Melanocortins and the cholinergic anti-inflammatory pathway.

Experimental evidence indicates that small concentrations of inflammatory molecules produced by damaged tissues activate afferent signals through ascending vagus nerve fibers, that act as the sensory arm of an "inflammatory reflex". The subsequent activation of vagal efferent fibers, which represent the motor arm of the inflammatory reflex, rapidly leads to acetylcholine release in organs of the reticuloendothelial system. Acetylcholine interacts with α7 subunit-containing nicotinic receptors in tissue macrophages and other immune cells and rapidly inhibits the synthesis/release of tumor necrosis factor-α and other inflammatory cytokines. This neural anti-inflammatory response called "cholinergic anti-inflammatory pathway" is fast and integrated through the central nervous system. Preclinical studies are in progress, with the aim to develop therapeutic agents able to activate the cholinergic anti-inflammatory pathway. Melanocortin peptides bearing the adrenocorticotropin/α-melanocyte-stimulating hormone sequences exert a protective and life-saving effect in animals and humans in conditions of circulatory shock. These neuropeptides are likewise protective in other severe hypoxic conditions, such as prolonged respiratory arrest, myocardial ischemia, renal ischemia and ischemic stroke, as well as in experimental heart transplantation. Moreover, experimental evidence indicates that melanocortins reverse circulatory shock, prevent myocardial ischemia/reperfusion damage and exert neuroprotection against ischemic stroke through activation of the cholinergic anti-inflammatory pathway. This action occurs via stimulation of brain melanocortin MC3/MC4 receptors. Investigations that determine the molecular mechanisms of the cholinergic anti-inflammatory pathway activation could help design of superselective activators of this pathway.

Neuroprotection in focal cerebral ischemia owing to delayed treatment with melanocortins.

In gerbils subjected to transient global cerebral ischemia, melanocortin peptides produce long-lasting protection with a broad time window, and through the activation of central nervous system melanocortin MC(4) receptors. Here we aimed to investigate whether melanocortins are neuroprotective also in a rat model of focal cerebral ischemia induced by intrastriatal microinjection of endothelin-1. The vasoconstrictor agent endothelin-1 caused a significant impairment in spatial learning and memory, as well as in sensory-motor orientation and limb use, associated with severe striatal morphological damage including intense neuronal death and an almost complete myelin degradation. Treatment of ischemic rats with a nanomolar dose (340 microg/kg/day i.p. for 11 days, beginning 3 h or 9 h after endothelin-1 microinjection) of the melanocortin analog [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) significantly reduced striatal damage, and improved subsequent functional recovery, with all scheduled NDP-alpha-MSH treatments. Pharmacological blockade of melanocortin MC(4) receptors prevented the protective effect of NDP-alpha-MSH. Our findings give evidence that melanocortins are neuroprotective, with a broad time window, also in a severe model of focal cerebral ischemia, and suggest that melanocortin MC(4) receptor agonists could produce neuroprotection in different experimental models of ischemic stroke.

Both early and delayed treatment with melanocortin 4 receptor-stimulating melanocortins produces neuroprotection in cerebral ischemia.

Ischemic stroke is one of the main causes of death and disability. We investigated whether melanocortin peptides, which have protective effects in severe hypoxic conditions, also produce neuroprotection in a gerbil model of ischemic stroke. A 10-min period of global cerebral ischemia, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory that was associated with activation of inflammatory and apoptotic pathways, including severe DNA damage and delayed neuronal death, in the hippocampus. Treatment with nanomolar doses of the melanocortin analog [Nle4, D-Phe7] alpha-MSH [which activates the melanocortin receptor subtypes (MC) mainly expressed in central nervous system, namely MC3 and MC4] modulated the inflammatory and apoptotic cascades and reduced hippocampus injuries even when delayed up to 9 h after ischemia, with consequent dose-dependent improvement in subsequent functional recovery. The selective MC3 receptor agonist gamma2-MSH had no protective effects. Pharmacological blockade of MC4 receptors prevented the neuroprotective effects of [Nle4, D-Phe7] alpha-MSH and worsened some ischemia outcomes. Together, our findings suggest that MC4 receptor-stimulating melanocortins might provide potential to develop a class of drugs with a broad treatment window for a novel approach to neuroprotection in ischemic stroke.

