The MC3 receptor binding affinity of melanocortins correlates with the nitric oxide production inhibition in mice brain inflammation model.

Melanocortins possess strong anti-inflammatory effects acting in the central nervous system via inhibition of the production of nitric oxide (NO) during brain inflammation. To shed more light into the role of melanocortin (MC) receptor subtypes involved we synthesized and evaluated some novel peptides, modified in the melanocyte-stimulating hormone (MSH) core structure, natural MCs and known MC receptor selective peptides - MS05, MS06. Since the study included both selective, high affinity binders and the novel peptides, it was possible to do the correlation analysis of binding activities and the NO induction-related anti-inflammatory effect of the peptides. beta-MSH, gamma1-MSH, gamma2-MSH, alpha-MSH, MS05, Ac-MS06 and Ac-[Ser12]MS06 caused dose dependent inhibition of the lipopolysaccharide (LPS)-induced increase of NO overproduction in the mice forebrain whereas MSH core modified peptides Ac-[Asp9,Ser12]MS06, [Asp9]alpha-MSH and [Asp16]beta-MSH were devoid of this effect in doses up to 10 nmol per mouse. When the minimal effective dose required for inhibition of NO production was correlated with the in vitro binding activity to MC receptor subtypes a strong and significant correlation was found for the MC3 receptor (r = 0.90; p = 0.0008), whereas weak correlation was present for the other receptors. Our results suggest that the MC3 receptor is the major player in mediating the anti-inflammatory activity of MCs in the central nervous system.

Mildronate, an inhibitor of carnitine biosynthesis, induces an increase in gamma-butyrobetaine contents and cardioprotection in isolated rat heart infarction.

The inhibition of gamma-butyrobetaine (GBB) hydroxylase, a key enzyme in the biosynthesis of carnitine, contributes to lay ground for the cardioprotective mechanism of action of mildronate. By inhibiting the biosynthesis of carnitine, mildronate is supposed to induce the accumulation of GBB, a substrate of GBB hydroxylase. This study describes the changes in content of carnitine and GBB in rat plasma and heart tissues during long-term (28 days) treatment of mildronate [i.p. (intraperitoneal) 100 mg/kg/daily]. Obtained data show that in concert with a decrease in carnitine concentration, the administration of mildronate caused a significant increase in GBB concentration. We detected about a 5-fold increase in GBB contents in the plasma and brain and a 7-fold increase in the heart. In addition, we tested the cardioprotective effect of mildronate in isolated rat heart infarction model after 3, 7, and 14 days of administration. We found a statistically significant decrease in necrotic area of infarcted rat hearts after 14 days of treatment with mildronate. The cardioprotective effect of mildronate correlated with an increase in GBB contents. In conclusion, our study, for the first time, provides experimental evidence that the long-term administration of mildronate not only decreases free carnitine concentration, but also causes a significant increase in GBB concentration, which correlates with the cardioprotection of mildronate.

Beta-MSH inhibits brain inflammation via MC(3)/(4) receptors and impaired NF-kappaB signaling.

The anti-inflammatory effects of melanocortin peptides have been demonstrated in different inflammation models. This is the first report describing the molecular mechanisms for the beta-MSH-induced suppression of bacterial lipopolisaccharide (LPS)-caused brain inflammation. We found that beta-MSH suppresses LPS-induced nuclear translocation of the transcription factor NF-kappaB, and inhibits the expression of inducible nitric oxide synthase, and the following nitric oxide overproduction in the brain, in vivo. Moreover, administering the preferentially MC(4) receptor selective antagonist HS014 blocked completely these effects, suggesting a tentative MC(4) receptor mediated mechanism of action for the beta-MSH. However, as HS014 shows quite low selectivity vis-à-vis the MC(3) receptor, a role for the MC(3) receptor cannot be excluded. In conclusion, our results show that beta-MSH is capable of inhibiting brain inflammation via activation of melanocortin receptors, of the subtypes 4 and/or 3.

Investigations on the pharmacology of the cardioprotective guanidine ME10092.

The guanidine compound ME10092 (1-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine), which possesses a strong cardioprotective effect to ischemia-reperfusion, was assessed for different pharmacological actions that may underlie its cardioprotective effect. In the living rat ME10092 decreased the blood pressure and heart rate in a dose-dependent manner. We found ME10092 to bind to alpha 1- and alpha 2-adrenoreceptors with moderate affinity (Ki values 1-4 microM), and to block adrenaline-elicited contractile responses in isolated guinea pig aortas. Our results indicate that ME10092 possesses a certain anti-oxidant profile. Thus, in a competitive manner and with low affinity it inhibited the bovine milk xanthine oxidase enzyme, as well as NAD(P)H oxidase driven oxyradical formation in membrane fractions isolated from the rat brain. By using electron paramagnetic resonance we here show that, after its systemic administration, ME10092 modulates the nitric oxide (NO) content in several tissues of the rat in a time-dependent manner. However, in vitro ME10092 inhibited the activities of nitric oxide synthases nNOS and eNOS, but not that of iNOS. Our data give evidence that the cardioprotective effect of ME10092 could be mediated through pharmacological mechanisms that include some modulation of NO production, as well as possible inhibition of radical formation during ischemia-reperfusion.

The methylester of gamma-butyrobetaine, but not gamma-butyrobetaine itself, induces muscarinic receptor-dependent vasodilatation.

Gamma-butyrobetaine (GBB) is known mostly as a bio-precursor of carnitine, a key molecule in the regulation of myocardial energy metabolism. The metabolites of carnitine and GBB were investigated for acetylcholine-like activity decades ago. The present study shows that the methylester of GBB (GBB-ME) exerts its biological activity by binding to muscarinic acetylcholine receptors. GBB-ME dose-dependently decreased the blood pressure in anaesthetised rats and also produced endothelium-dependent vasodilation in the isolated guinea-pig heart. The biological effects of GBB-ME were inhibited partially by the NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) and abolished by the acetylcholine receptor antagonist atropine, thus supporting the hypothesis that GBB-ME acts as muscarinic agonist. Moreover, we have shown here for the first time that GBB-ME binds directly to transfected human muscarinic (m) acetylcholine receptors, the potency order being m2>m5> or =m4> or =m1>m3. GBB itself showed neither biological activity nor significant affinity for the m1-5 receptors. We conclude that GBB-ME, but not the parent GBB, possesses acetylcholine-like activity in vivo and in vitro.

Beta- and gamma-melanocortins inhibit lipopolysaccharide induced nitric oxide production in mice brain.

The pro-opiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) mediates many diverse physiological actions, including anti-inflammatory and immunomodulatory effects. However, little is known about the physiological roles of the other melanocortins, beta- and gamma-MSH. Here, we investigated the effects of melanocortin peptides in an in vivo neuroinflammation model. Six hours following intracisternal (i.c.) administration of 10 microg lipopolysaccharide (LPS) to mice a five-fold increase in the nitric oxide (NO) level was seen in the animals' brains, when detected by electron paramagnetic resonance (EPR). All tested melanocortins, alpha-, beta-, gamma1- and gamma2-MSH (0.001-10 nmol/mouse i.c.), dose dependently reduced the LPS induced increases in brain NO, with an order of effectiveness: beta-MSH > or = gamma1-MSH=gamma2-MSH>alpha-MSH. Our results suggest specialized functions of beta- and gamma-MSH melanocortins in inflammatory signal modulation in the brain.