ORL1 Receptor Research Articles

Regulation of proinflammatory cytokines gene expression by nociceptin/orphanin FQ in the spinal cord and the cultured astrocytes

Peripheral inflammation induces central sensitization characterized by the development of allodynia and hyperalgesia to thermal stimuli. Recent evidence suggests that activation of glial cells and a subsequent increase in proinflammatory cytokines contribute to the development of behavioral hypersensitivity after nerve injury or peripheral inflammation. The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (ORL1 receptor), has been demonstrated to play an important role in modulation of nociceptive signals. In the present study, we investigated: (1) astrocyte activation and proinflammatory cytokine expression at the lumbar spinal cord following intraplantar administration of complete Freund’s adjuvant (CFA) in rats; (2) the mechanism of N/OFQ on nociception modulation, the relationship between N/OFQ and cytokines in the rat CNS in vivo and in vitro. The results showed: (1) CFA-induced peripheral inflammation evoked robust astrocyte activation and proinflammatory cytokines spinally; (2) down-regulation of cytokine mRNA transcripts by intrathecal administration of N/OFQ, the effects produced by N/OFQ were abolished by combination with ORL1 receptor-specific antagonist [Nphe 1]N/OFQ(1–13)NH 2; (3) ORL1 receptor was expressed on astrocytes of rat spinal cord; (4) cytokine gene expression was inhibited in astrocyte cultures exposed to N/OFQ, the inhibiting effects of N/OFQ were significantly blocked by [Nphe 1]N/OFQ(1–13)NH 2. The present data demonstrated that astrocyte activation and enhanced cytokine expression at the CNS had a role in eliciting behavioral hypersensitivity; the anti-nociception function of N/OFQ might be dependent on cytokines derived from astrocytes, the effects were attributable to the ORL1 receptor pathway.

Daily Intravesical Instillation of 1 mg Nociceptin/Orphanin FQ for the Control of Neurogenic Detrusor Overactivity: A Multicenter, Placebo Controlled, Randomized Exploratory Study

We studied the feasibility, safety and efficacy of daily intravesical instillation of 1 mg of the endogenous peptide nociceptin/orphanin FQ in a selected group of patients who perform clean intermittent self-catheterization for neurogenic detrusor overactivity incontinence. A total of 18 patients with neurogenic detrusor overactivity incontinence and on clean intermittent self-catheterization were prospectively randomized to receive 1 mg nociceptin/orphanin FQ in 10 cc saline (9) or placebo (saline) solution (9) at the first morning catheterization for 10 days. All patients completed a voiding diary using a frequency/volume chart according to International Continence Society guidelines, and reported daily incontinence frequency. Mean changes in incontinence episode frequency and voiding diary mean bladder capacity from baseline throughout treatment were the primary outcome variables. Urodynamic parameters (cystomanometric bladder capacity, maximum bladder pressure) were also recorded at baseline and during the study. The 2 groups were well balanced and all patients completed the study. The urodynamic parameters recorded during the study showed an increase in cystomanometric bladder capacity and a decrease in maximum bladder pressure compared to baseline only in patients assigned to the nociceptin/orphanin FQ group. Mean daily urine leakage episodes during nociceptin/orphanin FQ treatment were 0.94 vs a pretreatment baseline of 2.18 (p < 0.05), while no significant changes were reported in the placebo group (2.06 vs 2.43 baseline). The total mean voiding diary bladder capacity increased from 171 +/- 70 to 294 +/- 107 ml in patients receiving nociceptin/orphanin FQ, while voiding diary mean bladder capacity remained unchanged in patients receiving placebo (from 182 +/- 55 to 178 +/- 23 ml). No significant problems related to feasibility of the procedure as well as significant side effects were reported by patients. This study showed that intravesical nociceptin/orphanin FQ but not placebo inhibited the micturition reflex in patients with neurogenic detrusor overactivity incontinence, and demonstrated the clinical efficacy of nociceptin/orphanin FQ during 10 days of treatment. These findings support the use of nociceptin/orphanin FQ peptide receptor agonists as an innovative therapeutic approach for controlling detrusor overactivity incontinence.

