's protection can be added to that of either cariporide or PC, AMP 579's mechanism of protection probably differs from either of them. The combination of AMP 579 + cariporide was particularly efficacious and could be useful in the surgical setting.     

Pharmacological profile of SL 59.1227, a novel inhibitor of the sodium/hydrogen exchanger

1. The NHE1 isoform of the Na(+)/H(+) exchanger plays an important role in the regulation of intracellular pH and in cardiac cell injury caused by ischaemia and reperfusion. SL 59.1227 is a novel imidazolypiperidine Na(+)/H(+) antiport inhibitor which is structurally unrelated to previously described acylguanidine inhibitors such as cariporide. 2. Recovery of pH(i) following an intracellular acid load was measured in CCL39-derived PS120 variant cells, selectively expressing either NHE1 or NHE2 isoforms of the Na(+)/H(+) exchanger. pH(i) recovery was potently and selectively slowed by SL 59.1227 in NHE1-expressing cells (IC(50) 3.3+/-1.3 nM) versus NHE2-expressing cells (2.3+/-1.0 microM). The respective IC(50) values for cariporide were 103+/-28 nM (NHE1) and 73+/-46 microM (NHE2). 3. In anaesthetized rats following left coronary artery occlusion (7 min) and reperfusion (10 min) SL 59.1227 (10 - 100 microg kg(-1) min(-1) i.v.) inhibited ischaemia-mediated ventricular tachycardia (71 - 100%) and reperfusion-induced ventricular fibrillation (75 - 87%) and prevented mortality. Bolus i.v. administration of SL 59.1227 (1 mg kg(-1)) produced anti-arrhythmic effects when administered either before or during ischaemia. 4. Cardiac infarct size was determined in anaesthetized rabbits following left coronary artery occlusion (30 min) and reperfusion (120 min). Infarct size measured as a percentage of the area at risk was 36.2+/-3.4% (control group) versus 15.3+/-3.9% (SL 59.1227 0.6 mg kg(-1) i.v.). 5. SL 59.1227 is the first example of a potent and NHE1-selective non-acylguanidine Na(+)/H(+) exchanger inhibitor. It possesses marked cardioprotective properties.     

Cardioprotective effects of nicorandil in rabbits anaesthetized with halothane: potentiation of ischaemic preconditioning via KATP channels

1. The roles of ATP-sensitive K+ channels (KATP channels) in ischaemic or pharmacological preconditioning in the rabbit heart remain unclear. Infarct limitation by ischaemic preconditioning was abolished by the KATP channel blocker glibenclamide under ketamine/xylazine anaesthesia, but not under anaesthesia induced by pentobarbital. Infarct limitation by the KATP channel opener pinacidil was detected under ketamine/xylazine anaesthesia, but not under pentobarbital anaesthesia. Thus, these effects appear to be anaesthetic dependent. 2. In the present study, we examined whether nicorandil (a KATP channel opener nitrate) exhibits cardioprotective actions under halothane anaesthesia, another commonly used volatile anaesthetic. Control animals were subjected to 40 min coronary occlusion and 120 min reperfusion. Before 40 min ischaemia, the nicorandil group received nicorandil (100 microg/kg per min, i.v., for 10 min), the 5' preconditioning (PC) group received 5 min ischaemia/20 min reperfusion, the 2.5'PC group received 2.5 min preconditioning ischaemia/20 min reperfusion, the nicorandil +2.5'PC group received both nicorandil and 2.5 min ischaemia/20 min reperfusion, the nicorandil +2.5'PC + 5-hydroxydecanoate (5HD) group received both nicorandil and 2.5 min ischaemia/20 min reperfusion in the presence of 5-hydroxydecanoate (5HD; a KATP blocker) and the 5HD group received 5 mg/kg, i.v., 5HD alone. Myocardial infarct size in control (n = 7), nicorandil (n = 5), 5'PC (n = 8), 2.5'PC (n = 5), nicorandil + 2.5'PC (n = 5), nicorandil + 2.5'PC + 5HD (n = 5) and 5HD (n = 4) groups averaged 44.4 +/- 3.6, 41.7 +/- 5.7, 17.8 +/- 3.2,* 34.1 +/- 4.8, 21.3 +/- 4.2,* 39.1 +/- 5.6 and 38.9 +/- 5.0% of the area at risk, respectively (*P <0.05 vs control). 3. Thus, nicorandil alone did not have an infarct size-limiting effect in halothane-anaesthetized rabbits. However, the results suggest that even when nicorandil alone does not demonstrate a direct cardioprotective effect, it may enhance ischaemic preconditioning via KATP channels. Key words: ATP-sensitive K+ (KATP) channel, ischaemic preconditioning, myocardial infarction, nicorandil, rabbit.     