Activation of the cholinergic anti-inflammatory pathway reduces NF-kappab activation, blunts TNF-alpha production, and protects againts splanchic artery occlusion shock.

The cholinergic anti-inflammatory pathway has not yet been studied in splanchnic artery occlusion (SAO) shock. We investigated whether electrical stimulation (STIM) of efferent vagus nerves suppresses the inflammatory cascade in SAO shock. Animals were subjected to clamping of the splanchnic arteries for 45 min, followed by reperfusion. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham-operated animals were used as controls. Two minutes before the start of reperfusion, rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Application of constant voltage pulses to the caudal vagus ends (STIM: 5 V, 2 ms, 6 Hz for 15 min, 5 min after the beginning of reperfusion) increased survival rate (VGX + SAO + Sham STIM = 0% at 4 h of reperfusion; VGX + SAO + STIM = 90% at 4 h of reperfusion), reverted the marked hypotension, inhibited IkappaBalpha liver loss, blunted the augmented nuclear factor-kappaB activity, decreased hepatic tumor necrosis factor (TNF)-alpha mRNA (VGX + SAO + Sham STIM = 1.0 +/- 1.9 TNF-alpha/glyceraldehyde-3-phosphate dehydrogenase ratio; VGX + SAO + STIM = 0.3 +/- 0.2 TNF-alpha/glyceraldehyde-3-phosphate dehydrogenase ratio), reduced plasma TNF-alpha (VGX + SAO + Sham STIM = 118 +/- 19 pg/mL; VGX + SAO + STIM = 39 +/- 8 pg/mL), ameliorated leukopenia, and decreased leukocyte accumulation, as revealed by means of myeloperoxidase activity in the ileum (VGX + SAO + Sham STIM = 7.9 +/- 1 U/g tissue; VGX + SAO + STIM = 3.1 +/- 0.7 U/g tissue) and in the lung (VGX + SAO + Sham STIM = 8.0 +/- 1.0 U/g tissue; VGX + SAO + STIM = 3.2 +/- 0.6 U/g tissue). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Our results show a parasympathetic inhibition of nuclear factor-kappaB and TNF-alpha in SAO shock.

Broad therapeutic treatment window of [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone for long-lasting protection against ischemic stroke, in Mongolian gerbils.

Melanocortin peptides have been shown to produce neuroprotection in experimental ischemic stroke. The aim of the present investigation was to identify the therapeutic treatment window of melanocortins, and to determine whether these neuropeptides chronically protect against damage consequent to brain ischemia. A 10-min period of global cerebral ischemia in gerbils, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory (Morris test: four sessions from 4 to 67 days after the ischemic episode), associated with neuronal death in the hippocampus. Treatment with a nanomolar dose (340 microg/kg i.p., every 12 h for 11 days) of the melanocortin analog [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH), starting 3-18 h after the ischemic episode, reduced hippocampal damage with improvement in subsequent functional recovery. The protective effect was long-lasting (67 days, at least) with all schedules of NDP-alpha-MSH treatment; however, in the latest treated (18 h) gerbils, some spatial memory deficits were detected. Pharmacological blockade of melanocortin MC(4) receptors prevented the protective effects of NDP-alpha-MSH. Our findings indicate that, in conditions of brain ischemia, melanocortins can provide strong and long-lasting protection with a broad therapeutic treatment window, and with involvement of melanocortin MC(4) receptors, 18 h being the approximately time-limit for stroke late treatment to be effective.

Adrenocorticotropin reverses hemorrhagic shock in anesthetized rats through the rapid activation of a vagal anti-inflammatory pathway.

