The aim of the present study was to document the presence of cannabinoid receptors in the rat heart, and to assess the cardioprotective properties of CB(1)- and CB(2)-receptor agonists. Rat isolated hearts were exposed to low-flow ischemia and reperfusion, with selective cannabinoid agonists administered prior to and during the ischemic period. In some hearts, RT-PCR, Western blots, and immunohistological techniques were used to identify and localize both cannabinoid-receptor subtypes. The effect of cannabinoids on infarct size was evaluated in additional hearts using TTC staining. Protein and mRNA for both CB(1)- and CB(2)-receptors were found in rat heart extracts. CB(1)-receptors were localized almost exclusively on arterial and capillary endothelial cells in intact hearts, whereas CB(2)-receptors appeared on cardiomyocytes and endothelial cells of larger arteries. Both the CB(1)-agonist, ACEA (50 nM), and the CB(2)-agonist, JWH015 (50 nM), reduced infarct size. However, only the cardioprotective effect of the CB(1)-agonist was blocked by the NO-synthase inhibitor, N(G)-nitro-l-arginine (30 muM). In conclusion, CB(1)-receptors are present mainly on endothelial cells in the rat heart, and exert their effect through production of NO. In contrast, CB(2)-receptors present on cardiomyocytes exert a cardioprotective effect independent of this endothelial factor.

The aim of the present article is to review the cardioprotective properties of cannabinoids, with an emphasis on the signaling pathways involved. Cannabinoids have been reported to protect against ischemia in rat isolated hearts, as well as in rats and mice in vivo. Although these effects have been observed mostly with a pre-treatment of a cannabinoid, we report that the selective CB(2)-receptor agonist JWH133 is able to reduce infarct size when administered either before ischemia, during the entire ischemic period, or just upon reperfusion. Little is known about the signaling pathways involved in these cardioprotective effects. Likely candidates include protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) since they are activated during ischemia-reperfusion and contribute to the protective effect ischemic preconditioning. The use of pharmacological inhibitors suggests that PKC, p38 MAPK, and p42/p44 MAPK (ERK1/2) contribute to the protective effect of cannabinoids. In addition, perfusion with JWH133 in healthy hearts caused an increase in both p38 MAPK phosphorylation level and activity, whereas the CB(1)-receptor agonist ACEA was associated with an increase in the phosphorylation status of both ERK1 and ERK2 without any change in activity. During ischemia, both agonists doubled p38 MAPK activity, whereas ERK1/2 phosphorylation level and activity during reperfusion were enhanced only by the CB(1)-receptor agonist. Finally, although nitric oxide (NO) was shown to exert both pro and anti-apoptotic effects on cardiomyocytes, with an apparently controversial effect on myocardial survival, our data suggest that NO may contribute to the cardioprotective effect of some cannabinoids.

The pharmacological (and recreational) effects of cannabis have been known for centuries. However, it is only recently that one has identified two subtypes of G-protein-coupled receptors, namely CB1 and CB2-receptors, which mediate the numerous effects of delta9-tetrahydrocannabinol and other cannabinoids. Logically, the existence of cannabinoid-receptors implies that endogenous ligands for these receptors (endocannabinoids) exist and exert a physiological role. Hence, arachidonoylethanolamide (anandamide) and sn-2 arachidonoylglycerol, the first two endocannabinoids identified, are formed from plasma membrane phospholipids and act as CB1 and/or CB2 agonists. The presence of both CB1 and CB2-receptors in the rat heart is noteworthy. This endogenous cardiac cannabinoid system is involved in several phenomena associated with cardioprotective effects. The reduction in infarct size following myocardial ischemia, observed in rats exposed to either LPS or heat stress 24 hours before, is abolished in the presence of a CB2-receptor antagonist. Endocannabinoids and synthetic cannabinoids, the latter through either CB1 or CB2-receptors, exert direct cardioprotective effects in rat isolated hearts. The ability of cannabinoids to reduce infarct size has been confirmed in vivo in anesthetized mice and rats. This latter effect appears to be mediated through CB2-receptors. Thus, the endogenous cardiac cannabinoid system, through activation of CB2-receptors, appears to be an important mechanism of protection against myocardial ischemia.