The effect of body temperature on myocardial protection conferred by ischaemic preconditioning or the selective adenosine A1 receptor agonist GR79236, in an anaesthetized rabbit model of myocardial ischaemia and reperfusion

1 The cardioprotective effect of N-[(1S, trans)-2-hydroxycyclopentyl]adenosine (GR79236), an adenosine A1 receptor agonist, was compared with that produced by ischaemic preconditioning in an anaesthetized rabbit model of myocardial ischaemia and reperfusion. In addition, we examined the effect of different body core temperatures on GR79236- or ischaemic preconditioning-induced cardioprotection when administered prior to ischaemia, and on cardioprotection induced by GR79236 administered 10 min prior to the onset of reperfusion. 2 When rabbits were subjected to 30 min occlusion of the left coronary artery, followed by 2 h reperfusion, GR79236 (3 x 10(-8) mol kg-1 i.v. (10.5 microg kg-1 i.v.)) or ischaemic preconditioning (5 min ischaemia followed by 5 min reperfusion), administered or applied 10 min prior to the occlusion, significantly limited the development of infarction. The cardioprotective effect of ischaemic preconditioning was significantly greater than that seen after administration of GR79236. Pre-treatment with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 3.3 x 10(-6) mol kg-1 (1 mg kg-1 i.v.)), prevented the cardioprotective effect of GR79236, but not that of ischaemic preconditioning. 3 Maintaining body core temperature at 38.5 degrees C rather than at 37.0 degrees C did not influence infarct size in control groups of rabbits, but reduced the cardioprotective effect of GR79236 when administered 10 min prior to occlusion or 10 min prior to the onset of reperfusion. The cardioprotective effect of ischaemic preconditioning was not temperature-dependent. 4 In conclusion, myocardial protection conferred by GR79236 in anaesthetized rabbits is mediated via adenosine A1 receptors. Myocardial protection can be conferred when GR79236 is administered before the onset of ischaemia or reperfusion, and is reduced when body core temperature is maintained at 38.5 degrees C rather than at 37.0 degrees C. In contrast, myocardial protection conferred by ischaemic preconditioning is not reduced by adenosine A1 receptor blockade, or by maintaining body core temperature at 38.5 degrees C rather than at 37.0 degrees C. These findings point to distinct differences in the mechanisms of induction of myocardial protection by adenosine A1 receptor agonist and ischaemic preconditioning. They also highlight the need for careful control of body core temperature when investigating the phenomenon of cardioprotection.     

POSSIBLE MECHANISM OF CARDIOPROTECTIVE EFFECT OF ISCHAEMIC PRECONDITIONING IN ISOLATED RAT HEART

The present study is designed to investigate the mechanism of the cardioprotective effect of ischaemic preconditioning. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Four episodes of ischaemic preconditioning markedly reduced LDH and CK release in the coronary effluent and decreased myocardial infarct size. Administration of prazosin (alpha(1)adrenoceptor antagonist) before global ischaemia reduced the extent of ischaemia-reperfusion induced myocardial injury. The cardioprotective effect of ischaemic preconditioning was abolished by prazosin and colchicine (microtubule disaggregator). On the basis of these results, it may be concluded that the cardioprotective effects of ischaemic preconditioning may be mediated through stimulation of alpha(1)adrenoceptors and translocation of PKC.     

Possible role of adrenergic component and cardiac mast cell degranulation in preconditioning-induced cardioprotection.

The present study was designed to investigate the role of adrenergic component and cardiac mast cell degranulation in the cardioprotective effect of ischaemic preconditioning. Isolated rat hearts were subjected to 30 min of global ischaemia followed by 30 min of reperfusion. Ischaemic/norepinephrine (100 microm) preconditioning markedly reduced ischaemia-reperfusion-induced release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and the incidence of ventricular premature beats (VPBs) and ventricular tachycardia/fibrillation (VT/VF) during the reperfusion phase. Moreover, ischaemic/norepinephrine preconditioning significantly reduced ischaemia-reperfusion-induced release of mast cell peroxidase (MPO), a marker of mast cell degranulation. Prazosin (0.1 microm), a alpha(1)adrenoceptor blocker, administered during ischaemic/norepinephrine preconditioning attenuated the cardioprotective and antiarrhythmic effect of ischaemic/norepinephrine preconditioning. MPO release increased immediately after ischaemic/norepinephrine preconditioning and the release was found to be inhibited in hearts subjected to ischaemic/norepinephrine preconditioning in the presence of prazosin. However, prazosin (0.1 microm) treatment per se produced cardioprotective and antiarrhythmic effects and reduced ischaemia-reperfusion-induced MPO release. These findings tentatively suggest that ischaemic preconditioning produced cardioprotective and antiarrhythmic effect by activating alpha(1)adrenoceptors and consequent degranulation of cardiac mast cells. Prazosin administered during ischaemic preconditioning abolished its ameliorative effect. 1999 Academic Press@p$hr Copyright 1999 Academic Press.     

