Allison J Fulford

Nociceptin/orphanin FQ modulates human T cell function in vitro

Although nociceptin/orphanin FQ (N/OFQ) and its receptor (ORL-1) are widely distributed throughout the immune system, its role has yet to be elucidated. This study shows that N/OFQ (10(-14)-10(-12) M) modulates T cell activation by up-regulating activation marker expression, e.g. CD28, leading to enhanced proliferation and modulation of TNFalpha secretion. However, on re-stimulated T cells N/OFQ causes inhibition of proliferation, which could be linked with N/OFQ up-regulating CTLA-4 expression. We have also shown that some of these effects are partly prostaglandin-dependent and that N/OFQ induces prostaglandin synthesis. This report suggests that N/OFQ could exert a key modulatory role in human T cell functions.

Novel opioid peptides endomorphin-1 and endomorphin-2 are present in mammalian immune tissues

Endomorphin (EM)-1 and EM-2 are opioid tetrapeptides, reported within the central nervous system, which have very high specificity and affinity for the mu-opioid receptor. We have used newly developed and well-characterised radioimmunoassays (RIAs) in combination with reversed-phase high-performance liquid chromatography (HPLC) to detect EM-1 and EM-2 immunoreactivity (ir) in rat immune tissues. Endomorphins were detectable in extracts of rat spleen (total EM-1-ir/spleen: 440+/-73 pg, mean+/-SEM, a=group of eight rats; EM-2-ir: 150+/-12 pg) and thymus (EM-1-ir: 152+/-18 pg, mean+/-SEM n=8; EM-2-ir: 156+/-28 pg). EM-2-ir was detectable in extracts of human spleen (338+/-196 pg/g tissue, n=3). Multiple peaks of EM-1-ir and EM-2-ir were observed in rat spleen and thymus extracts, and multiple peaks of EM-2-ir were observed in extracts of human spleen, following reversed-phase HPLC and RIAs. This is the first report of endomorphin immunoreactivity in tissues of the rat and human immune systems.

Regulation of nociceptin/orphaninFQ secretion by immune cells and functional modulation of interleukin-2

Previous research has demonstrated that numerous populations of immune cell, including lymphocytes, synthesize nociceptin (N/OFQ) precursor mRNA although little is known regarding the immunological role of N/OFQ. In the present study we have demonstrated significant effects of mitogens, pro-inflammatory cytokines, cyclic AMP analogues, glucocorticoids and CRF on N/OFQ secretion by rat splenocytes in vitro. N/OFQ (10(-14) to 10(-10)M) was also shown to inhibit proliferation of Con A-activated splenocytes and production of IL-2 in vitro. In summary we have shown how a variety of stimuli relevant to inflammation can regulate endogenous N/OFQ secretion by splenocytes in vitro. We also suggest that N/OFQ may promote anti-inflammatory actions via suppression of IL-2 in vivo.

The nociceptin receptor antagonist [Nphe1,Arg14,Lys15]nociceptin/orphanin FQ-NH2 blocks the stimulatory effects of nociceptin/orphanin FQ on the HPA axis in rats

Nociceptin/orphanin FQ (N/OFQ) is an opioid-related peptide that stimulates corticosterone release after i.c.v. administration in non-stressed rats. We employed in situ hybridization histochemistry to investigate N/OFQ-stimulated activation of the HPA axis at the hypothalamic and pituitary level. We have demonstrated that N/OFQ-induced activation of the HPA axis is mediated via the central N/OFQ peptide receptor (NOP) using the recently described selective NOP antagonist [Nphe(1),Arg(14),Lys(15)]nociceptin/orphanin FQ-NH(2) (UFP-101). We found that, at 30 min post-i.c.v. injection, N/OFQ dose-dependently increased plasma adrenocorticotrophin hormone and corticosterone compared with the vehicle-injected controls. N/OFQ (1.0 microg) significantly increased CRF mRNA but not AVP mRNA within the parvocellular hypothalamic paraventricular nucleus compared with the control group, and significantly increased pro-opiomelanocortin (POMC) mRNA in the anterior pituitary. While UFP-101 (1.0 microg) alone had no significant effect on plasma corticosterone concentration it blocked the effect of N/OFQ (1.0 microg) on plasma corticosterone levels when compared with N/OFQ administered alone. UFP-101 also blocked the N/OFQ-induced increase in CRF mRNA and POMC mRNA. These results demonstrate that centrally administered N/OFQ activates the HPA axis via up-regulation of CRF and POMC mRNA and stimulation of corticosterone release in rats. Further, we have demonstrated for the first time that the selective NOP receptor antagonist UFP-101 blocks these effects indicating that N/OFQ-induced HPA axis activation is mediated via central NOP receptors.