3‐(4‐Piperidinyl)indoles and 3‐(4‐Piperidinyl)pyrrolo[2,3‐b]pyridines as Ligands for the ORL‐1 Receptor.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Nociceptin Levels in the Cerebrospinal Fluid of Chronic Pain Patients With or Without Intrathecal Administration of Morphine

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid-like receptor ORL-1 and is thought to be involved in pain transmission and modulation. Human studies have not yet defined its role in pain patients. The aims of this study were 1) to verify the presence of N/OFQ in the cerebrospinal fluid (CSF) of human controls and patients with chronic noncancer pain, including those treated with intrathecally administered morphine, and 2) to determine whether pain or treatment with long-term intrathecal morphine influences its levels. The CSF of 27 patients (nine controls and 18 with chronic noncancer pain, of whom 12 were treated chronically with intrathecally administered morphine and six were opioid naïve) was analyzed, blindly, with radioimmunoassay methods. N/OFQ was detected in all patients. Mean CSF concentrations were lowest in the morphine-treated group and highest in the untreated chronic pain patients (12.06 ± 1.19 and 57.41 ± 10.06 fmol/ml, respectively), and the difference between the morphine-treated group and controls was statistically significant (44.72 ± 13.56 fmol/ml, P < 0.05). The presence of N/OFQ peptide in human CSF may correlate with biological activities that are influenced by different pain states and long-term intrathecal-morphine treatment. Further studies should verify whether the determination of this peptide CSF level may provide information on opioid treatment efficacy and on the presence of opioid tolerance.

Coupling of ORL1 (NOP) receptor to G proteins is decreased in the nucleus accumbens of anxious relative to non-anxious mice

We studied the involvement of endogenous ORL1 (NOP) receptors in the anxiety state. In mice selected as “anxious” and “non-anxious”, ORL1 (NOP) receptor has been analysed by means of two autoradiographic approaches: [ 3H]nociceptin binding and nociceptin-stimulated [ 35S]GTPγS binding. We show that differences in anxiety state are associated with differences in G protein coupling efficiency of ORL1 (NOP) receptor in the nucleus accumbens, without any change in the density of the receptors.

Presynaptic opioid receptors on noradrenergic and serotonergic neurons in the human as compared to the rat neocortex

1. Electrically evoked release of [3H]-noradrenaline ([3H]-NA) or [3H]-5-hydroxytryptamine ([3H]-5-HT) in slices of human and the rat neocortex was used to characterize presynaptic opioid receptors. 2. Release of [3H]-NA in rat neocortical slices was reduced only by the mu-receptor agonist DAMGO (pIC50: 7.27, CI95: [7.22, 7.32]; Imax: 77.6+/-1.6%; antagonized by naloxone: pA2: 8.88, CI95: [8.78, 8.98]). 3. Release of [3H]-NA in human neocortical slices was unaffected by DAMGO, but inhibited by the delta-receptor agonist DPDPE (Imax: 25.7+/-2.2%) and the kappa-receptor agonist U-50,488H (19.7+/-2.7% inhibition at 1 microM). Both effects were antagonized by naltrindole (1 microM). 4. Release of [3H]-5-HT in rat neocortical slices, was inhibited by DAMGO (10 microM) and U-50,488H (1 and 10 microM) only in the presence of the 5-HT receptor antagonist methiotepin (1 microM). 5. Release of [3H]-5-HT in human neocortical slices was unaffected by DPDPE, but U-50,488H (Imax: 40.8+/-8.3%; antagonized by 0.1 microM norbinaltorphimine) and DAMGO (16.4+/-3.9% inhibition at 1 microM; antagonized by 0.1 microM naloxone) acted inhibitory. 6. Release of [3H]-5-HT in human neocortical slices was reduced by nociceptin/orphanin (0.1 and 1 microM). These effects were antagonized by the ORL1 antagonist J-113397 (1-[(3R,4R)-1-cyclo-octylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; 0.1 microM). 7. This study provides evidence for significant species differences in opioid receptor-mediated modulation of NA and 5-HT-release in human vs rat neocortex. In rats, mu-opioid receptors modulate NA release, but 5-HT release is only weakly affected by mu- and kappa-opioids. In contrast, NA release in human neocortex is modulated via delta-opioid receptors, but 5-HT release mainly via kappa-opioid receptors. In addition also the ORL1 receptor seems to be involved in 5-HT release modulation.

Synthesis and receptor binding properties of chimeric peptides containing a μ-opioid receptor ligand and nociceptin/orphanin FQ receptor ligand Ac-RYYRIK-amide

Four chimera peptides composed of ORL1 receptor ligand Ac-RYYRIK-NH2 and a μ-opioid receptor agonist dermorphin YAFGYPS-NH2 or YRFB-NH2, with a spacer linking the two pharmacophores, were synthesized and tested for their receptor binding properties. Chimera peptides with long spacers (a Lys and five or eight Gly residues) showed synergistically improved affinity for both the μ-opioid receptor and ORL1 receptor, while the chimera peptides with short spacers (Lys residue only) showed decreased or similar affinity compared to the monomeric receptor ligands. Chimera peptides containing long spacers may prove to be useful tools for studying ORL1 receptor/μ-opioid receptor heterodimers.

