Daniel Lamontagne

Endocannabinoids protect the rat isolated heart against ischaemia

The purpose of this study was to determine whether endocannabinoids can protect the heart against ischaemia and reperfusion. Rat isolated hearts were exposed to low‐flow ischaemia (0.5–0.6 ml min−1) and reperfusion. Functional recovery as well as CK and LDH overflow into the coronary effluent were monitored. Infarct size was determined at the end of the experiments. Phosphorylation levels of p38, ERK1/2, and JNK/SAPK kinases were measured by Western blots. None of the untreated hearts recovered from ischaemia during the reperfusion period. Perfusion with either 300 nM palmitoylethanolamide (PEA) or 300 nM 2‐arachidonoylglycerol (2‐AG), but not anandamide (up to 1 μM), 15 min before and throughout the ischaemic period, improved myocardial recovery and decreased the levels of coronary CK and LDH. PEA and 2‐AG also reduced infarct size. The CB2‐receptor antagonist, SR144528, blocked completely the cardioprotective effect of both PEA and 2‐AG, whereas the CB1‐receptor antagonist, SR141716A, blocked partially the effect of 2‐AG only. In contrast, both ACEA and JWH015, two selective agonists for CB1‐ and CB2‐ receptors, respectively, reduced infarct size at a concentration of 50 nM. PEA enhanced the phosphorylation level of p38 MAP kinase during ischaemia. PEA perfusion doubled the baseline phosphorylation level of ERK1/2, and enhanced its increase upon reperfusion. The cardioprotective effect of PEA was completely blocked by the p38 MAP kinase inhibitor, SB203580, and significantly reduced by the ERK1/2 inhibitor, PD98059, and the PKC inhibitor, chelerythrine. In conclusion, endocannabinoids exert a strong cardioprotective effect in a rat model of ischaemia–reperfusion that is mediated mainly through CB2‐receptors, and involves p38, ERK1/2, as well as PKC activation.

Involvement of cannabinoids in the cardioprotection induced by lipopolysaccharide

We have examined the involvement of the endocannabinoid system in the cardioprotection triggered by lipopolysaccharide (LPS). Rats were treated with saline or LPS (10 μg Kg−1). 24 h later, hearts were excised, retrogradely perfused, submitted to a low‐flow ischaemia (0.6 ml min−1) for 90 min and reperfused for 60 min. Some hearts were perfused with either SR 141716A (a cannabinoid CB1, receptor antagonist 1 μM), SR 144528 (a CB2 receptor anagonist μM), NNLA (3 μM) or sodium nitroprusside (1 μM) 5 min before ischaemia and during the ischaemic period. The cardioprotective effects of LPS treatment, in terms of infarction and functional recovery, were not altered by the perfusion of SR 141716A but abolished by both SR 144528 and NNLA. Finally, SR 144528 abolished the beneficial effects of SNP perfusion. Our results suggest an involvement of endocannabinoids, acting through the CB2 receptors, in the cardioprotection triggered by LPS against myocardial ischaemia. This could be attributed to a relationship between cannabinoids and NO.

Endocannabinoids are implicated in the infarct size-reducing effect conferred by heat stress preconditioning in isolated rat hearts

