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.

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.