Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC(3) receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH(2) (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC(3) receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC(3)/MC(4) receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(1-24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC(3) receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC(3) receptors, the C-terminal Arg-Phe structure being relevant.     

Tempol reduces reperfusion-induced arrhythmias in anaesthetized rats.

The generation of reactive oxygen species (ROS) contributes to reperfusion-induced arrhythmias. In the present study, the antiarrhythmic effects of tempol and tiron, two membrane-permeable radical scavengers, on reperfusion-induced arrhythmias in rats in vivo were investigated. The anaesthetized rats were subjected to 5 min of left descending coronary artery (LAD) occlusion followed by 30 min of reperfusion. All rats pretreated with saline developed ventricular tachycardia (VT) and ventricular fibrillation (VF) at the onset of reperfusion, and most of the rats died from irreversible VF at the end of reperfusion. However, pretreatment with tempol (30 or 100 mg kg(-1)) 5 min before reperfusion reduced mortality, arrhythmia score and the incidence and duration of VT and VF. In the rats pretreated with high dose of tempol (100 mg kg(-1)), no VF happened and all rats were alive at the end of the experiment. The arrhythmia score was also significantly decreased compared with that of rats pretreated with saline (0.80 +/- 0.4 versus 5.6 +/- 0.4, P < 0.01). Tiron also provided nearly complete protection against reperfusion-induced arrhythmias when given 2 min before reperfusion. On the other hand, intravenous administration of tempol induced decreases in mean arterial pressure (MAP), heart rate (HR) and pressure rate index (PRI), a relative indicator of myocardial oxygen consumption. In order to determine whether the antiarrhythmic effects of tempol were secondary to the reduction of myocardial oxygen consumption, continuous electrical stimulation of the aortic depressor nerve (3 V, 10 ms and 10 Hz) was carried out in a group of rats to induce decreases in MAP, HR and PRI similar to those in the high dose of Tempol group. However, these rats did not show significant changes in the severity of reperfusion-induced arrhythmias. We conclude that both tempol and tiron significantly reduce reperfusion-induced arrhythmias in rats, and this protective action is independent of hemodynamic effects.     

Suppression of reperfusion-induced arrhythmias with combined administration of 5-HT2 and thromboxane A2 antagonists.

1. The effects of the 5-HT2 antagonist, ICI 170,809 and the thromboxane A2 antagonist, ICI 192,605, given alone and in combination (n = 12 per group), were examined in anaesthetized rats. Haemodynamics and arrhythmias induced by permanent coronary artery occlusion or by reperfusion after 5 min of ischaemia were monitored. 2. In a study on reperfusion-induced arrhythmias, the only significant effect of ICI 170,809 (1 mg kg-1, i.v.) was a reduction in the number of ventricular premature beats (VPBs). ICI 192,605 (1 mg kg-1 min-1, i.v.) did not alter reperfusion-induced arrhythmias. However, in combination, when compared with controls, these drugs caused significant reductions in the incidence of ventricular tachycardia (VT), 100% to 58%; ventricular fibrillation (VF), 92% to 33%; and the mortality due to sustained VF, 67% to 17%. There was also a significant reduction in the number of VPBs following reperfusion. 3. In a second study with lower doses of drugs, ICI 170,809 (0.3 mg kg-1) and ICI 192,605 (0.3 mg kg-1 min-1) had no significant effects on reperfusion-induced arrhythmias either alone or in combination. 4. A third study examined the effects of the higher doses of the drugs on ischaemia-induced arrhythmias. Neither drug alone, nor in combination, altered the incidence of ischaemia-induced VT, VF, the mortality, or the number of VPBs. 5. These results indicate that, in contrast to the administration of either drug alone, combined administration of a 5-HT2 antagonist and a thromboxane A2 antagonist caused marked suppression of reperfusion-induced but not ischaemia-induced arrhythmias.     