Cytochrome P450 2J3/epoxyeicosatrienoic acids mediate the cardioprotection induced by ischaemic post-conditioning, but not preconditioning, in the rat

1. Cytochrome P450 (CYP) epoxygenases and their arachidonic acid metabolites play a protective role against ischaemia-reperfusion injury. In the present study, we investigated whether endogenous CYP2J3/epoxyeicosatrienoic acid (EET) mediates the cardioprotective effects of ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPost). 2. Male Wistar rats were subjected to two cycles of IPC, consisting of 5 min ischaemia and 5 min reperfusion, followed by 45 min occlusion and 2 h reperfusion; IPost consisted of three cycles of 30 s reperfusion and 30 s re-occlusion at the onset of reperfusion. The selective CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 3 mg/kg) was administered 10 min before ischaemia or during ischaemia 10 min before reperfusion started. Cardiac function was measured continuously with a angiocatheter connected to a fluid-filled pressure transducer and myocardial infarct size was assessed by triphenyl tetrazolium chloride staining at the end of the experiment. 3. Subjecting rats to IPC and IPost similarly improved cardiac function and reduced myocardial infarct size. Interestingly, IPost, but not IPC, significantly increased CYP2J3 mRNA (1.75 ± 0.22 vs 1.0; P < 0.05) and protein (1.62 ± 0.22 vs 1.0; P < 0.05), as well as 11,12-EET synthesis compared to I/R (6.2 ± 0.2 vs 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01). Administration of MS-PPOH before ischaemia significantly decreased 11,12-EET synthesis in both IPC and IPost compared with I/R rats (2.1 ± 0.2, 3.2 ± 0.3 and 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01), but decreased the cardioprotective effects, as evidenced by cardiac function and myocardial infarct size, of IPost only. 4. These data indicate that endogenous activation of CYP2J3/EET may be an essential trigger leading to the protective effects of IPost, but not IPC, in the rat heart.     

The time course of cardioprotection induced by GR79236, a selective adenosine A1-receptor agonist, in myocardial ischaemia-reperfusion injury in the pig

The cardioprotective effects of the selective adenosine A1-receptor agonist, GR79236 (N-[(1S, trans)-2-hydroxycyclopentyl]adenosine), were examined in a porcine model of myocardial ischaemia-reperfusion injury. When pigs were subjected to a 50-min coronary artery occlusion followed by 3-h reperfusion, GR79236 (10 nmol/kg, i.v.) significantly reduced infarct size whether given 10 min before the onset of ischaemia or reperfusion. This effect was independent of the bradycardia induced by GR79236, as it was also observed in animals in which heart rate was maintained by electrical pacing. However, GR79236 administered 10 min after reperfusion did not reduce infarct size. GR79236 had no effect on the incidence or outcome of ventricular dysrhythmias in this pig model of infarction. Similarly, ischaemic preconditioning (IPC, 2 x 10-min ischaemia and 10-min reperfusion) significantly reduced infarct size. The selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 3.3 micromol/kg, i.v.), abolished the haemodynamic and cardioprotective effects of GR79236 and the cardioprotective effects of IPC in anaesthetised pigs. In conclusion, GR79236 exerted a marked cardioprotective effect in a porcine model of myocardial ischaemia-reperfusion injury, provided that it was administered before reperfusion. This suggests that GR79236 may have clinical utility in the treatment of various aspects of ischaemic heart disease.     

In vitro and in vivo pharmacology of a structurally novel Na+-H+ exchange inhibitor, T-162559