The aim of this study was to determine whether endothelium-derived mediators and the endocannabinoid system were involved in the cardioprotective effects induced by exogenous kinins, namely bradykinin and its active metabolites, des-Arg(9)-bradykinin. Isolated rat hearts were submitted to a 20-min stabilisation period, followed by 90 min of low-flow ischemia (flow rate, 0.6 ml min(-1)) before a 60-min reperfusion period. Perfusion of bradykinin (BK, 30 nM) or des-Arg(9)-bradykinin (DBK, 30 nM) was initiated 1 min before the ischemia and maintained during the entire ischemic period. Perfusion with BK reduced infarct size, when measured at the end of the 60-min reperfusion. This effect was blocked by the B2-receptor antagonist, HOE140 (30 nM). Likewise, DBK reduced infarct size, effect that was blocked by the B1-receptor antagonist (30 nM Lys(0)-Leu(8)-DBK). The cardioprotective effect of both BK and DBK was abolished by the cannabinoid CB1-receptor antagonist (1 microM SR141716A), but not by the CB2-receptor antagonist (1 microM SR144528). Neither the NO synthase inhibitor, N-nitro-L-arginine (NNLA, 30 microM), the COX inhibitor, indomethacin (2.8 microM), nor the CYP450 inhibitor, clotrimazole (1 microM), prevented the cardioprotective effect of the kinins. However, a combined treatment with those three inhibitors abolished completely the ability of BK and DBK to reduce infarct size. In conclusion, exogenously administered BK and DBK exert a protective effect against ischemia in an isolated heart model. Endothelium-derived mediators such as nitric oxide, prostanoids, and endothelium-derived hyperpolarizing factor, as well as an SR141716A-sensitive mediator, appear to be involved in this beneficial effect.

1 The purpose of this study was to determine whether endocannabinoids can protect the heart against ischaemia and reperfusion. 2 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. 3 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 micro M), 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. 4 The CB(2)-receptor antagonist, SR144528, blocked completely the cardioprotective effect of both PEA and 2-AG, whereas the CB(1)-receptor antagonist, SR141716A, blocked partially the effect of 2-AG only. In contrast, both ACEA and JWH015, two selective agonists for CB(1)- and CB(2)- receptors, respectively, reduced infarct size at a concentration of 50 nM. 5 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. 6 In conclusion, endocannabinoids exert a strong cardioprotective effect in a rat model of ischaemia-reperfusion that is mediated mainly through CB(2)-receptors, and involves p38, ERK1/2, as well as PKC activation.

OBJECTIVE: The aim of this study was to evaluate the contribution of kinin B1 receptors in myocardial ischemia using both pharmacological blockade and gene knockout mice. MATERIAL AND METHODS: Hearts (n = 6-8 per group) from wild type or homozygous B1 receptor gene knockout mice were isolated and perfused using the Langendorff technique. After a 30-min stabilisation period, the left coronary artery was occluded for 30 min followed by 60 min of reperfusion. In two separate groups of wild type hearts, B1 and B2 receptors were blocked with 3 nM of (des-Arg9, Leu8)-bradykinin and 10 nM of Hoe 140, respectively,(started 15 min before ischemia and stopped before the reperfusion). RESULTS: Infarct size to risk zone (I/R) ratio was significantly reduced in hearts of knockout mice (11.3 +/- 2.1%) compared to those of wild type mice (25.7 +/- 1.7%). Furthermore, in wild type mice, I/R was significantly reduced in hearts perfused with the B1 receptor antagonist (12.8 +/- 2.4%) but not in hearts perfused with the B2 receptor antagonist (36.3 +/- 4.4%) compared to untreated hearts. Finally, a RT-PCR technique showed an activation of kinin B1 receptor gene transcription, in wild type hearts, subjected to the ischemia-reperfusion sequence. CONCLUSION: This study demonstrates that B1 receptors are induced during myocardial ischemia where they could play a detrimental role in mice.