REPERFUSION ARRHYTHMIAS IN CLOSED-CHEST RATS: THE EFFECT OF MYOCARDIAL NORADRENALINE DEPLETION AND Ca2+-ANTAGONISM

1. The influence of myocardial noradrenaline depletion on the incidence and severity of reperfusion arrhythmias in closed-chest anaesthetized rats was investigated. Five-minute left coronary artery occlusion was carried out via an implanted occluder. Four groups of rats were studied: controls, rats treated with reserpine (5 mg/kg, intraperitoneally) 24 h before occlusion, and rats receiving 0.2 mg/kg gallopamil intravenously 5 min before occlusion either with or without reserpine pretreatment. 2. In the control group all animals had ventricular tachycardia (VT) and fibrillation (VF) immediately after reperfusion. Gallopamil reduced VT to 50% and VF to 25% (P less than 0.05 versus control). In the reserpine group all had VT, and 67% had VF, this being not significantly different from controls. Additional treatment with gallopamil markedly reduced VT and totally prevented VF (P less than 0.05 versus control). 3. Thus, total depletion of myocardial noradrenaline stores neither prevented the occurrence nor reduced the severity of reperfusion arrhythmia in rats, while gallopamil treatment was effective.     

1-(2,6-二甲基苯氧基)-2-(3,4-二甲氧基苯乙胺基)丙烷盐酸盐对麻醉猫、大鼠心肌缺血再灌注所致心律失常的作用

DDPH是具有α肾上腺素受体阻断作用和较弱抗钙作用的新化合物。本实验研究了DDPH对麻醉猫、大鼠心肌缺血再灌注所致心律失常的作用和对猫血压的影响。结果表明,DDPH iv 0.5～3 mg/kg可降低麻醉猫血压,减少麻醉猫冠状动脉左前降支结扎时心肌缺血产生的VEB,减少麻醉猫和大鼠再灌注所致VEB和VT,VF的持续时间,降低VT,VF的发生率。对动物再灌注时由VF引起的死亡率也有降低的趋势。与哌唑嗪比较,两者具有相似的抗麻醉猫和大鼠心肌缺血再灌注所致心律失常的作用。     

Occlusion and reperfusion-induced arrhythmias in rats: involvement of platelets and effects of calcium antagonists.

The role of blood platelets in ischemia- and reperfusion-induced arrhythmias and the efficacy of three calcium blocking drugs (verapamil, diltiazem, and nicardipine) in preventing the arrhythmias were investigated. Using anesthetized rats, we measured platelet count (Pc) continuously in vivo with a Technicon autocounter. Thromboxane B2 (TxB2) and 6-keto-PGF1 alpha levels in blood from coronary sinus were determined by radioimmunoassay (RIA). Myocardial ischemia and arrhythmias were monitored from lead I ECG during and after occlusion of the left anterior descending coronary artery (LAD) for 7 min. Ischemia-induced arrhythmias were mainly ventricular ectopic contractions (VECs), whereas reperfusion produced VECs, ventricular tachycardia (VT), and reversible and irreversible ventricular fibrillation (VF). Both ischemia and reperfusion decreased platelet count and increased TxB2 level in blood from the coronary sinus. The effects of the CEBs were determined at two dose levels (0.1 and 0.3 mg/kg). Each calcium entry blocker (CEB), at both dose levels, significantly inhibited ischemia-induced arrhythmias. Verapamil and diltiazem significantly reduced reperfusion-induced VECs, prevented VT and irreversible VF, and reduced the number of animals with reversible VF. Nicardipine in preventing arrhythmias was not very effective at either dose. The CEBs also inhibited both ischemia- and reperfusion-induced decreases in PC with a moderate increase (up to 7%) as compared with levels in sham-operated controls. The CEBs also significantly reduced TxB2 levels in blood from the coronary sinus. These results indicate that ischemia and postischemic reperfusion both induce platelet aggregation in rats. Aggregating platelets release biologically active substances including thromboxane A2 (TxA2) which exacerbates existing ischemia and facilitates generation of arrhythmias. CEBs inhibit platelet aggregation and TxA2 release and enhance PGI2 synthesis, thereby preventing arrhythmias.     

CDP-choline prevents cardiac arrhythmias and lethality induced by short-term myocardial ischemia–reperfusion injury in the rat: involvement of central muscarinic cholinergic mechanisms