OBJECTIVE We have investigated the involvement of the endocannabinoid system in the delayed cardioprotection conferred by heat stress preconditioning in the isolated rat heart. METHODS Rats were divided into eight groups (n=7 in each group), subjected to either heat stress (42 degrees C for 15 min, HS groups) or sham anaesthesia (Sham groups). Twenty-four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30-min occlusion of the left coronary artery followed by 120 min of reperfusion. Some hearts were perfused with either SR 141716 (a cannabinoid CB(1) receptor antagonist, 1 microM), SR 144528 (a CB(2) receptor antagonist, 1 microM) or L-NAME (a NOS inhibitor, 3 microM) 5 min before ischaemia and during the ischaemic period. RESULTS The infarct size-reducing effect conferred by heat stress (35.7+/-1.8% in Sham to 14.1+/-0.6% in HS groups) was not altered by the perfusion of SR 141716 (11.2+/-1.5%) but was abolished by both SR 144528 (36.6+/-1.6%) and L-NAME (32.0+/-4.4%). In hearts from non-heat-stressed rats, perfusion with SR 141716 (32.8+/-1.6%), SR 144528 (33.4+/-2.2%) and L-NAME (31.6+/-2.9%) had no effect on infarct size. CONCLUSION These results suggest an involvement of endocannabinoids, acting through CB(2) receptors, and NO in the cardioprotection conferred by heat stress against myocardial ischaemia. The possible interaction between both mediators of the heat stress response remains to be determined.

Contribution of endocannabinoids in the endothelial protection afforded by ischemic preconditioning in the isolated rat heart

The aim of the present study was to assess the contribution of endogenous cannabinoids in the protective effect of ischemic preconditioning on the endothelial function in coronary arteries of the rat. Isolated rat hearts were exposed to a 30-min low flow ischemia (1 ml/min) followed by 20-min reperfusion, after which the response to the endothelium-dependent vasodilator, serotonine (5-HT), was compared with that of the endothelium-independent vasodilator, sodium nitroprusside (SNP). In untreated hearts, ischemia-reperfusion diminished selectively 5-HT-induced vasodilatation, compared with time-matched sham hearts, the vasodilatation to SNP being unaffected. A 5-min zero-flow preconditioning ischemia in untreated hearts preserved the vasodilatation produced by 5-HT. Blockade of either CB(1)-receptors with SR141716A or CB(2)-receptors with SR144528 abolished the protective effect of preconditioning on the 5-HT vasodilatation. Perfusion with either palmitoylethanolamide or 2-arachidonoylglycerol 15 min before and throughout the ischemia mimicked preconditioning inasmuch as it protected the endothelium in a similar fashion. This protection was blocked by SR144528 in both cases, whereas SR141716A only blocked the effect of PEA. The presence of CB(1) and CB(2)-receptors in isolated rat hearts was confirmed by Western blots. In conclusion, the data suggest that endogenous cannabinoids contribute to the endothelial protective effect of ischemic preconditioning in rat coronary arteries.

Contribution of angiotensin-converting enzyme to the cardiac metabolism of bradykinin: an interspecies study