The nociceptin/orphanin FQ antagonist UFP-101 differentially modulates the glucocorticoid response to restraint stress in rats during the peak and nadir phases of the hypothalamo–pituitary–adrenal axis circadian rhythm

The involvement of nociceptin (N/OFQ) and the nociceptin/orphanin FQ peptide (NOP) receptor in behavior associated with stress and anxiety has been established but their role in the regulation of the hypothalamo-pituitary-adrenal (HPA) axis under conditions of stress has not been fully investigated. We used the selective NOP receptor antagonist UFP-101 to examine the contribution of endogenous N/OFQ to HPA axis control under conditions of restraint stress in the morning and the evening. We found that in the morning during the HPA axis circadian nadir rats exposed to restraint stress in both the presence and absence of UFP-101 exhibited significantly elevated plasma corticosterone at 30 min post-i.c.v. injection compared to the home cage control group. Additionally, rats treated with UFP-101 and exposed to restraint had significantly elevated corticosterone levels at 60 min post-i.c.v. injection compared to all other treatment groups. Interestingly, while there was a significant increase in the expression of CRF mRNA in the paraventricular nucleus (PVN) of rats exposed to restraint stress only, there was no comparable increase in those co-treated with UFP-101. There was no change in the expression of AVP or POMC mRNA in any of the treatment groups. In contrast, when carried out in the evening we observed significantly elevated plasma corticosterone in the vehicle-treated restraint group only at 30 min post-i.c.v. injection. There was no significant difference between the UFP-101-treated restraint group and either of the home cage control groups or the vehicle-treated restraint group. Additionally, in contrast to the morning study, UFP-101 did not prolong glucocorticoid release at the 60 min time-point. These results demonstrate for the first time a differential effect of UFP-101 on restraint stress-induced HPA axis activity characterized by significant prolongation of stress-induced activity in the morning but no significant effect on the response to restraint in the evening.

Role of Nociceptin/Orphanin FQ and NOP Receptors in the Response to Acute and Repeated Restraint Stress in Rats