In the present study, we aimed to determine whether cytidine-5'-diphosphatecholine (CDP-choline or citicoline) can improve the outcome of short-term myocardial ischemia-reperfusion injury in rats. Ischemia was produced in anesthetized rats by ligature of the left anterior descending coronary artery for 7 min followed by a reperfusion period of 7 min. Reperfusion-induced ventricular tachycardia (VT), ventricular fibrillation (VF), survival rate, and changes in arterial pressure were evaluated. Saline (1 ml/kg), CDP-choline (100, 250,and 500 mg/kg), or lidocaine (5 mg/kg) was intravenously injected in the middle of the ischemic period. Intracerebroventricular (i.c.v.) mecamylamine (50 microg) or atropine sulfate (10 microg) pretreatments were made 10 min before the coronary occlusion period. Pretreatment with intravenous (i.v.) atropine methylnitrate (2 and 5 mg/kg; i.v.) or bilateral vagotomy was performed 5 min before the induction of ischemia. An in vivo microdialysis study was performed in the nucleus ambiguus area (NA); choline and acetylcholine levels were measured in extracellular fluids. In control rats, VT, VF, and lethality were observed in 85%, 60% and 50% of the animals, respectively. Intravenous CDP-choline produced a short-term increase in blood pressure and reduced the incidence of VT, VF, and lethality dose-dependently when injected in the middle of the ischemic period. CDP-choline at doses of 250 and 500 mg/kg completely prevented death. Intracerebroventricular atropine sulfate pretreatment completely abolished the protective effect of CDP-choline, while mecamylamine pretreatment had no effect on the drug. CDP-choline increased the levels of extracellular choline and acetylcholine in the NA area. Bilateral vagotomy completely abolished the protective effect of CDP-choline in the reperfusion period. Moreover, the intravenous pretreatment with atropine methylnitrate produced dose-dependent blockade in the reduction of VT, VF, and mortality rates induced by CDP-choline. Neither of these pretreatments except mecamylamine affected the pressor effect of CDP-choline. Intracerebroventricular mecamylamine attenuated the increase in blood pressure induced by CDP-choline. In conclusion, intravenously injected CDP-choline prevents cardiac arrhythmias and death induced by short-term myocardial ischemia-reperfusion injury. Activation of central muscarinic receptors and vagal pathways mediates the protective effect of CDP-choline. The protective effect of CDP-choline is not related to its pressor effect.     

Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts

Effects of the calcineurin inhibitor FK506, the platelet-activating factor (PAF) antagonist, and free radical scavenger Ginkgo biloba extract, EGb 761, and their combination on reperfusion-induced ventricular fibrillation (VF), ventricular tachycardia (VT), and recovery of cardiac function were studied after 30 min of global ischemia followed by 2 h of reperfusion in isolated rat hearts. In the first series of studies, rats received a daily (oral) dose of 0, 1, 5, 10, 20, or 40 mg/kg/day FK506 for 10 days. FK506 dose-dependently reduced the incidence of reperfusion-induced total (irreversible plus reversible) VF from a value of 92% for untreated animals to 92% (NS), 83% (NS), 67% (NS), 33% (p<0.05), and 25% (p<0.05), for doses of 1-40 mg/kg/day, respectively, with effects on incidence of VT showing the same pattern. FK506, between 20 and 40 mg/kg/day, also resulted in significant recovery of postischemic cardiac function. In the second series of studies, rats were treated with EGb 761 alone or in combination with FK506. Whereas no significant reduction in arrhythmias or improvement in cardiac function resulted from a single intervention of EGb 761 at 25 mg/kg/day, combined treatment of rats with 25 mg/kg/day of EGb 761 and 1 or 5 mg/kg/day of FK506 resulted in a reduction in total and irreversible VF of 92% and 92% to 42% (p<0.05) and 33% (p<0.05), 25% (p<0.05) and 8% (p<0.05), respectively, versus untreated control animals, paralleled by similar effects on the incidence of VT and accompanied by significant improvements in postischemic cardiac function. Our results demonstrate a novel cardioprotective characteristic of FK506 and suggest that combination therapy by using FK506 plus EGb 761 synergistically improves postischemic cardiac function, while reducing the incidence of reperfusion-induced VF and VT, which may expand the clinical utility of FK506 and allow therapy with FK506 at lower doses than are currently useful.     

R56865, a potent new antiarrhythmic agent, effective during ischemia and reperfusion in the rat heart.

