Studies Of The Biological Activities Induced By Hemopressin Related Peptides,the Endogenous Cannabinoid Peptideic Agonists

Cannabinoid system plays a critical role in pain modulation,and a growing number of studies have shown that two types of the cannabinoid receptors,namely cannabinoid CB₁ receptor and CB₂ receptor,become the attractive targets for analgesics development.It is well known that the cannabinoid receptors are traditionally thought to be modulated by phytocannabinoids,lipid endocannabinoids and synthetic agonists.Recently,hemopressin and related peptides,the bioactive peptides derived from hemoglobin,have shown to function as the endogenous ligands or the regulator of cannabinoid CB₁ and CB₂ receptors.Hemopressin was reported to function as an endogenous inverse agonist or antagonist of the CB₁ receptor.The efforts have largely focused on the behavioral and physiological effects of hemopressin.However,the biological functions of the novel cannabinoid agonist,hemopressin related petides,remain poorly understood.Our recent data demonstrated that?m?VD-hemopressin???was involved in pain modulation,cardiovascular action,food intake,locomotor activity,gastrointestinal and hypothermic modulation.On the basis of the previous research in our lab,the biological activities of the endogenous cannabinoid agonists hemopressin related peptides were inveastigated.Firstly,according to the amino acid sequence analyses of hemoglobin subunit ?,?r?VD-hemopressin???was predicted as a cannabinoid peptide derived from rat ?-hemoglobin.And the modulating activities of?r?VD-hemopressin???on pain,body temperature,locomotor activity and CNS side-effects were evaluated.Secondly,?m?VD-hemopressin???was reported to exert CB₁-mediated central antinociception in the mouse tail-flick assay.And?m?VD-hemopressin???was to further investigate the analgesic properties of supraspinal?m?VD-hemopressin???in a range of preclinical rodent pain models.At last,the antinociceptive profiles of?m?VD-hemopressin???were investigated in different pain models of the mouse tail-flick test,the acetic acid-induced writhing test and the formalin test.On the basis of the amino acid sequence analyses of hemoglobin,?r?VD-hemopressin???was predicted as a cannabinoid peptide derived from rat ?-hemoglobin.Thus,?r?VD-hemopressin???was synthesized and characterized in a series of in vitro and in vivo assays.Our results demonstrated that?r?VD-hemopressin???induced neurite outgrowth in Neuro 2A cells via CB₁ receptor.In the tail-flick assay,?r?VD-hemopressin???dose-dependently exerted central antinociception through CB₁ receptor,but not CB₂ and opioid receptors.In mice,supraspinal administration of?r?VD-hemopressin???produced dose-dependent hypothermia,which was partially reduced by the CB₁ receptor antagonist AM251,but not by the antagonists of CB₂ and opioid receptors.In addition,?r?VD-hemopressin???caused hypoactivity after intracerebroventricular injection,and this effect was insensitive to the antagonists of cannabinoid and opioid receptors.Further assessment of the side-effects demonstrated that?r?VD-hemopressin???evoked the limited effects on gastrointestinal transit and reward effect at antinociceptive doses,but repeated i.c.v.injection of?r?VD-hemopressin???induced development of antinociceptive tolerance.Taken together,these data suggest that the predicted peptide?r?VD-hemopressin???produces antinociception,hypothermia and hypoactivity via different pharmacological mechanisms,at least partially.Moreover,it implies that?r?VD-hemopressin???has therapeutic potential in pain management with limited side-effects.Moreover,the analgesic properties of supraspinal?m?VD-hemopressin???were characterized in a range of preclinical rodent pain models,and the classical cannabinoid agonist WIN 55212-2 was used as a positive control.In animal models of post-operative pain,supraspinal administration of?m?VD-hemopressin???dose-dependently reduced injury-induced mechanical allodynia and the effects were not reversed by pretreatment with the selective antagonists of CB₁ and CB₂ receptors.Similarly,in phase I of formalin test and acetic acid-induced visceral pain model,intracerebroventricular administration of?m?VD-hemopressin???induced dose-related analgesia in mice,which was not antagonized by the selective antagonists of CB₁ and CB₂ receptors.However,central injection of mouse VD-Hp? did not cause significant effect in phase II of formalin test.By contrast,in these rodent preclinical models of nociception,WIN55,212-2 induced supraspinal analgesia via CB₁ receptor,independently of CB₂ receptor.Taken together,our data indicate that the CB₁ receptor peptidic agonist?m?VD-hemopressin???produces analgesic action in the preclinical models of pain via non-CB₁,non-CB₂ mechanism.Therefore,?m?VD-hemopressin???could exert antinociceptive effects in different pain models via different pharmacological mechanisms.At last,the antinociceptive profiles?m?VD-hemopressin???,a novel endogenous agonist of cannabinoid CB₁ and CB₂ receptors,was investigated in different pain models.In the mouse tail-flick test,supraspinal,intrathecal,subcutaneous and intraperitoneal administrations of?m?VD-hemopressin???produced dose-dependent antinociception,which were completely blocked by the CB₁ receptor antagonist AM251,but not by the CB₂ receptor antagonist AM630,given at the same levels.Furthermore,our results indicated that the systemic antinociception of?m?VD-hemopressin???was selectively antagonized by supraspinal,intrathecal or subcutaneous injections of AM251.These findings suggest that?m?VD-hemopressin???induces CB₁ receptor-mediated antinociception in the tail-flick test when given various routes.In the acetic acid-induced writhing test,i.c.v.injection of?m?VD-hemopressin???markedly inhibited the pain-related behaviors and this antinociceptive action was partially prevented by AM251.In the formalin test,i.c.v.injection of?m?VD-hemopressin???reduced licking times in the phase I,but did not affect phase II pain behaviors,which were insensitive to the antagonists of cannabinoid receptors.Therefore,these data suggest that?m?VD-hemopressin???produces analgesic activities in the preclinical pain models,which are at least partially mediated by other receptors besides cannabinoid receptors.In summary,these findings have shown the antinociceptive activities of the cannabinoid peptidic agonists including?r?VD-hemopressin???,?m?VD-hemopressin???and?m?VD-hemopressin???,which might be mediated by the complex mechanisms,and are at least partially mediated by other receptors besides cannabinoid receptors.Thus,the detailed mechnisms will need further elaboration in the futher.