OBJECTIVE: Several melanocortin peptides have a prompt and sustained resuscitating effect in conditions of hemorrhagic shock. The transcription nuclear factor kappaB (NF-kappaB) triggers a potentially lethal systemic inflammatory response, with marked production of tumor necrosis factor-alpha (TNF-alpha), in hemorrhagic shock. Here we investigated whether the hemorrhagic shock reversal produced by the melanocortin ACTH-(1-24) (adrenocorticotropin) depends on the activation of the recently recognized, vagus nerve-mediated, brain "cholinergic anti-inflammatory pathway". METHODS AND RESULTS: Anesthetized rats were stepwise bled until mean arterial pressure (MAP) stabilized at 20-25 mm Hg. The severe hypovolemia was incompatible with survival, and all saline-treated animals died within 30 min. In rats intravenously (i.v.) treated with ACTH-(1-24), neural efferent activity along vagus nerve (monitored by means of a standard system for extracellular recordings) was markedly increased, and the restoration of cardiovascular and respiratory functions was associated with blunted NF-kappaB activity and with decreased TNF-alpha mRNA liver content and TNF-alpha plasma levels. Bilateral cervical vagotomy, pretreatment with the melanocortin MC(4) receptor antagonist HS014, atropine sulfate or chlorisondamine, but not with atropine methylbromide, prevented the life-saving effect of ACTH-(1-24) and the associated effects on NF-kappaB activity and TNF-alpha levels. HS014 and atropine sulfate prevented, too, the ACTH-(1-24)-induced increase in neural efferent vagal activity, and accelerated the evolution of shock in saline-treated rats. CONCLUSIONS: The present data show, for the first time, that the melanocortin ACTH-(1-24) suppresses the NF-kappaB-dependent systemic inflammatory response triggered by hemorrhage, and reverses shock condition, by brain activation (in real-time) of the "cholinergic anti-inflammatory pathway", this pathway seeming to be melanocortin-dependent.

Evidence for a role of nuclear factor-kappaB in acute hypovolemic hemorrhagic shock.

BACKGROUND: In acute hypovolemic shock, a rapid systemic release of the inflammatory cytokine tumor necrosis factor (TNF-alpha) contributes to vascular failure. Nuclear factor kappaB (NF-kappaB) is an ubiquitous rapid-response transcription factor involved in inflammatory reactions and exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. The purpose of this study was to evaluate the role of NF-kappaB in acute hypovolemic hemorrhagic shock. METHODS: Hemorrhagic shock was induced in anesthetized male rats by intermittently withdrawing blood from an iliac catheter for 20 minutes (bleeding period) until mean arterial blood pressure (MAP) decreased and stabilized within the range of 20 to 30 mm Hg. Two minutes after bleeding was discontinued the rats received tacrolimus (100 microg/kg), an inhibitor of NF-kappaB activation, or its vehicle. We then evaluated survival rate and survival time, liver NF-kappaB activation by means of electrophoretic mobility shift assay, liver IkappaBalpha protein in the cytoplasm, hepatic TNF-alpha messenger RNA expression, plasma TNF-alpha, arterial blood pressure, and the contractile response of aortic rings to phenylephrine. RESULTS: Rats that underwent hemorrhagic shock died 28+/-2 minutes after bleeding was discontinued, experienced marked hypotension (MAP, 20-30 mm Hg), and had enhanced plasma levels of TNF-alpha (218 +/- 28 pg/mL 20 minutes after bleeding was discontinued). Aortas taken 20 minutes after bleeding was discontinued in rats that underwent hemorrhagic shock showed marked hyporeactivity to phenylephrine (1 nmol/L-10 micromol/L) compared with aortas harvested from sham shocked rats. Rats that underwent hemorrhagic shock also had increased levels of TNF-alpha messenger RNA in the liver. Furthermore, electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus, and Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm decreased. Tacrolimus (100 microg/kg, administered 2 minutes after bleeding was discontinued) inhibited the loss of IkappaBalpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Moreover, tacrolimus increased survival time (118 +/- 7 minutes; P <.01) and survival rate (vehicle = 0 and tacrolimus = 90% 240 minutes after bleeding was discontinued), reverted the marked hypotension, decreased liver messenger RNA for TNF-alpha reduced plasma TNF-alpha (35 +/- 6 pg/mL), and restored the hyporeactivity to phenylephrine to control values. CONCLUSIONS: Our results suggest that acute blood loss (50% of the estimated total blood volume during a 20-minute period) causes activation of NF-kappaB and that tacrolimus, by inhibiting this transcription factor, protects against acute hypovolemic shock.