The antiarrhythmic effects of R56865 were characterized both in vivo and in vitro. Four groups (n = 12 per group) of anesthetized rats, subjected to 5- or 30-min coronary artery ligation and reperfusion, were studied: saline, dimethyl sulfoxide (DMSO) carrier, and R56865 (0.5 or 2 mg/kg) were administered as an intravenous (i.v.) bolus before ligation. After 5 min of ischemia, the incidences of reperfusion-induced ventricular tachycardia (VT) and fibrillation (VF), which were high in the saline (100 and 75%, respectively) and DMSO (100 and 82%, respectively) control groups, were abolished with both doses of R56865. With 30 min of ischemia, R56865 (2 mg/kg) significantly reduced the incidences of ischemia-induced VT and VF (from 100 and greater than 50% to 25 and 8%, respectively). For in vitro studies, five groups (n = 12 per group) of isolated rat hearts subjected to 10- or 30-min coronary ligation and reperfusion were studied: unmodified buffer and buffer containing DMSO or R56865 (10(-7), 10(-8), 10(-9) M). After 10 min of ischemia, R56865 (10(-7) M) decreased reperfusion-induced VT and VF (from 100 and 75% in buffer controls to 42 and 8%, respectively) when administered throughout the experiment. With 30 min of ischemia, R5685 (10(-7) M) reduced the incidences of ischemia-induced VT and VF (from 75 and 67% in the buffer controls to 25 and 25%, respectively). Although reperfusion after 30 min of ischemia did not induce VF in any of the groups studied, VT and other arrhythmias did occur and their incidences were reduced significantly by R56865. To investigate whether calcium overload might mediate the effects of R56865, hearts were perfused aerobically with a high-calcium/low-sodium medium. VT and VF occurred in 80% of control hearts; R56865 (10(-7) M) did not prevent these arrhythmias. In conclusion, R56865 exerts a potent effect against ischemia- and reperfusion-induced arrhythmias through a mechanism which appears to operate during ischemia.     

Effect of iloprost on reperfusion-induced arrhythmias and myocardial ion shifts in isolated rat hearts.

Isolated hearts excised from normotensive (NT) and spontaneously hypertensive (SH) rats subjected to transient normothermic global ischemia were used to study the effect of chronic treatment with iloprost on reperfusion-induced arrhythmias and myocardial ion shifts. After 30 min of ischemia, iloprost given s.c. in doses of 10, 50, 100 and 200 micrograms/kg per day for 14 days reduced the incidence of reperfusion-induced ventricular fibrillation (VF) in isolated hearts from the control value of 91 to 83, 75, 50 (P less than 0.05) and 25% (P less than 0.01) respectively, in NT rats. In the SH groups, the incidence of VF was also reduced from 100 to 75, 58, 33 (P less than 0.01) and 17% (P less than 0.001), respectively, with 10, 50, 100 and 200 micrograms/kg per day of iloprost. A similar reduction was observed in the incidence of reperfusion-induced ventricular tachycardia (VT). Ischemia and reperfusion caused significant changes in myocardial ion contents, i.e. an increase in Na+ and Ca2+ and a decrease in K+ and Mg2+ concentrations. The myocardial water content was also increased in parallel to the Na+ gain. The effect of iloprost given s.c. in doses of 50 and 200 micrograms/kg per day for 14 days was also measured on myocardial ion contents after 15- or 30-min ischemia and 30-min ischemia plus 10-min reperfusion. The higher iloprost dose significantly reduced the myocardial Na+, Ca2+ and water gains and the loss of K+ induced by ischemia and reperfusion in the NT and SH groups, while the decrease in Mg2+ content was alleviated only in SH rats. The results suggest that long-term iloprost treatment reduces the incidence of reperfusion-induced VF and VT by preventing Na+, Ca2+ and water accumulation as well as K+ and Mg2+ loss from myocardial tissue.     

Oxytocin: an extremely potent inducer of penile erection and yawning in male rats

The intracerebroventricular (i.c.v.) injection of oxytocin, in doses ranging from 5 to 90 ng (5–90 pmol) induced penile erection and yawning in male rats. Such response was not induced by doses of the peptide higher than 100 ng, nor by equimolar doses of i.c.v. [Arg⁸]vasopressin, ACTH-(1–24), -MSH, rat corticotropin-releasing factor (rCRF), delta sleep-inducing peptide, neurotensin or substance P. Oxytocin-induced penile erection and yawning were prevented by atropine and morphine, but not by methylatropine or the opiate antagonist naloxone. Haloperidol, a dopamine receptor antagonist, was ineffective at low doses; it partially prevented penile erection but not yawning at high doses. Since oxytocin is present not only in the neurohypophysis but also in other brain areas, our results suggest that oxytocin is implicated in the regulation of penile erection and yawning, and provide further evidence that oxytocin acts as a neuropeptide in the central nervous system.     