1. We investigated the inhibitory effects of a non-acylguanidine Na(+)-H(+) exchange (NHE) inhibitor, T-162559 ((5E,7S)-[7-(5-fluoro-2-methylphenyl)-4-methyl-7,8-dihydro-5(6H)-quinolinylideneamino] guanidine dimethanesulphonate), on NHE-1, and its cardioprotective effect against ischaemia and reperfusion injury in rats and rabbits. 2. T-162559 inhibited human platelet NHE-1 in a concentration-dependent manner, with an IC(50) value of 13+/-3 nmol l(-1), making it 16 and three times more potent than cariporide IC(50): 209+/-75 nmol l(-1), P<0.01) and eniporide (IC(50): 40+/-11 nmol l(-1), P=0.066), respectively. T-162559 also inhibited rat NHE-1 with an IC(50) value of 14+/-2 nmol l(-1), which was five and three times lower than that of cariporide (IC(50): 75+/-7 nmol l(-1), P<0.01) and eniporide (IC(50): 44+/-2 nmol l(-1), P<0.01), respectively. 3. T-162559 inhibited, in a concentration-dependent manner, the reduction in cardiac contractility, progression of cardiac contracture, and increase in lactate dehydrogenase release after global ischaemia and reperfusion in perfused rat hearts. The inhibitory effects of T-162559 were observed at a lower concentration range (10 - 100 nmol l(-1)) than with cariporide and eniporide. T-162559 did not alter basal cardiac contractility or coronary flow after reperfusion, suggesting that it exerts direct cardioprotective effects on the heart. 4. Intravenous administration of T-162559 (0.03 and 0.1 mg kg(-1)) significantly inhibited the progression of myocardial infarction induced by left coronary artery occlusion and reperfusion in rabbits; the infarct size normalized by area at risk was 74+/-6% in the vehicle group, and 47+/-5% and 51+/-7% in the T-162559-0.03 mg kg(-1) and T-162559-0.1 mg kg(-1) groups (both P<0.05), respectively. 5. These results indicate that the new structural NHE-1 inhibitor T-162559 is more potent than cariporide and eniporide and possesses a cardioprotective effect against ischaemia and reperfusion injury in rat and rabbit models.     

Effects of Bordetella pertussis toxin pretreatment on the antiarrhythmic action of ischaemic preconditioning in anaesthetized rats.

1. Bordetella pertussis toxin, which catalyses the ADP-ribosylation of certain guanine nucleotide binding proteins (G proteins), thus functionally uncoupling them from associated receptors, was examined to determine whether it modified the antiarrhythmic effect of ischaemic preconditioning in anaesthetized rats. 2. Pertussis toxin (25 micrograms kg-1, i.p., 48 h prior to heart isolation) attenuated the negative chronotropic effect of acetylcholine (ACh) in rat isolated Langendorff perfused hearts. ACh (10 microM) reduced heart rate by 4% in hearts taken from pertussis toxin-treated animals, compared to a reduction of 57% in hearts taken from animals treated only with vehicle. 3. In anaesthetized rats, ischaemic preconditioning (a single 3 min occlusion of the left main coronary artery followed by 10 min reperfusion) had a pronounced antiarrhythmic effect during a subsequent 30 min period of regional myocardial ischaemia. Compared to hearts receiving only a 30 min period of left coronary occlusion, there was a reduced mortality (67% and 0% for control and preconditioned groups, respectively; P < 0.01) and decreased incidences of ventricular tachycardia (VT) and ventricular fibrillation (VF). Pretreatment with pertussis toxin (25 micrograms kg-1, i.p., 48 h previously) did not modify the arrhythmias associated with a 30 min period of regional myocardial ischaemia, neither did it modify the reduction in mortality (from 56% to 0%; P < 0.05) associated with preconditioning. Furthermore, the decrease in total ventricular premature beat count induced by preconditioning seen in controls (from 427 +/- 130 to 95 +/- 45) was also seen in pertussis toxin-treated rats (from 252 +/- 190 to 57 +/- 25).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of protein kinase C in the reduction of infarct size by N-methyl-1-deoxynojirimycin, an alpha-1,6-glucosidase inhibitor.

Preischaemic treatment with N-methyl-1-deoxynojirimycin (MOR-14), an alpha-1,6-glucosidase inhibitor, attenuates glycogenolysis and lactate accumulation during ischaemia and markedly reduces infarct size in rabbit hearts. In the present study, we have investigated whether protein kinase C (PKC), a principal mediator of ischaemic preconditioning, is also involved in the cardioprotective effect of MOR-14. To assess the effect of PKC inhibition on infarct size in MOR-14-treated hearts, 38 rabbits were subjected to 30 min of ischaemia followed by 48 h of reperfusion. Infarct size, as a per cent of area at risk, was significantly smaller in rabbits administered 100 mg kg(-1) of MOR-14 10 min before ischaemia (17+/-2%, n=10), than in a control group (46+/-5%, n=10). This beneficial effect of MOR-14 was abolished when 5 mg kg(-1) of chelerythrine, a PKC inhibitor, was given 10 min prior to MOR-14 injection (39+/-4%, n=10), although chelerythrine alone did not alter infarct size (43+/-4%, n=8). Further, chelerythrine had no effect on MOR-14-induced attenuation of glycogen breakdown and lactate accumulation in hearts excised at 30 min of ischaemia. Immunoblot analysis of PKC in homogenates of Langendorff-perfused rabbit hearts revealed that MOR-14 significantly increased levels of PKC-epsilon in the particulate fraction at 20 and 30 min of ischaemia and in the cytosolic fraction at 30 min of ischaemia. Taken as a whole, our data suggest that PKC acts downstream of the inhibition of glycogenolysis by MOR-14 to reduce infarct size. Thus, activation of PKC is a more direct mediator of the cardioprotection afforded by MOR-14 than is inhibition of glycogenolysis.     