The mechanism involved in the induction of kinin B1 receptors in pathological situations is not completely defined. In this study, we evaluated whether p42/p44 mitogen activated protein (MAP) and p38 stress activated protein (SAP) kinases were implicated in the activation of the gene encoding for the B1 receptor after heat stress in rat vascular smooth muscle cells (SMCs). Rat vascular SMCs were incubated with either vehicle, or 4(4-fluorophenyl)-2-(4 methylsulfinylphenyl)-5-(4pyridil)imidaz (SB 203580)(10 microM), a selective inhibitor of the p38 SAP kinase pathway or 2-(2amino-3-methoxyphenyl)4H-1-benzopyran-4-one (PD 98059)(25 microM), a selective inhibitor of the p42/p44 MAP kinase pathway and submitted or not to heat stress (42 degrees C, 20 min). Five hours later, B1 receptor mRNA was detected using a semi-quantitative RT-PCR technique. In the meantime, we characterised p42/p44 MAP kinase activation after heat stress by immunodetection. A basal expression of B1 receptor mRNA was detected in rat vascular SMCs. This expression was increased by heat stress. However, in cells previously incubated with either SB 203580 or PD 98059 and submitted to heat stress, this increase in B1 receptor mRNA was not detected. Moreover, we showed by immunodetection that heat stress was followed by a transient phosphorylation of p42/p44 MAP kinases. In conclusion, both p42/p44 and p38 kinases play a crucial role in the mechanism leading to B1 receptor mRNA induction after heat stress.

We have examined the involvement of the endocannabinoid system in the cardioprotection triggered by lipopolysaccharide (LPS). Rats were treated with saline or LPS (10 microg x Kg(-1)). 24 h later, hearts were excised, retrogradely perfused, submitted to a low-flow ischaemia (0.6 ml x min(-1)) for 90 min and reperfused for 60 min. Some hearts were perfused with either SR 141716A (a cannabinoid CB(1), receptor antagonist 1 microM), SR 144528 (a CB2 receptor anagonist microM), NNLA (3 microM) or sodium nitroprusside (1 microM) 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.

Kinin B(1)-receptors are inducible-receptors. They are absent under basal conditions but expressed following pathophysiological stresses. This study was designed to examine a possible role of nitric oxide (NO) in the mechanism underlying B(1)-receptor induction after heat stress (HS). Rats were divided into six groups, subjected or not to HS (42 degrees C internal temperature, 20 min) without or with treatment with nitro-L-arginine-methylester (L-NAME), a nonselective inhibitor of NO synthase (NOS) isoforms, or L-N(6)-(1-imino-ethyl)lysine (L-NIL), a selective inhibitor of the inducible NOS. Twenty-four hours after HS, rats were injected with bradykinin and [des-Arg(9)]-bradykinin and hypotensive responses were recorded. In six additional groups, B(1)-receptor mRNAs were detected in aorta 5 h after HS or sham treatment. Bradykinin, a B(2)-receptor agonist, induced a hypotension of a similar magnitude in all the groups studied.[des-Arg(9)]-bradykinin, a B(1)-receptor agonist, induced no response in sham rats. In rats previously subjected to hyperthermia, this agonist induced a hypotensive response, which was, respectively, decreased and increased by pretreatment with L-NAME and with L-NIL prior to hyperthermia. RT-PCR results confirmed these in vivo observations. In conclusion, this study suggests a role for NO in B(1)-receptor induction after HS as well as a possible interaction between NOS isoforms.

B1 receptors are inducible receptors expressed only in stressful conditions. The aim of this study was to determine if, in (mREN2)27 transgenic rats, hypertension is associated with the presence of B1 receptors in the cardiovascular system and if a heat stress inducible effect is preserved during hypertension. Age-matched (16 weeks old) heterozygous hypertensive transgenic (mREN2)27 rats (HT rats) and the normotensive control animals (homozygous Sprague-Dawley rats, NT rats) were used. The study was conducted in two parts: in the first part the responsiveness of B1 receptors was studied in rats submitted to heat stress (42 degrees C rectal temperature, 20 min) or sham anaesthesia 24 h before, by recording changes in isometric tension in aortic rings in response to [des-Arg9]-bradykinin, a B1 receptor agonist. In the second part, we studied whether B1 receptor mRNA was present in aorta, heart and kidneys, using a semi-quantitative RT-PCR technique.[des-Arg9]-Bradykinin induced a concentration-dependent relaxation of aortic rings only from animals submitted to prior heat stress. This response was significantly higher in aortic rings from heat stressed HT rats than from heat stressed NT ones. B1 receptor mRNA was undetectable in organs from rats not submitted to heat stress but they were present 5 h after heat stress in aorta, heart and kidneys from both NT and HT rats. In conclusion, arterial hypertension observed in (mREN2)27 rats is not associated with the presence of B1 receptors. However, after heat stress, we observed an increase in responsiveness from HT rat aortas compared to NT ones.