The role of angiotensin-converting enzyme (ACE) in the metabolism of bradykinin (BK) has been studied in several tissues. However, and contrary to angiotensin I, the metabolism of BK at the cardiac level has not been investigated. In this study, we define the participation of ACE in the carboxy-terminal degradation of BK in heart membranes of the dog, human, rabbit, and rat. The calculation of the kinetic parameters characterizing the metabolism of BK and the generated des-Arg9-BK can be summarized as follows: the half-life ( t 1/2) of BK [dog (218 ± 32 s) > human (143 ± 9 s) = rat (150 ± 4 s) > rabbit (22 ± 2 s)] and of des-Arg9-BK [dog (1,042 ± 40 s) > human (891 ± 87 s) > rat (621 ± 65 s) > rabbit (89 ± 8 s)] both showed significant differences according to species. Enalaprilat, an ACE inhibitor, significantly prevented the rapid degradation of BK and des-Arg9-BK in all species studied, whereas retrothiorphan, a neutral endopeptidase inhibitor, and losartan, an angiotensin II type I receptor antagonist, did not affect this metabolism. The relative importance of ACE in the cardiac metabolism of BK was species related: dog (68.4 ± 3.2%) = human (72.2 ± 2.0%) > rabbit (47.7 ± 5.0%) = rat (45.3 ± 3.9%). ACE participation in the metabolism of des-Arg9-BK was as follows: rabbit (57.0 ± 4.0%) > dog (39.9 ± 8.8%) = human (25.4 ± 5.5%) = rat (36.0 ± 7.0%). The participation of cardiac kininase I (carboxypeptidase M) in the transformation of BK into des-Arg9-BK was minor: human (2.6 ± 0.1%) > dog (0.9 ± 0.1%) = rabbit (1.0 ± 0.1%) = rat (1.0 ± 0.1%). These results demonstrate that ACE is the major BK-degrading enzyme in cardiac membranes. However, the metabolism of exogenous BK by heart membranes is species dependent. Our observations could explain some discrepancies regarding the contribution of kinins in the cardioprotective effects of ACE inhibitors.The role of angiotensin-converting enzyme (ACE) in the metabolism of bradykinin (BK) has been studied in several tissues. However, and contrary to angiotensin I, the metabolism of BK at the cardiac level has not been investigated. In this study, we define the participation of ACE in the carboxy-terminal degradation of BK in heart membranes of the dog, human, rabbit, and rat. The calculation of the kinetic parameters characterizing the metabolism of BK and the generated des-Arg9-BK can be summarized as follows: the half-life (t1/2) of BK [dog (218 +/- 32 s) > human (143 +/- 9 s) = rat (150 +/- 4 s) > rabbit (22 +/- 2 s)] and of des-Arg9-BK [dog (1,042 +/- 40 s) > human (891 +/- 87 s) > rat (621 +/- 65 s) > rabbit (89 +/- 8 s)] both showed significant differences according to species. Enalaprilat, an ACE inhibitor, significantly prevented the rapid degradation of BK and des-Arg9-BK in all species studied, whereas retrothiorphan, a neutral endopeptidase inhibitor, and losartan, an angiotensin II type I receptor antagonist, did not affect this metabolism. The relative importance of ACE in the cardiac metabolism of BK was species related: dog (68.4 +/- 3.2%) = human (72.2 +/- 2.0%) > rabbit (47.7 +/- 5.0%) = rat (45.3 +/- 3.9%). ACE participation in the metabolism of des-Arg9-BK was as follows: rabbit (57.0 +/- 4.0%) > dog (39.9 +/- 8.8%) = human (25.4 +/- 5.5%) = rat (36.0 +/- 7.0%). The participation of cardiac kininase I (carboxypeptidase M) in the transformation of BK into des-Arg9-BK was minor: human (2.6 +/- 0.1%) > dog (0.9 +/- 0.1%) = rabbit (1.0 +/- 0.1%) = rat (1.0 +/- 0.1%). These results demonstrate that ACE is the major BK-degrading enzyme in cardiac membranes. However, the metabolism of exogenous BK by heart membranes is species dependent. Our observations could explain some discrepancies regarding the contribution of kinins in the cardioprotective effects of ACE inhibitors.

Effects of chronic N-acetylcysteine treatment on the actions of peroxynitrite on aortic vascular reactivity in hypertensive rats.

Background Peroxynitrite (ONOO−), the product of superoxide and nitric oxide, seems to be involved in vascular alterations in hypertension. Objectives To evaluate the effects of ONOO− on endothelium-dependent and independent aortic vascular responsiveness, oxidized/reduced glutathione balance (GSSG/GSH), malondialdehyde aortic content, and the formation of 3-nitrotyrosine (3-NT), a stable marker of ONOO−, in N-acetylcysteine (NAC)-treated normotensive Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Results In SHR only, NAC significantly reduced heart rate and systolic, but not diastolic, blood pressure. It also improved endothelium-dependent aortic relaxation in SHR, but not after exposure to ONOO−. Endothelium-dependent and independent aortic relaxations were markedly impaired by ONOO− in both strains of rat. NAC partially protected SHR against the ONOO−-induced reduction in endothelium-independent relaxation. Aortic GSSG/GSH ratio and malondialdehyde, which were higher in SHR than in WKY rats, showed a greater increase in SHR after exposure to ONOO−. NAC decreased GSSG/GSH and malondialdehyde in both strains of rat before and after exposure to ONOO−. The 3-NT concentration, which was similar in both strains of rat under basal conditions, was greater in SHR than in WKY rats after the addition of ONOO−, with a reduction only in NAC-treated SHR. Conclusions These findings suggest an increased vulnerability of SHR aortas to the effects of ONOO− as compared with those of WKY rats. The selective improvements produced by NAC, in systolic arterial pressure, heart rate, aortic endothelial function, ONOO−-induced impairment of endothelium-independent relaxation, aortic GSSG/GSH balance, malondialdehyde content and 3-NT formation in SHR suggest that chronic administration of NAC may have a protective effect against aortic vascular dysfunction in the SHR model of hypertension.