Possible involvement of insulin, endogenous opioids and calcitonin gene-related peptide in remote ischaemic preconditioning of the brain

The present study has been designed to investigate the role of insulin, endogenous opioids and calcitonin gene related peptide (CGRP) on remote mesenteric ischaemic preconditioning induced reversal of global cerebral ischaemia-reperfusion injury in mice. Bilateral carotid artery occlusion of 10 min followed by reperfusion for 24 hour was employed in present study to produce ischaemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Short-term memory was evaluated using elevated plus maze. Inclined beam walking and resistance to lateral push response, tests were employed to assess motor incoordination. Bilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired short-term memory, motor co-ordination and lateral push response. A preceding episode of mesenteric artery occlusion for 15 min and reperfusion of 15 min (remote mesenteric ischaemic preconditioning) prevented markedly, ischaemia-reperfusion-induced cerebral injury measured in terms of infarct size, loss of short-term memory, motor coordination and lateral push response. Anti-insulin serum, naloxone (an opioid receptor antagonist) and alpha-CGRP (8-37) (a selective CGRP receptor blocker) attenuated the neuroprotective effect of remote mesenteric ischaemic preconditioning. It may be concluded that neuroprotective effect of remote mesenteric ischaemic preconditioning probably is mediated through insulin, endogenous opioids and CGRP released as a consequence of mesenteric ischaemia and reperfusion in mice.     

Pharmacological evidence that inducible nitric oxide synthase is a mediator of delayed preconditioning

Brief periods of myocardial ischaemia preceding a subsequent more prolonged ischaemic period 24-72 h later confer protection against myocardial infarction ('delayed preconditioning' or the 'second window' of preconditioning). In the present study, we examined the effects of pharmacological modifiers of inducible nitric oxide synthase (iNOS) induction and activity on delayed protection conferred by ischaemic preconditioning 48 h later in an anaesthetized rabbit model of myocardial infarction. Rabbits underwent a myocardial preconditioning protocol (four 5 min coronary artery occlusions) or were sham-operated. Forty-eight hours later they were subjected to a sustained 30 min coronary occlusion and 120 min reperfusion. Infarct size was determined with triphenyltetrazolium staining. In rabbits receiving no pharmacological intervention, the percentage of myocardium infarcted within the risk zone was 43.9+5.0% in sham-operated animals and this was significantly reduced 48 h after ischaemic preconditioning with four 5 min coronary occlusions to 18.5+5.6% (P<0.01). Administration of the iNOS expression inhibitor dexamethasone (4 mg kg(-1) i.v) 60 min before ischaemic preconditioning completely blocked the infarct-limiting effect of ischaemic preconditioning (infarct size 48.6+/-6.1%). Furthermore, administration of aminoguanidine (300 mg kg(-1), s.c.), a relatively selective inhibitor of iNOS activity, 60 min before sustained ischaemia also abolished the delayed protection afforded by ischaemic preconditioning (infarct size 40.0+/-6.0%). Neither aminoguanidine nor dexamethasone per se had significant effect on myocardial infarct size. Myocardial risk zone volume during coronary ligation, a primary determinant of infarct size in this non-collateralized species, was not significantly different between intervention groups. There were no differences in systolic blood pressure, heart rate, arterial blood pH or rectal temperature between groups throughout the experimental period. These data provide pharmacological evidence that the induction of iNOS, following brief periods of coronary occlusion, is associated with increased myocardial tolerance to infarction 48 h later.     