Pravastatin prevents arrhythmias induced by coronary artery ischemia/reperfusion in anesthetized normocholesterolemic rats

HMG-CoA reductase inhibitors (statins) have been shown to decrease cardiovascular mortality. Since ventricular tachyarrhythmias are closely related to cardiovascular mortality, we tested effects of the hydrophilic statin pravastatin and the lipophilic statin fluvastatin in a rat arrhythmia model of ischemia/reperfusion and simultaneously measured serum total cholesterol level. Anesthetized rats were subjected to 5-min ischemia and 10-min reperfusion after chronic administration of oral pravastatin (0.02, 0.2, or 2 mg/kg), fluvastatin (0.2, 2, or 4 mg/kg), or vehicle for 22 days, once daily. The acute effect of pravastatin (0.2 or 2 mg/kg, once orally) was also observed. Chronically administrated pravastatin significantly reduced the incidence of ischemia-induced ventricular tachycardia (VT) from 70% (control) to 9% at 2 mg/kg, and it reduced the incidence of reperfusion-induced lethal ventricular fibrillation (VF) from 90% (control) to 20% at 0.2 mg/kg. Acute pravastatin and chronically administrated fluvastatin had no significant effect on these arrhythmias. There were no significant changes in blood pressure, heart rate, QT interval, and serum cholesterol among pravastatin-, fluvastatin-, and vehicle-treated groups. Hydrophilic pravastatin prevented reperfusion-induced lethal VF in anesthetized rats by chronic administration independent of its cholesterol lowering effect. This may be a new beneficial role of pravastatin in decreasing cardiovascular mortality.     

Effects of captopril and losartan on myocardial ischemia-reperfusion induced arrhythmias and necrosis in rats.

Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II type 1 (AT (1)) receptor blockers improve ischemia-reperfusion induced arrhythmias and infarct size in several animal models. However, the effects of pretreatment with ACEIs or AT (1) receptor blockers on acute myocardial infarct size and arrhythmias are controversial. Thus, we sought to assess the comparative effects of pretreatment with ACEI captopril and AT (1)-receptor blocker losartan on myocardial infarct size and arrhythmias in a rat model of ischemia-reperfusion. We randomly assigned 92 male Wistar rats for arrhythmias ( n= 60) and necrosis ( n= 32) experiments. To produce arrhythmia, the left main coronary artery was occluded for 7 min, followed by 7 min of reperfusion and to produce necrosis, the the left main coronary artery was occluded for 30 min, followed by 120 min of reperfusion. Captopril (3 mg kg (-1)) and losartan (0.2 and 2 mg kg (-1)) were given intravenously 10 min before occlusion. Captopril reduced the incidences of ventricular fibrillation (VF) and mortality associated with irreversible VF, whereas the studied doses of losartan did not. Captopril also decreased the number of ventricular beats on reperfusion. Losartan 2 mg kg (-1) reduced both the number of ventricular premature beats and the incidence of ventricular tachycardia (VT) on reperfusion, while losartan at dose of 0.2 mg kg (-1) had no effect on these arrhythmias. Compared to the control group, both captopril and losartan reduced myocardial infarct size in the rat model of ischemia-reperfusion, but this was statistically significant for captopril only. In this experimental model, although captopril did not reduce the incidence of reperfusion-induced VT, it was more effective than the AT (1)-receptor blocker losartan at preventing mortality associated with irreversible VF and to reduce myocardial infarct size in rat model of ischemia-reperfusion.     

Minocycline attenuates ischemia-induced ventricular arrhythmias in rats

Minocycline has been shown to protect against myocardial ischemia–reperfusion injury. This study investigated the effects of minocycline on ischemia-induced ventricular arrhythmias in rats. Anesthetized male rats were once treated with minocycline (45 mg/kg, i.p.) 1 h before ischemia in the absence and/or presence of 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002, 0.3 mg/kg, i.v., a PI3K inhibitor) and 5-hydroxydecanoic acid [5-HD, 10 mg/kg, i.v., a specific inhibitor of mitochondrial ATP-sensitive potassium (K_ATP) channels] which were once injected 10 min before ischemia and then subjected to ischemia for 30 min. Ventricular arrhythmias were assessed. L-type Ca²⁺ current was measured by the patch-clamp technique. During the 30-minute ischemia, minocycline significantly reduced the incidence of ventricular fibrillation (VF) (P < 0.05). The duration of VT + VF, the number of VT + VF episodes and the severity of arrhythmias were all significantly reduced by minocycline compared to those in myocardial ischemia group (P < 0.05 for all). Administration of LY294002 or 5-HD abolished the protective effects of minocycline on VF incidence, the duration of VT + VF, the number of VT + VF episodes and the severity of arrhythmias (P < 0.05 for all). In addition, minocycline inhibited L-type Ca²⁺ currents of normal myocardial cell membrane in a dose-dependent manner. This study suggested that minocycline could attenuate ischemia-induced ventricular arrhythmias in rats in which PI3K/Akt signaling pathway, mitochondrial K_ATP channels and L-type Ca²⁺ channels may be involved_.     