Role of kinins in the endothelial protective effect of ischaemic preconditioning.

The aim of this study was to assess whether the protective effect of ischaemic preconditioning on endothelial function in coronary arteries of the rat involves kinins. Isolated hearts of the rat were exposed to a 30‐min low‐flow ischaemia (flow rate of 1 ml min−1) followed by 20‐min reperfusion, after which coronaries were precontracted with 0.1 μM U‐46619, and the response to the endothelium‐dependent vasodilator, 5‐hydroxytryptamine (5‐HT, 10 μM), compared to that of the endothelium‐independent vasodilator, sodium nitroprusside (SNP, 3 μM). In untreated hearts, ischaemia‐reperfusion diminished selectively 5‐HT‐induced vasodilatation, compared with time‐matched sham hearts. The vasodilatation to SNP was unaffected after ischaemia‐reperfusion. Preconditioning (5 min of zero‐flow ischaemia followed by 10 min reperfusion) in untreated hearts preserved the vasodilatation produced by 5‐HT. Blockade of B1 and B2 receptors with either 3 nM [Lys0, Leu8, des‐Arg9]‐bradykinin (LLDBK) or 10 nM Hoe 140 (icatibant), respectively, (started 15 min before ischaemic preconditioning or a corresponding sham period and stopped just before the 20‐min reperfusion period) had no effect on the vasodilatation produced by either 5‐HT or SNP in sham hearts. Pretreatment with Hoe 140 did not block the protective effect of ischaemic preconditioning on the 5‐HT vasodilatation. In contrast, LLDBK halved the protective effect of ischaemic preconditioning on endothelium‐dependent vasodilatation. Perfusion with either bradykinin or des‐Arg9‐bradykinin (1 nM) 30 min before and lasting throughout the ischaemia protected the endothelium. In conclusion, ischaemic preconditioning affords protection to the endothelial function in coronary resistance arteries of the rat partly by activation of B1 receptors. Although exogenous BK perfusion can protect the endothelium, B2 receptors do not play an important role in this protection in the rat isolated heart.

Effect of enalaprilat on bradykinin and des‐Arg9‐bradykinin release following reperfusion of the ischaemic rat heart

1 The release of bradykinin (BK) and its metabolite, des‐Arg9‐bradykinin (des‐Arg9‐BK), was studied following reperfusion of a globally ischaemic rat heart. 2 BK‐like immunoreactivity increased from 13±3 (preischaemic value) to 48±12 fmol min−1 g−1 (P<0.05, n = 14) 30 s after reperfusion. No difference in BK release was found between control hearts and hearts pretreated with the angiotensin converting enzyme (ACE or kininase II) inhibitor, enalaprilat (50 ng ml−1). 3 No significant change in des‐Arg9‐BK‐like immunoreactivity during reperfusion was observed in control hearts. In contrast, des‐Arg9 ‐ BK ‐ like immunoreactivity rose from 44 ± 15 to 177 ± 61 fmol min−1 g−1 (P<0.05, n = 7) 30 s after reperfusion in enalaprilat‐treated hearts. 4 In conclusion, BK is released upon reperfusion of the globally ischaemic rat heart. ACE inhibitors, through the inhibition of kininase II, increase the formation of the active metabolite, des‐Arg9‐BK.