Opioid Receptor-Like (ORL1) Receptor Utilizes Both GoA and GoB for Signal Transductio

The ORL1 receptors stably expressed in HEK 293 cells can utilize PTX-resistant mutants of Galpha(oA/B) to inhibit adenylyl cyclase (AC) and stimulate extracellular signal-regulated protein kinases (ERKs). However, development of AC superactivation and loss of ERK1/2 responsiveness induced by chronic activation of the ORL1 receptors remained PTX-sensitive.

3-(4-Piperidinyl)indoles and 3-(4-piperidinyl)pyrrolo-[2,3-b]pyridines as ligands for the ORL-1 receptor

A novel series of indoles and 1H-pyrrolo[2,3-b]pyridines having a piperidine ring at the 3-position were synthesized and found to bind with high affinity to the ORL-1 receptor. Structure–activity relationships at the piperidine nitrogen were investigated in each series. Substitution on the phenyl ring and nitrogen atom of the indole and 1H-pyrrolo[2,3-b]pyridine cores generated several selective high-affinity ligands that were agonists of the ORL-1 receptor.

Pharmacological characterization of inhibitory effects of postsynaptic opioid and cannabinoid receptors on calcium currents in neonatal rat nucleus tractus solitarius

1. The profile of opioid and cannabinoid receptors in neurons of the nucleus tractus solitarius (NTS) has been studied using the whole-cell configuration of the patch clamp technique. 2. Experiments with selective agonists and antagonists of opioid, ORL and cannabinoid receptors indicated that mu-opioid, kappa-opioid, ORL-1 and CB1, but not delta-opioid, receptors inhibit VDCCs in NTS. 3. Application of [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; mu-opioid receptor agonist), Orphanin FQ (ORL-1 receptor agonist) and WIN55,122 (CB1 receptor agonist) caused inhibition of I(Ba) in a concentration-dependent manner, with IC50's of 390 nM, 220 nM and 2.2 microM, respectively. 4. Intracellular dialysis of the G(i)-protein antibody attenuated DAMGO-, Orphanin FQ- and WIN55,122-induced inhibition of I(Ba). 5. Both pretreatment with adenylate cyclase inhibitor and intracellular dialysis of the protein kinase A (PKA) inhibitor attenuated WIN55,122-induced inhibition of I(Ba) but not DAMGO- and Orphanin FQ-induced inhibition. 6. Mainly N- and P/Q-type VDCCs were inhibited by both DAMGO and Orphanin FQ, while L-type VDCCs were inhibited by WIN55,122. 7. These results suggest that mu- and kappa-opioid receptors and ORL-1 receptor inhibit N- and P/Q-type VDCCs via G alpha(i)-protein betagamma subunits, whereas CB1 receptors inhibit L-type VDCCs via G alpha(i)-proteins involving PKA in NTS.

Preparation of 3‐Spirocyclic Indolin‐2‐ones as Ligands for the ORL‐1 Receptor.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access the actual ChemInform Abstract, please click on HTML or PDF.

Distribution of nociceptin and Ro64-6198 activated [35S]-GTPγS binding in the rat brain

The peptide, nociceptin, was discovered as the endogenous ligand for the opioid-like receptor, ORL1. Since its discovery, this peptide has been shown to modulate the perception of pain, modulate feeding and produce behavioral effects in rodent models of mood disorders. Recently, the non-peptide agonist {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one} (Ro64-6198) of the ORL1 receptor has been reported in the literature. In the present study, we compared the distribution and potency of Ro64-6198 with nociceptin for their ability to stimulate [(35)S]-GTPgammaS binding to sections of rat brain. In initial studies, Ro64-6198 inhibited (125)I-nociceptin binding to the hORL1 receptors with a K(i) of 1.75 nM compared with 0.20 nM for nociceptin. To assess agonist potency in a whole cell assay, a cell line expressing the hORL1 receptor and G(alpha15) was created and used for calcium mobilization studies. In this assay system, Ro64-6198 increased intracellular calcium with an EC(50) of 52nM compared with 24 nM for nociceptin. Having verified the agonist properties of Ro64-6198, we then assessed the potency and distribution of ORL1 receptor activation in rat brain sections. In dose-response studies, Ro64-6198 increased [(35)S]-GTPgammaS binding to a variety of brain regions with EC(50) values ranging from 84.9 to 2,143 nM depending on the brain regions evaluated. These potencies were similar to that seen for nociceptin, but substantially lower than values established using [(125)I] nociceptin binding to the cloned human ORL1 receptor. In general, the brain distribution of agonist stimulated [(35)S]-GTPgammaS binding was similar when either Ro64-6198 or nociceptin were used. Using these techniques, we have demonstrated, for the first time that Ro64-6198 activates [(35)S]-GTPgammaS binding to rat brain sections and this compound stimulates a similar population of receptors as nociceptin.