Cardioprotection and kallikrein-kinin system in acute myocardial ischaemia in mice

1. Acute myocardial ischaemia and reperfusion trigger cardioprotective mechanisms that tend to limit myocardial injury. These cardioprotective mechanisms remain for a large part unknown, but can be potentiated by performing ischaemic preconditioning or by administering drugs such as angiotensin-I-converting enzyme (kininase II) inhibitors (ACEI). 2. This brief review summarizes the findings concerning the role of tissue kallikrein (TK), a major kinin-forming enzyme, kinins and kinin receptors in the cardioprotection afforded by ischaemic preconditioning (IPC) or by pharmacological postconditioning by drugs originally targeted at the renin-angiotensin system, ACEI and type 1 angiotensin-II receptor blockers (ARB) in acute myocardial ischaemia. Myocardial ischaemia was induced by left coronary occlusion and was followed after 30 min by a 3 h reperfusion period (IR), performed in vivo in mice. The role of the kallikrein-kinin system (KKS) was studied by using genetically engineered mice deficient in TK gene and their wild-type littermates, or by blocking B1 or B2 bradykinin receptors in wild-type mice using selective pharmacological antagonists. 3. Ischaemic preconditioning (three cycles: 3 min occlusion/5 min reperfusion) enhances the ability of the heart of wild-type mice to tolerate IR. Tissue kallikrein plays a major role in the cardioprotective effect afforded by IPC, which is largely reduced in TK-deficient mice. The B2 receptor is the main kinin receptor involved in the cardioprotective effect of IPC. 4. Tissue kallikrein is also required for the cardioprotective effects of pharmacological postconditioning with ACEI (ramiprilat) or ARB (losartan), which are abolished for both classes of drugs in TK-deficient mice. The B2 receptor mediates the cardioprotective effects of these drugs. Activation of angiotensin-II type 2 (AT2) receptor is involved in the cardioprotective effects of losartan, suggesting a functional coupling between AT2 receptor and TK during angiotensin-II type 1 (AT1) receptor blockade. 5. The demonstration of a cardioprotective effect of the KKS in acute myocardial ischaemia involving TK and the B2 receptor and playing a major role in IPC or pharmacological postconditioning by ACEI or ARB, suggests a potential therapeutic approach based on pharmacological activation of the B2 receptor.     

Combined pharmacological preconditioning with a G-protein-coupled receptor agonist,a mitochondrial KATP channel opener and a nitric oxide donor mimics ischaemic preconditioning

1. Although pharmacological preconditioning (PPC) has emerged as an alternative to ischaemic preconditioning (IPC) in cardioprotection, the efficacy of PPC compared with IPC has not been investigated. Because IPC is mediated by complex signalling cascades arising from multiple triggers, we have hypothesized that combined PPC is necessary to mimic IPC. 2. Isolated and perfused rat hearts underwent IPC by three cycles of 5 min ischaemia and 5 min reperfusion before 30 min global ischaemia followed by 120 min reperfusion. Adenosine (30 micromol/L), diazoxide (50 micromol/L) and s-nitroso-N-acetylpenicillamine (SNAP; 50 micromol/L) were added for 25 min just before (pretreatment modality) or 45 min before (PPC modality) the index ischaemia. 3. Ischaemic preconditioning significantly improved isovolumic left ventricular (LV) function and reduced infarct size. Although pretreatment with adenosine, diazoxide or SNAP alone was capable of reducing infarct size, PPC with each drug alone or in a combination of two drugs except for diazoxide plus SNAP failed to reduce infarct size. In contrast, PPC in combination with adenosine, diazoxide and SNAP (triple combination PPC) conferred significant improvement of LV function and reduction of infarct size that was as effective as IPC. 4. Cardioprotection afforded by triple combination PPC was abolished by the Gi/o-protein inhibitor pertussis toxin, the mitochondiral KATP channel inhibitor 5-hydroxydecanoate or the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). 5. Protein kinase C (PKC)-epsilon in the particulate fraction was activated throughout preconditioning ischaemia and reperfusion. Although PKC-epsilon was activated during treatment with adenosine, diazoxide or SNAP alone, it was inactivated after washout. In contrast, PKC-epsilon remained activated after triple combination PPC. The PKC inhibitor chelerythrine abolished activation of PKC-epsilon and cardioprotection afforded by IPC and triple combination PPC. 6. These results demonstrate that combined PPC with a G-protein-coupled receptor agonist, a mitochondrial KATP channel opener and an NO donor is necessary to mimic IPC and such synergistic cardioprotection is associated with enhanced and sustained activation of PKC-epsilon.     

The role of mitochondrial K(ATP) channels in antiarrhythmic effects of ischaemic preconditioning in dogs