Vasorelaxant effects of the chronic treatment with melatonin on mesenteric artery and aorta of spontaneously hypertensive rats

Objective To investigate the effect of a chronic treatment with melatonin on arterial pressure and a possible improvement of the vascular muscarinic and NO synthase (NOS) pathways in spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats. Design and methods Mean arterial pressure (MAP), systolic (SBP), diastolic blood pressure (DBP), and heart rate (HR) were evaluated in conscious rats treated with 30 mg/kg per day of melatonin during 4 weeks. Changes in MAP were evaluated following an intravenous injection of the NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME). Relaxant effects of acetylcholine (Ach), sodium nitroprusside (SNP), and the calcium ionophore A23187 were examined on mesenteric beds and aortic rings with or without treatment with melatonin. Results Melatonin produced a significant reduction of MAP, SBP, DBP and HR in SHR (P < 0.05). l-NAME increased the MAP of melatonin-treated SHR by the same magnitude as that of WKY rats which was significantly higher than that of non-treated SHR (P < 0.05). Melatonin treatment improved the maximal relaxation of mesenteric arteries to A23187 in SHR (P < 0.001) to the WKY level and caused a slight increament in Ach- and A23187-induced vasodilations in aorta from SHR and WKY rats (P < 0.05). Conclusion The present study showed that melatonin exerted a bradycardic and an antihypertensive action in SHR. The enhancement by melatonin of the endothelium-dependent vasodilation (Ach and/or A23187) in mesenteric artery and aorta from SHR and WKY rats and the higher increase in MAP following l-NAME treatment in melatonin-treated SHR suggest the contribution of an improved vascular NOS pathway activity in the hypotensive effect of melatonin.

Mediation by B1 and B2 receptors of vasodepressor responses to intravenously administered kinins in anaesthetized dogs

1 Vasodepressor responses to intravenous (i.v.) injection of bradykinin (BK) and des‐Arg9‐BK, a selective B1 kinin receptor agonist, were characterized following i.v. pretreatment with selective B1 ([Leu8]‐des‐Arg9‐BK) and B2 (Hoe 140) kinin receptor antagonists in anaesthetized dogs. 2 Des‐Arg9‐BK (0.05–3.3 nmol kg−1) produced dose‐dependent decreases in mean arterial blood pressure with a ED50 0.4 nmol kg−1. The vasodepressor effects evoked by des‐Arg9‐BK (0.6 nmol kg−1) and BK (0.2 nmol kg−1) were greater after i.v. and i.a. injections, respectively. 3 The vasodepressor response to BK (0.6 nmol kg−1) but not to des‐Arg9‐BK (0.6 nmol kg−1) was significantly (P < 0.001) blocked by pretreatment with the B2 receptor antagonist, Hoe 140. 4 The vasodepressor response to des‐Arg9‐BK (0.6 nmol kg−1) but not to BK (0.6 nmol kg−1) was significantly (P < 0.001) reduced by pretreatment with the selective B1 receptor antagonist, [Leu8]‐des‐Arg9‐BK. Although both B1 and B2 receptor antagonists caused a transient fall in blood pressure, their inhibitory action was unlikely to be related to a desensitization mechanism. 5 Inhibition of prostaglandin synthesis with indomethacin prevented the vasodepressor response induced by arachidonic acid (1 mg kg−1, i.v.) but not that to BK or des‐Arg9‐BK (0.6 nmol kg−1). 6 These results suggest, firstly, that the vasodepressor responses to i.v. BK and des‐Arg9‐BK are mediated by the activation of B2 and B1 receptors, respectively; secondly, that prostaglandins are not involved in the vasodepressor responses to kinins. These findings provide pharmacological evidence for the existence of functionally active B1 receptors in canine cardiovascular homeostasis.