Calcium Channels Brought Inside by Opioid-Like Receptor

Nociceptin is an endogenous pain-alleviating peptide that binds to the receptor ORL1, which is structurally similar to opioid receptors. ORL1 was recently found to interact directly with N-type calcium channels, resulting in tonic, agonist-independent inhibition. Altier et al. extend these observations and report that extended exposure to nociceptin stimulated the internalization of both ORL1 and N-type calcium channels, thereby decreasing calcium signaling. Initial experiments performed with transfected cells--imaging analysis and biochemical assays for cell surface versus internal pools of the receptor and channel--indicated that prolonged nociceptin exposure of cells expressing tagged forms ORL1 and Cav2.2 resulted in their internalization and their accumulation in acidic, lysosomal, and endosomal structures. The internalization was specific for the Cav2.2 channel and the ORL1 receptor and did not occur if ORL1 was coexpressed with other types of channels or if Cav2.2 was expressed with other opioid receptors or the G protein-coupled receptor angiotensin receptor 1 (ATR1). Analysis of dorsal root ganglion (DRG) neurons indicated that nociceptin treatment promoted the redistribution of N-type calcium channels in a subset of neurons (presumably the 10% in which ORL1 was present) from the cell surface to punctate internal structures. In the transfected cells, N-type channel current density was decreased by prolonged exposure to nociceptin. In cultured DRG neurons, one population of cells exhibited a pronounced inhibition of calcium signal after 30-minute nociceptin exposure. The abundance of the pool of cells that showed inhibition was the same as the abundance of ORL1-positive cells in the culture, which is consistent with the inhibited cells being the ones expressing ORL1. This cointernalization represents a novel mechanism for regulation of calcium channel activity and has implications for pain management and understanding the molecular mechanisms underlying tolerance to opioid pain relievers. C. Altier, H. Khosravani, R. M. Evans, S. Hameed, J. B. Peloquin, B. A. Vartian, L. Chen, A. M. Beedle, S. S. G. Ferguson, A. Mezghrani, S. J. Dubel, E. Bourinet, J. E. McRory, G. W. Zamponi, ORL1 receptor-mediated internalization of N-type calcium channels. Nat. Neurosci. 9 , 31-40 (2005). [PubMed]

Effects of ORL1 Receptor Agonists and Antagonists in Nociception

AbstractAbstractNociceptin/orphanin FQ (N/OFQ), a 17-amino acid neuropeptide structurally similar to opioid peptides, selectively binds to opioid receptor-like (ORL1) receptor, which has the structure related to classical opioid receptors, but it does not bind opioid ligands. The functions of the N/OFQ system are still under active investigation. The N/OFQ, similarly to traditional opioids, produces membrane hyperpolarization through the opening of potassium channels and activation the ORL1 receptor inhibits adenylyl cyclase activity. In the central nervous system, N/OFQ affects feeding, anxiolytic activity, memory and locomotion but particularly influences sensory perception. In the periphery, N/OFQ inhibits neurogenic contractions in various tissues and affects the function of the cardiovascular and renal systems. N/OFQ is localized in the central nervous system in structures related to nociceptive processes and plays a role in peptidergic signaling of the primary afferent neurons which may be particularly relevant to pain control. Many studies have shown that exogenous application of N/OFQ into the central and peripheral nervous system of rats and mice induces both pro- and antinociceptive effects depending on the drug dose, route of administration, pain stimulus or animal species. The aim of the present review was to demonstrate contribution of both central and peripheral ORL1 receptors as well as various ORL1 ligands to the acute nociceptive stimulation and in chronic pain processes, e.g., inflammation and neuropathy. Moreover, intensive search for both agonists and antagonists of ORL1 receptor opened new experimental and therapeutic possibilities. The novel ligands of ORL1 receptor with high potency and selectivity corroborated a well-documented contribution of this receptor to pain perception, and their potential use in clinical practice has been postulated.Key Words: Nociceptin/orphanin FQopioid receptor-like receptor (ORL1)ORL1 ligandsacute paininflammatory painneuropathic painanalgesia

