Roles of nitric oxide synthase inhibitor on antinociceptive effects of mu-opioid agonist in mice.

In the present study, it was found that intraperitoneal (i.p.) pre-injection of N(G)-nitro-L-arginine methyl ester (L-NAME) significantly influenced the endomorphin-1 (EM-1) and endomorphin-2 (EM-2) induced antinociception. These effects could be inhibited or reversed by L-Arg or naloxone. Our results suggest that the modulatory effect of NO system on the mu-receptor evoked analgesia is different between the two mu receptor subtypes.

Thymopentin (TP5), an immunomodulatory peptide, suppresses proliferation and induces differentiation in HL-60 cells.

Thymopentin (Arg-Lys-Asp-Val-Tyr, TP5) has shown immuno-regulatory activities in humans. In the present study, we investigated the effects of TP5 on the proliferation and differentiation of a human promyelocyte leukemia cell line, HL-60. It is noteworthy that TP5 displayed concentration-dependent inhibitory effects on the proliferation and colony formation of HL-60 cells. Furthermore, the decrease or even disappearance of AgNORs from nucleoli was observed in HL-60 cells after the treatment with TP5. The suppression induced by TP5 was accompanied by an accumulation of cell cycle in the G0/G1 phase. Moreover, TP5 significantly increased the NBT-reduction activity of HL-60 cells. Cytofluorometric and morphologic analysis indicated that TP5 had induced differentiation along the granulocytes lineage in HL-60 cells. d-tubocurarine (TUB) significantly antagonized the inhibitory effects induced by TP5, whereas atropine did not exhibit such effect. All the results indicated that TP5 was able to significantly inhibit proliferation and induce differentiation in HL-60 cells. Our observations also implied that TP5 not only acted as an immunomodulatory factor in cancer chemotherapy, but is also a potential chemotherapeutic agent in the human leukemia therapy.

Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor.

Nociceptin/orphanin FQ (N/OFQ) and its receptor system modulate a variety of biological functions and further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor. Two series of N/OFQ related analogues were synthesized to investigate the relationship of different modifications. We combined modifications including: (a) Phe(4)-->(pF)Phe(4); (b) Ala(7), Ala(11)-->Aib(7), Aib(11); (c) Leu(14), Ala(15)-->Arg(14), Lys(15). Compared with the first series, N-terminus of the second series was changed from Phe(1) to Nphe(1). All the analogues were amidated at C-terminus. These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. Results indicated that the compounds of the first series showed higher affinity and potency than N/OFQ (pK(i)=9.33; pEC(50)=7.50). In particular, [(pF)Phe(4), Aib(7), Aib(11), Arg(14), Lys(15)] N/OFQ-NH(2) was found to be a highly potent agonist with pK(i)=10.78 in binding studies and pEC(50)=9.37 in mouse vas deferens assay. The second series all competitively antagonized the effects of N/OFQ in mouse vas deferens assay. [Nphe(1), (pF)Phe(4), Aib(7), Aib(11), Arg(14), Lys(15)] N/OFQ-NH(2) was the best antagonist with pA(2)=8.39 and showed high binding affinity with pK(i)=9.99. Thus modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist.

Endomorphin 1[psi] and endomorphin 2[psi], endomorphins analogues containing a reduced (CH2NH) amide bond between Tyr1 and Pro2, display partial agonist potency but significant antinociception.

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are highly potent and selective mu-opioid receptor agonists and have significant antinociceptive action. In the mu-selective pocket of endomorphins (EMs), Pro2 residue is a spacer and directs the Tyr1 and Trp3/Phe3 side chains into the required orientation. The present work was designed to substitute the peptide bond between Tyr1 and Pro2 of EMs with a reduced (CH2NH) bond and study the agonist potency and antinociception of EM1[psi] (Tyr[psi(CH2NH)]Pro-Trp-Phe-NH2) and EM2[psi] (Tyr[psi(CH2NH)]Pro-Phe-Phe-NH2). Both EM1[psi] and EM2[psi] are partial mu opioid receptor agonists showing significant loss of agonist potency in GPI assay. However, EMs[psi] exhibited potent supraspinal antinociceptive action in vivo. In the mice tail-flick test, EMs[psi] (1, 5, 10 nmol/mouse, i.c.v.) produced potent and short-lasting antinociception in a dose-dependent and naloxone (1 mg/kg) reversed manner. At the highest dose of 10 nmol, the effect of EM2[psi] was prolonged and more significant than that of EM2. In the rat model of formalin injection induced inflammatory pain, EMs[psi] (0.1, 1, 10 nmol/rat, i.c.v.), like EMs, exerted transient but not dose-dependent antinociception. These results suggested that in the mu-selective pocket of EMs, the rigid conformation induced by the peptide bond between Tyr1 and Pro2 is essential to regulate their agonist properties at the mu opioid receptors. However, the increased conformational flexibility induced by the reduced (CH2NH) bond made less influence on their antinociception.

Endomorphins, endogenous opioid peptides, provide antioxidant defense in the brain against free radical-induced damage.

Oxidative stress has been considered to be a major cause of cellular injuries in a variety of chronic health problems, such as carcinogenesis and neurodegenerative disorders. The brain appears to be more susceptible to oxidative damage than other organs. Therefore, the existence of antioxidants may be essential in brain protective systems. The antioxidative and free radical scavenging effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, have been investigated in vitro. The oxidative damage was initiated by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrocholoride) (AAPH) and hydrogen peroxide (H2O2). The linoleic acid peroxidation, DNA and protein damage were monitored by formation of hydroperoxides, by plasmid pBR 322 DNA nicking assay and single-cell alkaline electrophoresis, and by SDS-polyacrylamide gel electrophoresis. Endomorphins can inhibit lipid peroxidation, DNA strand breakage, and protein fragmentation induced by free radical. Endomorphins also reacted with galvinoxyl radicals in homogeneous solution, and the pseudo-first-order rate constants were determined spectrophotometrically by following the disappearance of galvinoxyl radicals. In all assay systems, EM1 was more potent than EM2 and GSH, a major intracellular water-soluble antioxidant. We propose that endomorphins are one of the protective systems against free radical-induced damage in the brain.

Endomorphins inhibit contractile responses of rat thoracic aorta rings induced by phenylephrine and angiotensin II in vitro.

AIM: To study the effects of opioid receptor agonists endomorphin-1 and -2 on contractile responses of rat thoracic aorta rings to phenylephrine (PE) and angiotensin II (Ang II), and their possible mechanism in vitro. METHODS: Isometric tension recording was progressed in thoracic aorta rings from Wistar rats. RESULTS: Pretreatment of morphine, endomorphin-1 and -2 (0.1, 1, and 10 micromol/L) could inhibit the contractile responses of the endothelium-intact aorta rings to PE (0.1 micromol/L) and Ang II ( 1 micromol/L) in a concentration-dependent manner (P < 0.01), but could not inhibit the contraction of rings without endothelium (P > 0.05). Naloxone (1 micromol/L) could partially antagonize the effects of endomorphine-1 and -2 (P < 0.01). N(omega)-nitro-L-arginine (L-NNA, 10 micromol/L) or endothelial rubbing could completely blocked the effects of morphine, endomorphine-1 and -2 (P < 0.01). CONCLUSION: Endomorphin-1 and -2 could inhibit PE- and Ang II-induced contractions of rat aorta rings, which was partially by naloxone-sensitive mechanism and related to the release of nitric oxide from vascular endothelium.