1. In the canine a single brief (5 min) coronary artery occlusion protects the myocardium against the severe ventricular arrhythmias and reduces the ischaemic changes that result from a subsequent, more prolonged (25 min) occlusion. The main purpose of the present study was to examine whether mitochondrial K(ATP) channels are involved in this protection. 2. In chloralose-urethane anaesthetized dogs, preconditioning (PC) was induced by a single 5 min period occlusion of the left anterior descending (LAD) coronary artery, 20 min prior to a 25 min occlusion of the same artery. In some of these PC dogs 5-hydroxydecanoate (5-HD; 150 micro g kg(-1) min(-1) by intracoronary infusion) was given over a period of 30 min either before, or after PC. In other dogs the mitochondrial K(ATP) channel opener diazoxide (1 mg kg(-1); i.c.) was given, either alone or in the presence of 5-HD. Control dogs (infused with saline) were simply subjected to a 25 min occlusion and reperfusion. 3. Compared to controls, both PC and diazoxide significantly reduced the number of ventricular premature beats (VPBs; 295+/-67 to 89+/-28 and 19+/-11, respectively; P<0.05), the number of episodes of ventricular tachycardia (VT; 8.3+/-4.2 to 1.6+/-0.9 and 0.2+/-0.1; P<0.05) and the incidences of VT (100 to 43 and 33%; P<0.05) and ventricular fibrilation (VF; 60 to 0 and 17%; P<0.05) during the 25 min occlusion of the LAD. Further, 43% of the PC dogs and 58% of the diazoxide treated dogs survived the combined ischaemia-reperfusion insult (cp. 0% in the controls; P<0.05). The protection afforded by PC and diazoxide was abolished by 5-HD, especially when it was given prior to the PC occlusion. In the presence of 5-HD, three out of 10 dogs fibrillated during the PC occlusion and another three dogs died following reperfusion. Furthermore, there were no survivors in this group from the prolonged ischaemia/reperfusion insult. 5-HD given after PC only attenuated the antiarrhythmic protection. 4. Opening of mitoK(ATP) channels prior to ischaemia by preconditioning and diazoxide protects the myocardium against ischaemia and reperfusion-induced arrhythmias. This protection is abolished if the opening of these channels is prevented by the prior administration of 5-HD but only attenuated if 5-HD is given after preconditioning. The results indicate that opening of mitoK(ATP) channels prior to ischaemia is mandatory for protection against ischaemia and reperfusion-induced arrhythmias.     

Kallidin-like peptide mediates the cardioprotective effect of the ACE inhibitor captopril against ischaemic reperfusion injury of rat heart

1. The potential cardioprotective effect of ACE inhibitors has been attributed to the inhibition of bradykinin degradation. Recent data in rats documented a kallidin-like peptide, which mimics the cardioprotective effect of ischaemic preconditioning. This study investigates in isolated Langendorff rat heart the effect of the ACE inhibitor captopril, the role of bradykinin, kallidin-like peptide, and nitric oxide (NO). 2. The bradykinin level in the effluent of the control group was 14.6 pg ml(-1) and was not affected by captopril in the presence or absence of kinin B2-receptor antagonist, HOE140. 3. The kallidin-like peptide levels were approximately six-fold higher (89.8 pg ml(-1)) and increased significantly by treatment with captopril (144 pg ml(-1)), and simultaneous treatment with captopril and HOE140 (197 pg ml(-1)). 4. Following 30 min ischaemia in the control group, the creatine kinase activity increased from 0.4 to 53.4 U l(-1). In the captopril group and in the captopril+L-NAME group, the creatine kinase activity was significantly lower (18.5 and 22.8 U l(-1)). This beneficial effect of captopril was completely abolished by the kinin B2-receptor antagonist, HOE140, as well as by the kallidin antiserum. 5. Perfusion of the hearts with kallidin before the 30 min ischaemia, but not with bradykinin, yielded an approximately 50% reduction in creatine kinase activity after reperfusion. 6. Pretreatment with L-NAME alone and simultaneously with captopril, and with kallidin, respectively, suggests a kinin-independent action of NO before the 30 min ischaemia on coronary flow and a kinin-dependent action after ischaemia. 7. These data show that captopril increases kallidin-like peptide in the effluent. Kallidin-like peptide via kinin B2 receptor seems to be the physiological mediator of cardioprotective actions of captopril against ischaemic reperfusion injury. HOE140 as well as the kallidin antiserum abolished the cardioprotective effects of captopril.     

Effects of acute and chronic treatment with the sodium hydrogen exchanger 1 (NHE-1) inhibitor cariporide on myocardial infarct mass in rabbits with hypercholesterolaemia