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage

BACKGROUND AND PURPOSE: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. EXPERIMENTAL APPROACH: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP--MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. Key results: All shocked rats treated with saline died within 30-35 min. Treatment with NDP--MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. Conclusions and Implications: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.     

MC3-R as a novel target for antiinflammatory therapy

To date five melanocortin receptors (MC-R) have been cloned, identified and shown to have a wide distribution throughout the body and likely many diverse functions. MC1-R, found on melanocytes, is involved in pigmentation, while MC2-R is the classic adrenocorticotropic (ACTH) receptor found on the adrenal cortex and adipocytes. MC3-R, MC4-R and MC5-R are in their infancy with regard to their characterization. MC4-R has generated wide interest for its involvement in obesity, whereas our own studies have indicated a role for MC3-R in experimental inflammation. An ACTH fragment unable to alter circulating corticosterone, ACTH-4-10, acts at murine MC3-R present on peritoneal macrophage to inhibit cytokine formation and subsequent neutrophil extravasation. These findings were confirmed using agonists with a higher degree of selectivity toward MC3-R, such as gamma-2-MSH and the synthetic mixed MC3/4-R agonist MTII. In vitro, all these agents were able to affect macrophage functions, including phagocytosis and production of the CXC chemokine KC. Besides using RT-PCR and cAMP formation assays, the involvement of MC3-R in the antiinflammatory actions of these melanocortins was validated with the antagonist SHU-9119. Together these experimental data support the notion that agonism at MC3-R can be used for the design of novel therapeutics for inflammatory conditions.     

The differential influences of melanocortins on nociception in the formalin and tail flick tests

Melanocortins exert multiple physiological effects that include the modulation of immune responses, inflammation processes, and pain transmission. In the present study we investigated the peripheral activity of natural melanocortins - alpha-, beta-, gamma1- and gamma2-melanocyte stimulating hormone (MSH) - and melanocortin receptor subtypes 3 and 4 (MC3/4 receptor) antagonist HS014 in pain (formalin and tail flick) tests after peptide subcutaneous administration in mice. In the formalin test, among all substances tested only alpha-MSH (1 micromol/kg) statistically significantly inhibited the formalin-induced first phase pain response, however, all tested peptides (except gamma1-MSH) at the dose of 1 micromol/kg produced a pronounced inhibitory effect on nociceptive behavior in the second phase and this activity was comparable with that of indomethacin (reference drug, 5 mg/kg intraperitoneally); beta-MSH was also active at a dose 0.1 micromol/kg. In the tail flick test, alpha-MSH (1 micromol/kg) showed algesic, whereas HS014 (0.5 micromol/kg) and indomethacin (10 mg/kg) exerted analgesic activity. Other peptides did not exert any activity in the tail flick test. These data indicate that peripherally administered melanocortin receptor agonists alpha-MSH, beta-MSH and gamma2-MSH, as well as MC3/4 receptor antagonist HS014 induced antinociception on pain/inflammatory events caused by formalin suggesting a predominant anti-inflammatory role of these peptides.     

Involvement of the melanocortin MC4 receptor in stress-related behavior in rodents