Central nociceptin/orphanin FQ (N/OFQ)‐expressing neurones are abundantly expressed in the hypothalamus and limbic system and are implicated in the regulation of activity of the hypothalamic‐pituitary‐adrenal axis (HPA) and stress responses. We investigated the role of the endogenous N/OFQ receptor (NOP) system using the nonpeptidic NOP antagonist, JTC‐801 [N‐(4‐amino‐2‐methylquinolin‐6‐yl)‐2‐(4‐ethylphenoxy‐methyl)benzamide monohydrochloride], during the HPA axis response to acute physical/psychological stress (60 min of restraint). Although i.v. JTC‐801 (0.05 mg/kg in 100 μl) had no significant effect on restraint‐induced plasma corticosterone release at 30 or 60 min post‐injection, i.v. JTC‐801 (0.05 mg/kg in 100 μl) in quiescent rats significantly increased basal plasma corticosterone at the 30‐min time‐point compared to i.v. vehicle (1% dimethysulphoxide in sterile saline). Central injection of JTC‐801 i.c.v. was associated with increased Fos expression in the parvocellular paraventricular nucleus 90 min after infusion compared to vehicle control. These findings contrast to the effects of i.c.v. UFP‐101, a NOP antagonist that we have previously shown to have no effect on HPA activity in quiescent rats. To determine whether restraint stress was associated with compensatory changes in N/OFQ precursor (ppN/OFQ) or NOP receptor mRNAs, in a separate study, we undertook reverse transcriptase‐polymerase chain reaction and in situ hybridisation analysis of ppN/OFQ and NOP transcripts in the brains of male Sprague–Dawley rats. In support of an endogenous role for central N/OFQ in psychological stress, we found that acute restraint significantly decreased preproN/OFQ transcript expression in the hippocampus 2 h after stress compared to unstressed controls. PpN/OFQ mRNA was also reduced in the mediodorsal forebrain 4 h after stress. NOP mRNA was reduced in the hypothalamus 2 h after restraint and at 4 h in mediodorsal forebrain and hippocampus. In situ hybridisation analysis showed that acute restraint significantly decreased ppNN/OFQ in the central amygdala, with significantly increased expression in bed nucleus and reticular thalamus associated with repeated restraint. There was a strong trend for reduced NOP mRNA in the bed nucleus of acute and repeated restraint groups, although there were no other significant changes seen. Although the exact mechanisms require elucidation, the findings obtained in the present study provide evidence indicating that the endogenous N/OFQ system is involved in both acute and chronic restraint stress responses. In summary, our findings confirm the significant role of endogenous NOP receptors and tonic N/OFQ function in the response to the psychological stress of restraint.

Endogenous nociceptin / orphanin FQ system involvement in hypothalamic-pituitary-adrenal axis responses: relevance to models of inflammation.

Nociceptin/orphanin FQ (N/OFQ) peptide and its receptor (NOP) function in the neuromodulation of anxiety, stress and hypothalamic‐pituitary‐adrenal (HPA) axis activity. We investigated the endogenous NOP system using the selective NOP antagonist, UFP‐101, during the HPA axis response to bacterial endotoxin, lipopolysaccharide (LPS). Although i.c.v. N/OFQ (1 μg/rat) had no significant effect on LPS‐induced (250 μg/rat i.p) plasma corticosterone release at 30 or 60 min post‐i.c.v. injection, i.c.v. UFP‐101 (1 μg/rat)/LPS significantly attenuated plasma adrenocorticotrophic hormone and corticosterone at the 30‐min time‐point compared to i.c.v saline (0.9%)/LPS. Parvocellular paraventricular nucleus (PVN) corticotrophin‐releasing factor (CRF) and corticotrophic pro‐opiomelanocortin (POMC), but not parvocellular PVN arginine vasopressin (AVP), mRNA expression was significantly increased by LPS compared to non‐LPS control. Intracerebroventricular UFP‐101/LPS treatment was associated with increased POMC mRNA expression 4 h after injection and a clear trend towards increased parvocellular CRF mRNA. Furthermore, i.c.v. UFP‐101 was selectively associated with an LPS‐induced increase in parvocellular AVP mRNA, an effect that was absent in the i.c.v saline/LPS group. To determine whether LPS challenge was associated with compensatory changes in N/OFQ precursor or NOP receptor mRNAs, in a separate study, we undertook reverse transcriptase‐polymerase chain reaction analysis of preproN/OFQ and NOP transcripts. In support of an endogenous role for central N/OFQ in inflammatory stress, we found that LPS significantly increased preproN/OFQ transcript expression in the hypothalamus 4 h after injection compared to the saline control. No changes in preproN/OFQ mRNA level in the hippocampus or basal forebrain (including bed nucleus of stria terminalis) were seen, albeit at 4 h. LPS was associated with a significant attenuation of NOP mRNA in the basal forebrain at 4 h, possibly as a compensatory response to increased N/OFQ release. Although the exact mechanisms require elucidation, the findings obtained in the present study provide evidence indicating that the endogenous NOP system is involved in the acute HPA axis response to immune challenge.

