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Background and Purpose Intrathecally (i.t.) administered nociceptin/orphanin FQ ( N/OFQ) evokes antinociceptive effects in rodents. Recent studies in monkeys demonstrated that i.t. co-application of N/OFQ and morphine elicits synergistic antinociceptive actions suggesting mixed N/OFQ peptide ( NOP) and μ opioid receptor agonists as innovative spinal analgesics. Thus, novel N/OFQ related peptides were synthesized in order to identify and pharmacologically characterize a mixed NOP/ μ opioid receptor agonist. Experimental Approach The following in vitro assays were used: calcium mobilization in cells expressing the human NOP or classical opioid receptors and chimeric G proteins, receptor and [35S]-GTPγS binding, [35S]-GTPγS binding in rat spinal cord membranes, guinea pig ileum bioassay. In vivo experiments were performed in monkeys using the tail withdrawal assay. Key Results From calcium mobilization studies [Dmt1]N/OFQ(1-13)-NH2 was selected as the most potent and least selective compound. The mixed NOP/opioid full agonist activity and high affinity of [Dmt1]N/OFQ(1-13)-NH2 was confirmed at human recombinant receptors in receptor binding, calcium mobilization and/or [35S]-GTPγS binding studies, at rat spinal cord receptors in [35S]-GTPγS binding experiments, and at guinea pig receptors inhibiting neurogenic contractions in the ileum. In vivo in the tail withdrawal assay in monkeys i.t. [Dmt1]N/OFQ(1-13)-NH2 was able to elicit robust and long-lasting antinociceptive effects. Conclusions and Implications Collectively, these results demonstrate that [Dmt1]N/OFQ(1-13)-NH2 behaves as NOP/opioid receptor universal agonist and substantiate the suggestion that such mixed ligands are worthy of development as innovative spinal analgesics. [ABSTRACT FROM AUTHOR]

Background and Purpose: Neuropeptide S (NPS) was recently identified as the endogenous ligand of an orphan receptor, now referred to as the NPS receptor. In vivo, NPS produces a unique behavioural profile by increasing wakefulness and exerting anxiolytic-like effects. In the present study, we further evaluated the effects of in vivo supraspinal NPS in mice.Experimental Approach: Effects of NPS, injected intracerebroventricularly (i.c.v.), on locomotor activity (LA), righting reflex (RR) recovery and on anxiety states (measured with the elevated plus maze (EPM) and stress-induced hyperthermia (SIH) tests) were assessed in Swiss mice.Key Results: NPS (0.01-1 nmol per mouse) caused a significant increase in LA in naive mice, in mice habituated to the test cages and in animals sedated with diazepam (5 mg kg(-1)). In the RR assay, NPS dose dependently reduced the proportion of animals losing the RR in response to diazepam (15 mg kg(-1)) and their sleeping time. In the EPM and SIH test, NPS dose dependently evoked anxiolytic-like effects by increasing the time spent by animals in the open arms and reducing the SIH response, respectively.Conclusions and Implications: We provide further evidence that NPS acts as a novel modulator of arousal and anxiety-related behaviours by promoting a unique pattern of effects: stimulation associated with anxiolysis. Therefore, NPS receptor ligands may represent innovative drugs for the treatment of sleep and anxiety disorders. [ABSTRACT FROM AUTHOR]

1 GPI-80, a glycosylphosphatidylinositol (GPI)-anchored protein initially identified on human neutrophils, plays a role(s) in the regulation of β2 integrin function. Previous studies have shown that GPI-80 is sublocated in secretory vesicles. It is also found in soluble form in the synovial fluid of rheumatoid arthritis patients, and in the culture supernatant of formyl-methionyl-leucylphenylalanine-stimulated neutrophils. To understand the behaviour of GPI-80 under conditions of stimulation, we investigated the effects of turnout necrosis factor (TNF)-α on its expression and release. We also probed the mechanism of its release with various pharmacologic tools. 2 TNF-α induced the release of GPI-80 from human neutrophils in a concentration-and timedependent manner (in the range of 1 100 u ml[sup -1] and 30 120 min, respectively), but did not affect surface GPI-80 levels. 3 Cytochalasin B, genistein, and SB203580 but not PD98059 inhibited TNF-α-stimulated GPI-80 release and neutrophil adherence at the same concentration. In addition, TNF-α-induced GPI-80 release was inhibited by blocking monoclonal antibodies specific to components of Mac-1 (CD1lb and CD18). 4 Antioxidants (pyrrolidine dithiocarbamate and N-acetyl-L-cysteine) inhibited GPI-80 release by TNF-α stimulation, but superoxide dismutase did not. Antioxidants but not superoxide dismutase reduced an intracellular oxidation state. 5 These findings indicate that TNF-α-stimulated GPI-80 release from human neutrophils depends upon adherence via β2 integrins. They also suggest that cytochalasin B, genistein, and SB203580 inhibit GPI-80 release by suppressing signals for cell adherence, rather than by a direct effect on its secretion. Finally, we suggest that GPI-80 release involves an intracellular change in a redox state. [ABSTRACT FROM AUTHOR]

Abstract: Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, the NPSR ligand [ t Bu-d-Gly5]NPS was generated and in vitro characterized as a pure antagonist at the mouse NPSR. In the present study the pharmacological profile of [ t Bu-d-Gly5]NPS has been investigated. [ t Bu-d-Gly5]NPS activity was evaluated in vitro in the calcium mobilization assay at the rat NPSR and in vivo in the locomotor activity and righting reflex tests in mice and in the elevated plus maze and defensive burying assays in rats. In vitro, [ t Bu-d-Gly5]NPS was inactive per se while it inhibited the calcium mobilization induced by 30nM NPS (pKB 7.42). In Schild analysis experiments [ t Bu-d-Gly5]NPS (0.1–10μM) produced a concentration-dependent rightward shift of the concentration–response curve to NPS, showing a pA2 value of 7.17. In mouse locomotor activity experiments, supraspinal injection of [ t Bu-d-Gly5]NPS (1–10nmol) dose dependently counteracted NPS (0.1nmol) stimulant effects. In the mouse righting reflex assay [ t Bu-d-Gly5]NPS (0.1–10nmol) fully prevented the arousal-promoting action of the natural peptide (0.1nmol). Finally, [ t Bu-d-Gly5]NPS (3–30nmol) was able to completely block NPS (1nmol) anxiolytic-like actions in rat elevated plus maze and defensive burying assays. Collectively, the present results demonstrated that [ t Bu-d-Gly5]NPS behaves both in vitro and in vivo as a pure and potent NPSR antagonist. This compound represents a novel and useful tool for investigating the pharmacology and neurobiology of the NPS/NPSR system. [Copyright &y& Elsevier]