Cannabinoid CB(1) receptor blockade enhances the protective effect of melanocortins in hemorrhagic shock in the rat.

Activation of peripheral cannabinoid CB(1) receptors contributes to hemorrhagic hypotension, and endocannabinoids produced by macrophages and platelets may be mediators of this effect. A number of studies have provided evidence that functional links exist in the mechanisms of action of cannabinoids and opioid peptides; and opioids too play an important role in the pathophysiology of hemorrhagic hypotension and shock. On the other hand, melanocortin peptides, which are the main endogenous functional antagonists of opioid peptides, have an antishock effect in animals and humans. Thus, we investigated whether an interaction exists between endocannabinoids and the endogenous opioid/antiopioid system also in a condition of hemorrhagic shock and, particularly, whether the blockade of cannabinoid CB(1) receptors potentiates the antishock effect of melanocortins. Urethane-anesthetized rats were stepwise bled until mean arterial pressure decreased to, and stabilized at, 21-23 mm Hg. In this model of hemorrhagic shock, which caused the death of all control rats within 30 min after vehicle (tween 80, 5% in saline) injection, the intravenous (i.v.) bolus injection of the cannabinoid CB(1) receptor antagonist N-piperidino-5-[4-chlorophenyl]-1-[2,4 dichlorophenyl]-4-methyl-3-pyrazolecarboxamide (SR141716A) increased mean arterial pressure, pulse pressure, respiratory rate and survival rate in a dose-related manner (0.1-3 mg/kg), an almost complete recovery of mean arterial pressure, pulse pressure and respiratory rate, and 100% survival at the end of the observation period (2 h), occurring with the dose of 3 mg/kg. The melanocortin ACTH-(1-24) (adrenocorticotropin) also produced in a dose-related manner (0.02-0.16 mg/kg i.v.) a restoration of cardiovascular and respiratory functions, and increased survival rate, an almost complete recovery and 100% survival at the end of the observation period (2 h) occurring with the dose of 0.16 mg/kg. When a subactive dose of SR141716A (0.2 mg/kg; 30% survival) was associated with a subactive dose of ACTH-(1-24) (0.02 mg/kg; 12% survival), a complete reversal of the shock condition was obtained with 100% survival at the end of the 2-h observation period. The present results show that the concurrent inhibition of both endogenous opioid and cannabinoid systems produces a reversal of hemorrhagic shock more effective than that produced by the inhibition of either of them. These data suggest that functional interactions between endocannabinoids and opioid/antiopioid are at work also in the pathophysiology of hemorrhagic shock.

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.

MC(3) receptors are involved in the protective effect of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias.

Myocardial ischemia/reperfusion induces ventricular tachycardia (VT), ventricular fibrillation (VF) and a high degree of lethality. Since ACTH-(1-24) (adrenocorticotropin) protects against such injuries in rats, we investigated which melanocortin MC receptor is involved. Ischemia was produced in anesthetized rats by ligature of the left anterior descending coronary artery (5 min), and reperfusion-induced VT, VF, lethality and time-course of arterial blood pressure within the 5 min following reperfusion were evaluated. I.v. administration of the selective MC(3) receptor agonist gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), as well as of an equimolar dose (162 nmol/kg) of both the non-selective agonist ACTH-(1-24) and alpha-MSH, significantly prevented VT and VF, and increased survival. Coronary reperfusion was followed by an abrupt and massive fall in mean arterial pressure and pulse pressure, in saline-treated rats. Treatment either with ACTH-(1-24) or gamma(1)-MSH completely prevented such fall. The protective effect of ACTH-(1-24) against the occurrence of VT, VF and lethality was neither affected by adrenalectomy, nor by i.v. pretreatment with the selective MC(4) receptor antagonist HS014 and the MC(4)-MC(5) antagonist HS059. On the other hand, the MC(3)-MC(4) receptor antagonist SHU 9119 prevented such protective effect. Moreover, the selective MC(1) receptor agonist MS05 (162 nmol/kg i.v.) failed to reduce the incidence of arrhythmias and lethality. These data demonstrate that MC(3) receptors mediate the protective effect of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias, in rats.