An intact dopaminergic system is required for context-conditioned release of 5-HT in the nucleus accumbens of postweaning isolation-reared rats

We investigated the effect of the tyrosine hydroxylase inhibitor, alpha-methyl-para-tyrosine (AMPT) on extracellular dopamine and 5-HT levels in the nucleus accumbens of group- and isolation-reared rats. Microdialysis with high-performance liquid chromatography-electrochemical detection was used to quantify dopamine and 5-HT efflux in the nucleus accumbens following foot shock and in association with a conditioned emotional response (CER). Isolation- and group-reared rats received i.p. injections of either saline (0.9%) or AMPT (200 mg/kg) 15 h and 2 h prior to sampling. There was no significant difference between saline-treated isolation- or group-reared rats for basal efflux of dopamine or 5-HT, however as expected, AMPT-treatment significantly reduced dopamine efflux in both groups to an equivalent level (50-55% saline-treated controls). Exposure to mild foot shock stimulated basal dopamine efflux in saline-treated groups only, although the effect was significantly greater in isolation-reared rats. In AMPT-treated rats, foot shock did not affect basal dopamine efflux in either rearing group. Foot shock evoked a prolonged increase in 5-HT efflux in both isolation- and group-reared saline-treated rats but had no effect on 5-HT efflux in AMPT-treated rats. In response to CER, isolation-rearing was associated with significantly greater efflux of both dopamine and 5-HT in saline-treated rats, compared to saline-treated, group-reared controls. However in AMPT-treated rats, efflux of dopamine or 5-HT did not change in response to CER. These data suggest that unconditioned or conditioned stress-induced changes in 5-HT release of the nucleus accumbens are dependent upon intact catecholaminergic neurotransmission. Furthermore, as the contribution of noradrenaline to catecholamine efflux in the nucleus accumbens is relatively minor compared to dopamine, our findings suggest that dopamine efflux in the nucleus accumbens is important for the local regulation of 5-HT release in this region. Finally, these findings implicate the isolation-enhanced presynaptic dopamine function in the accumbens with the augmented ventral striatal 5-HT neurotransmission characterized by isolation-reared rats.

An introduction to the HPA Axis

Abstract ntegrity of the hypothalamo–pituitary–adrenal (HPA) axis is essential to survival of vertebrate species. This neuroendocrine axis functions to coordinate neural, endocrine and immune responses to diverse stressful stimuli that threaten homeostasis. The final products of activation of the HPA axis are the glucocorticoids that exert widespread effects on body functions, including cellular metabolism and immune function. Inappropriate secretion of endogenous glucocorticoids is potentially damaging and may predispose to disease. Homeostatic regulation of the HPA axis is complex and involves coordination of multiple systems of the body, in part mediated by the bi-directional communication network between the brain, endocrine and immune systems. Health and integrity of the individual relies on the appropriate integration of stress signals, including pro-inflammatory messages, generated at central and peripheral sites. Functional balance between pro- and anti-inflammatory mediators is fundamental to the appropriate control of the HPA axis and the prevention of dysregulation in its activity, a characteristic of numerous stress-related disorders including chronic inflammatory disease.

Antisense inhibition of pro-opiomelanocortin and proenkephalin A messenger RNA translation alters rat immune cell function in vitro

Pro-opiomelanocortin (POMC) and proenkephalin A (PEA) antisense oligodeoxynucleotides respectively reduced and enhanced proliferation of rat splenocytes incubated with concanavalin A in vitro. Nonsense base sequences used as controls were without effect. Coincubation with the exogenous synthetic opioid peptides, ACTH, beta-endorphin, met-enkephalin or [D-ala,D-leu]-enkephalin did not significantly alter either the POMC or PEA antisense response, indicating potential differences in bioactivity of immunocyte opioid peptides compared with synthetic equivalents. Levels of the POMC opioid products, ACTH and beta-endorphin, were significantly reduced in splenocytes incubated with POMC antisense probes. These data provide evidence for functional effects of endogenous opioid peptides on rat splenocyte proliferation in vitro.