The melanocortin subtype 4 (MC4) receptor has been postulated to be involved in stress and stress-related behavior. We made use of melanocortin MC4 receptor agonists and antagonist to investigate the relationship between the melanocortin MC4 receptor and stress related disorders. The nonspecific melanocortin receptor agonist alpha-melanocyte stimulating hormone (alpha-MSH) and the melanocortin MC4 receptor agonist, Ac-[Nle4,Asp5,D-Phe7,Lys10]alpha-MSH-(4-10)-NH2 (MT II) dose-dependently and significantly reduced the number of licking periods in the rat Vogel conflict test, suggesting that stimulation of the melanocortin MC4 receptor causes anxiogenic-like activity in rats. We synthesized a peptidemimetic melanocortin MC4 receptor selective antagonist, Ac-D-2Nal-Arg-2Nal-NH2 (MCL0020), which has high affinity for the melanocortin MC4 receptor with IC50 values of 11.63 +/- 1.48 nM, in contrast, the affinities for melanocortin MC1 and MC3 receptors were negligible. In addition, MCL0020 significantly attenuated the cAMP formation induced by alpha-MSH in COS-1 cells expressing the melanocortin MC4 receptor without affecting basal cAMP contents. Thus, we considered MCL0020 to be a selective melanocrotin MC4 receptor antagonist among melanocortin receptors. Restraint stress significantly reduced food intake in rats, and i.c.v. administration of MCL0020 dose-dependently and significantly attenuated restraint stress-induced anorexia without affecting food intake. Swim stress induced reduction in the time spent in the light area in the mouse light/dark exploration test, and MCL0020 significantly prevented it. Taken together our findings suggest that the melanocortin MC4 receptor might be related to stress-induced changes in behavior, and blockade of the melanocortin MC4 receptor may prevent stress-induced disorders such as anxiety.     

Evidence for separate peptide sequences related to the lipolytic and magnesium-accumulating activities of ACTH. Analogy with adrenergic receptors.

Native adrenocorticotropin [ACTH-(1-39)] and ACTH-(1-24) stimulate both lipolysis and magnesium accumulation in rat adipocyte plasma membrane vesicles. ACTH-(1-20) retains full lipolytic activity but has a minimal effect on magnesium accumulation. In contrast ACTH-(11-24) stimulates magnesium accumulation but not lipolysis. These findings indicate that within the ACTH molecule the peptide sequence responsible for stimulation of magnesium accumulation is distinctly separate from the core sequence (residues 4-10) essential for stimulation of adenylyl cyclase activity and cAMP mediated lipolysis. Phentolamine, an alpha-adrenergic antagonist, blocks the bulk of magnesium accumulation stimulated by native ACTH and norepinephrine; propranolol, a beta-adrenergic antagonist, blocks the earliest phase of Mg2+ uptake by these hormones but has little effect on net uptake. Isoproterenol, a beta-adrenergic agonist, stimulates magnesium uptake only minimally. The pattern of uptake stimulated by methoxamine, an alpha-adrenergic agonist, or ACTH-(11-24) is quite similar to that produced by native ACTH in the presence of propranolol. The receptor through which ACTH mediates stimulation of the bulk of magnesium appears to be analogous to the alpha-adrenergic receptor through which norepinephrine stimulates this same process.     

Melanocortin MC₄ receptor agonists counteract late inflammatory and apoptotic responses and improve neuronal functionality after cerebral ischemia

Indirect evidence indicates that, in cerebral ischemia, melanocortins have neuroprotective effects likely mediated by MC? receptors. To gain direct insight into the role of melanocortin MC? receptors in ischemic stroke, we investigated the effects of a highly selective MC? receptor agonist. Gerbils were subjected to transient global cerebral ischemia by occluding both common carotid arteries for 10 min. In saline-treated stroke animals, an impairment in learning and memory occurred that, at day 11 after stroke, was associated with hippocampus up-regulation of tumor necrosis factor-α (TNF-α), BAX, activated extracellular signal-regulated kinases (ERK1/2), c-jun N-terminal kinases (JNK1/2) and caspase-3, down-regulation of Bcl-2, and neuronal loss. Treatment for 11days with the selective melanocortin MC? receptor agonist RO27-3225, as well as with the well known non-selective [Nle?,D-Phe?]α-melanocyte-stimulating hormone (NDP-α-MSH) as a reference non-selective melanocortin, counteracted the inflammatory and apoptotic responses, as indicated by the changes in TNF-α, BAX, ERK1/2, JNK1/2, caspase-3 and Bcl-2 protein expression. Furthermore, melanocortin treatment reduced neuronal loss and dose-dependently improved learning and memory. These positive effects were associated with overexpression of Zif268, an immediate early gene involved in injury repair, synaptic plasticity and memory formation. Pharmacological blockade of MC? receptors with the selective MC? receptor antagonist HS024 prevented all effects of RO27-3225 and NDP-α-MSH. These data give direct evidence that stimulation of MC? receptors affords neuroprotection and promotes functional recovery from stroke, by counteracting prolonged and/or recurrent inflammatory and apoptotic responses, and likely by triggering brain repair pathways.