Involvement of the central nervous system in the protective effect of melanocortins in myocardial ischaemia/reperfusion injury.

Melanocortin peptides exert, in rats, a protective effect in myocardial ischaemia followed by reperfusion, or permanent occlusion of a coronary artery. Moreover, melanocortins have an anti-shock effect. Since the mechanism of the life-saving effect of these peptides in haemorrhagic shock includes an essential brain loop, we aimed to determine whether the central nervous system (CNS) is also involved in the protective effect against the outcome of short-term myocardial ischaemia followed by reperfusion. Ischaemia was produced in anaesthetized rats by ligature of the left anterior descending coronary artery for 5 min. Reperfusion-induced ventricular tachycardia (VT), ventricular fibrillation (VF) and lethality, and the time-course of arterial blood pressure over 5 min following reperfusion were evaluated. Groups of 8-14 rats were used. Intravenous (i.v.) injection of ACTH-(1-24) (0.16-0.48 mg/kg) during the ischaemic period dose dependently reduced the incidence of VT, VF and of lethality. In saline-treated rats, coronary reperfusion caused VT in 100% animals, VF in 86%, and death in 86%. The highest dose of ACTH-(1-24) (0.48 mg/kg) completely prevented the occurrence of VT, VF and death in all rats (P<0.005). Moreover, the melanocortin peptide prevented the fall in mean arterial pressure (MAP) occurring during reperfusion. Treatment with ACTH-(1-24) by the intracerebroventricular (i.c.v.) route also reduced the incidence of VT, VF and lethality, and prevented the fall in MAP in a dose dependent manner. Complete (100%) protection occurred with an i.c.v. dose (0.048 mg/kg) 10 times less than that needed by the i.v. route. The present data show that in the protective effect of melanocortin peptides against the injury after myocardial ischaemia/reperfusion, the i.c.v. route of administration is more effective than the i.v. route. They suggest that a CNS mechanism, not yet identified, may be involved.

Activation of an efferent cholinergic pathway produces strong protection against myocardial ischemia/reperfusion injury in rats.

OBJECTIVE: A vagus nerve-mediated, brain cholinergic protective mechanism activated by melanocortin peptides is operative in conditions of circulatory shock; moreover, there is anatomical evidence of dual vagal-cardiac efferent pathways in rats, which could play different roles in controlling heart function. Therefore, we investigated the role and functional mechanism of such vagal efferent pathway(s) in an experimental model of ischemic heart disease. DESIGN: Randomized experimental study. SETTING: Research laboratory. SUBJECTS: Adult Wistar rats of either sex. INTERVENTIONS: After bilateral cervical vagotomy (with or without pretreatment with atropine), efferent vagal fibers were electrically stimulated in rats subjected to coronary artery occlusion (5 mins) followed by reperfusion (5 mins). Other rats (intact, vagotomized, or pretreated with atropine) were treated with nanomolar doses of melanocortin peptides. MEASUREMENTS AND MAIN RESULTS: Electrical stimulation of efferent vagal fibers (5 V, 2 m secs, 1-9 Hz, for the whole period of ischemia/reperfusion) strongly reduced the high incidence of severe arrhythmias and lethality, reduced the increase in free radical blood levels and left-ventricle histologic alterations, and augmented the extracellular signal-regulated kinase activation. Treatment with the melanocortin peptides adrenocorticotropin and gamma2-melanocyte-stimulating hormone (162 nmol/kg intravenously or 16.2 nmol/kg intracerebroventricularly, during coronary occlusion) produced the same protective effects of electrical stimulation and with the same muscarinic acetylcholine receptor-dependent mechanism, seemingly through brain activation (mediated by melanocortin MC3 receptors, as previously described) of such efferent vagal pathway. CONCLUSIONS: The present results give evidence for the identification of a protective, melanocortin-activated, efferent vagal cholinergic pathway, operative in conditions of myocardial ischemia/reperfusion. These data suggest that melanocortins and pertinent compounds able to activate such a pathway could provide the potential for development of a new class of drugs for a novel approach to management of ischemic heart disease.