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In the present study, it was found that intraperitoneal (i.p.) pre-injection of NG-nitro-L-arginine methyl ester (LNAME) 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.

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are highly potent and selective μ-opioid receptor agonists and have significant antinociceptive action. In the μ-selective pocket of endomorphins (EMs), Pro 2 residue is a spacer and directs the Tyr 1 and Trp 3 /Phe 3 side chains into the required orientation. The present work was designed to substitute the peptide bond between Tyr 1 and Pro 2 of EMs with a reduced (CH 2 NH) bond and study the agonist potency and antinociception of EM1[ψ] (Tyr[ψ(CH 2 NH)]Pro-Trp-Phe-NH 2 ) and EM2[ψ] (Tyr[ψ(CH 2 NH)]Pro-Phe-Phe-NH 2 ). Both EM1[ψ] and EM2[ψ] are partial μ opioid receptor agonists showing significant loss of agonist potency in GPI assay. However, EMs[ψ] exhibited potent supraspinal antinociceptive action in vivo. In the mice tail-flick test, EMs[ψ] (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[ψ] was prolonged and more significant than that of EM2. In the rat model of formalin injection induced inflammatory pain, EMs[ψ] (0.1, 1, 10 nmol/rat, i.c.v.), like EMs, exerted transient but not dose-dependent antinociception. These results suggested that in the μ-selective pocket of EMs, the rigid conformation induced by the peptide bond between Tyr 1 and Pro 2 is essential to regulate their agonist properties at the μ opioid receptors. However, the increased conformational flexibility induced by the reduced (CH 2 NH) bond made less influence on their antinociception.

Nocistatin was synthesized by the solid-phase peptide synthesis method. Its effects on rat systemic arterial pressure; rat hindquarter vascular bed resistance; tension of rabbit pectoral, abdominal, femoral aorta muscle strips without endothelium; and nociceptin induced decreases of rat systemic arterial pressure were determined. The results showed that nocistatin can increase the systemic arterial pressure, increase the hindquarter vascular bed resistance and induce the contraction significantly of abdominal, femoral aorta muscle strips without endothelium; it has no significant effect on tension of pectoral aorta muscle strips, it cannot antagonize significantly the decrease of rat systemic arterial pressure induced by nociceptin. These results suggest that nocistatin has some important effects on blood vessel activities.

Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly, NH2, Del II), an endogenous linear heptapeptide, is a highly selective agonist of the δ-opioid receptor. To study the effect of the position 4 residue (Glu) on the opioid activity of Del II, we designed and synthesized three analogues of Del II by solid-phase peptide synthesis. They were [Val4, Glu5]Del II, [Val4, Glu6]Del II and [Gly4, Glu7]Del II. To study the effect of spin labeling on peptide bioactivities, all the peptides were labeled using a free radical. The labeling material was a stable nitrogen-oxygen free radical which was linked to the N-terminal via an amide bond. We investigated the opioid bioactivities of these analogues both in vivo and in vitro, and concluded that the differences in opioid activity of Del II and its analogues were due to structural differences. When the Glu residue is at position 5 or 6, the internal hydrogen bonds in Del II are affected and there is a change in three-dimensional structure and opioid activity. The antinociceptive activity of all the peptides decreased after spin labeling. This indicates that the stable nitrogen-oxygen free radical is a dual-function spin-labeling molecule.

The second zinc finger fragment of Sp1 (Sp1-ZF2), its mutant (Sp1-ZF2/HT. E(20) --H, R(23) --T), and two mimic analogues (ZF20 and ZF15) were synthesized by stepwise solid phase technique. The CD spectra and UV-visible spectrum with CoC1(2) indicated that the formation of zinc finger structure was affected not only by the hydrophobic amino acids but also by the change of the distance between Cys and His. Gel-retardat ion electrophoresis assays indicated that the Glu and Arg residues are very important for recognition. A single zinc finger like Sp1-ZF2 is able to bind DNA sequence specifically.

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.

Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2, Del II) is a highly selective agonist of δII opioid receptor [1], which was isolated from the frog Phyllmedusa Sauvagei in 1989 [2]. DeltorphinII and dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-SerNH2, DRM), which is the μ-receptor selective agonist [3], share common generalized N-terminal tripeptide sequences, H-Tyr1-D-Ala2-Phe3, which comprise the message domains of these peptides. The critical structural differences between these two peptides lie in the C-terminal tetrapeptide region, the address domain, adjacent to the N-terminal message domain. Deltorphin II analogs were selected to test the hypothesis of Schwyzer [4] that flexible .peptide hormones contain “synchnologic organization”, i.e., the polypeptide sequence is composed of proximal regions which exist as distinct message and address domains. The message domain provides information for signal transduction leading to a biological response, while the address domain’ primarily influences binding affinities and accommodates the elements of selectivity without necessarily affecting transduction. In order to expand our understanding of the characteristics of the Cterminal tetrapeptide address domain of deltorphin II on opioid receptor affinities and selectivities, studies were conducted with several analogs of the peptide in which the spatial distribution of the negatively charged residues was repositioned relative to the hydrophobic residues. Its position 4 is a negative charged amino acid Glu. To study the effect of Glu on the function of deltorphin II, we designed and synthesized a series of analogs of deltorphin II by SPPS (solid phase peptide synthesis), they were: (fig. 1).

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 (p K 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 p K 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 p A 2 = 8.39 and showed high binding affinity with p K 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.

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.Isometric tension recording was progressed in thoracic aorta rings from Wistar rats.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 (P0.01), but could not inhibit the contraction of rings without endothelium (P0.05). Naloxone (1 micromol/L) could partially antagonize the effects of endomorphine-1 and -2 (P0.01). N(omega)-nitro-L-arginine (L-NNA, 10 micromol/L) or endothelial rubbing could completely blocked the effects of morphine, endomorphine-1 and -2 (P0.01).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.

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.