We investigated the cardioprotective effect of acute and chronic sodium hydrogen exchanger 1 (NHE-1) inhibition with cariporide under pathological conditions in rabbits fed an atherogenic diet (0.25% cholesterol, 3% coconut oil), an experimental model of atherosclerosis. New Zealand White rabbits were fed over 4 weeks with normal diet or with atherogenic diet and randomized in 3 subgroups (n=7 in each group); placebo, acute cariporide (0.3 mg/kg, 10 min. before occlusion of left anterior descending coronary artery and chronic cariporide (4 weeks 0.1% in chow). In the final infarction experiments the animals were subjected to 30 min. of myocardial ischaemia by occlusion of a branch of the left anterior descending coronary artery followed by 2 hr of reperfusion. Infarct mass was evaluated by triphenyl-tetrazolium chloride staining and the infarct size expressed as a percentage of area at risk. Besides the assessment of aortic endothelium-dependent function aortic and cardiac vessels were inspected for atherosclerotic lesions. In cholesterol-fed rabbits, the infarct size was significantly increased when compared with normal diet animals (63+/-3% versus 41+/-3%). Acute cariporide treatment reduced the infarct size in normal diet rabbits to 14%+/-3% (66% decrease, P<0.05) as well as in atherogenic diet rabbits to 22+/-3% (65% decrease, P<0.05). Chronic treatment with cariporide also reduced the infarct size significantly: normal diet 19+/-2% (53% decrease, P<0.05), atherogenic diet 32+/-3% (49% decrease, P<0.05). Total cholesterol serum levels in rabbits with atherogenic diet were significantly higher (15.3+/-2.7 mmol/l) than those on a standard diet (0.65+/-0.08 mmol/l). Chronic cariporide treatment significantly attenuated the increase of serum cholesterol (7.9+/-1.9 mmol/l) and improved the lipoprotein pattern. Although the aortas and heart vessels of hypercholesterolaemic animals were without any histological evidence of atherosclerosis they developed endothelial dysfunction (reduced endothelium-dependent relaxation by ACh), which was prevented by chronic cariporide treatment. Acute and chronic treatment with the NHE-1 inhibitor cariporide significantly reduced infarct mass. This effect was associated with improved endothelial function.     

Synergistic protective effect of caspase inhibitors and bFGF against brain injury induced by transient focal ischaemia

We tested the hypothesis that combined use of trophic factors and caspase inhibitors increases brain resistance to ischaemia in mice. Intracerebroventricular administration of bFGF (>10 ng) 30 min after MCA occlusion decreased infarct size and neurological deficit in a dose-dependent manner following 2 h ischemia and reperfusion (20 h). Combined administration of the subthreshold doses of bFGF (3 ng) and caspase inhibitors (z-VAD.FMK, 27 ng or z-DEVD.FMK, 80 mg) reduced infarct volume by 60%, and reduced neurological deficit. Treatment with a subthreshold dose of bFGF (3 ng) extended the therapeutic window for z-DEVD.FMK (480 ng) from 1 to 3 h after reperfusion. Caspase-3 activity in the ischaemic brain was increased 30 min and 2 h after reperfusion but, was significantly reduced in bFGF-treated animals by 29 and 16%, respectively. Caspase-3 activity was not reduced by a direct bFGF effect because addition of bFGF (10 nM - 2 microM) did not decrease recombinant caspase-3 activity, in vitro. Our data show that combining caspase inhibitors and bFGF lengthens the treatment window for the second treatment, plus lowers the dosage requirements for neuroprotection. These findings are important because low doses of caspase inhibitors or bFGF reduce the possibility of side effects plus extend the short treatment window for ischaemic stroke.     

Effects of Y-27632, a selective Rho-kinase inhibitor, on myocardial preconditioning in anesthetized rats

The objective of this study was to examine the effects of Y-27632, a selective Rho-kinase inhibitor, on ischemic preconditioning (IP) and carbachol preconditioning (CP) in anesthetized rats. Administration of Y-27632 (0.1 mg/kg) produced slight, but not significant, reduction in mean arterial blood pressure and suppressed the total number of ventricular ectopic beats (VEBs). IP, induced by 5 min coronary artery occlusion and 5 min reperfusion, decreased the incidence of ventricular tachycardia (VT) from 100 (n=30) to 25% (n=24) and abolished the occurrence of ventricular fibrillation (VF) (40% in control group) during 30 min of ischemia. The incidences of VT and VF in Y-27632+IP group were found to be similar to IP group. Carbachol (4 microg/kg/min for 5 min) induced marked depressions in mean arterial blood pressure, heart rate and attenuated the total number of VEBs, but significant reductions in VT and VF incidences were noted in Y-27632+CP group. Y-27632 infusion for 5 min abolished VF occurrence. Marked reductions in plasma lactate levels were observed in all treatment and preconditioning groups. IP led to marked decrease in malondialdehyde levels. Decreases in infarct size were also observed with all groups when compared to control. These results suggest that infusion of Y-27632 was able to produce cardioprotective effects on myocardium against arrhythmias, infarct size or biochemical parameters and mimic the effects of ischemic preconditioning in anesthetized rats. Therefore, it is likely that inhibition of Rho-kinase is involved in the signaling cascade of myocardial preconditioning.