ORL1 and opioid receptor preferences of nociceptin and dynorphin A analogues with Dmp substituted for N-terminal aromatic residues

Nociceptin (NOC) and dynorphin A (DYN) analogues containing 2′,6′-dimethylphenylalanine (Dmp) in place of Phe or Tyr in position 1 and/or 4 were synthesized and their metabolic stability and receptor-binding properties were investigated. [Dmp 1]NOC(1–13)-NH 2 ( 1) possessed high ORL1 receptor affinity comparable to that of the parent peptide with substantially improved affinities for κ-, μ-, and δ-opioid receptors. However, Dmp 4 substitution of NOC peptide ( 2) reduced ORL1 receptor affinity. [Dmp 1]DYN(1–13)-NH 2 ( 4) and its Dmp 4 analogue ( 5) possessed a 3-fold greater κ-opioid receptor affinity and improved κ-receptor selectivity compared to the parent peptide. Analogue 4 however exhibited an unexpectedly low in vitro bioactivity (GPI assay), suggesting, the phenolic hydroxyl group at the N-terminal residue in DYN peptide is extremely important for activation of the κ-opioid receptor. Analogue 5 possessed an improved κ-opioid receptor selectivity with an IC 50 ratio of 1(κ)/509(μ)/211598(δ); thus, this peptide may serve as a highly selective κ-receptor agonist for pharmacological study. Dmp 1 substitution in both the NOC and DYN peptides improved metabolic stability toward these peptides, while Dmp 4 substitution provided no additional metabolic stability.

N-terminally Truncated Variant of the Mouse GAIP/RGS19 Lacks Selectivity of Full-length GAIP/RGS19 Protein in Regulating ORL1 Receptor Signaling

The regulators of G protein signaling (RGS) are a family of proteins with conserved RGS domains and play essential roles in regulating G protein-mediated signal transduction and physiological events. GAIP/RGS19 (G alpha interacting protein, also classified as RGS19), a member of the RGS family, has been shown to negatively regulate the signaling of many G protein-coupled receptors, including the opioid receptors. Two GAIP/RGS19 mRNA variants, resulted from an alternative splicing of exon 2 of the GAIP/RGS19 gene, were identified in multiple mouse tissues. One of the transcripts consists of a complete set of exons and encodes a full-length GAIP/RGS19 protein, and the other does not have exon 2 and therefore encodes an N-terminal 22 residue truncated short GAIP/RGS19 protein. When co-expressed with either the opioid-receptor-like (ORL1) receptor or one of the mu, delta, and kappa opioid receptors, by transfecting dual-expression plasmids into COS-7 cells, the full-length GAIP/RGS19 was more effective than the N-terminally truncated variant and was more selective in regulating the ORL1 receptor signaling than in regulating the mu, delta, and kappa opioid receptors, as measured by the effectiveness to increase the agonist-stimulated GTPase activity and to reverse the agonist-induced inhibition of cyclic AMP accumulation. In the same assays, the N-terminally truncated GAIP/RGS19 did not distinguish ORL1 from the mu, delta, and kappa opioid receptors. In contrast, co-expression of RGS4 with either ORL1 or opioid receptors showed the selectivity of RGS4 for regulating opioid receptors was mu > kappa > delta > ORL1, an order completely different from that of GAIP/RGS19. The results suggest that GAIP/RGS19 prefers regulating ORL1 receptor signaling over other opioid receptors, and that the N-terminal domain of GAIP/RGS19 plays a crucial role in its receptor preference.

Preparation of 3-spirocyclic indolin-2-ones as ligands for the ORL-1 receptor

A novel series of indolin-2-ones having a spirocyclic piperidine ring at the 3-position was synthesized and found to bind with high affinity to the ORL-1 receptor. Structure–activity relationships at the piperidine nitrogen were investigated. Substitution on the phenyl ring and nitrogen atom of the indolin-2-one core generated several selective high-affinity ligands that were antagonists of the ORL-1 receptor.

Involvement of nociceptin/orphanin FQ in release of hypothalamic GnRH mediated by ORL1 receptor in ovariectomized rats

To investigate effect of the nociceptin/orphanin FQ (OFQ) on hypothalamus gonadotropin-releasing hormone (GnRH) release in ovariectomized (OVX) rats. GnRH radioimmunoassay (RIA) was used to study the effect of OFQ on GnRH release in hypothalamus slices in vitro. Push-pull perfusion and intracerebroventicular (icv) injection were used to examine the effect of OFQ on GnRH release in the hypothalamus medial preoptic area (POA) in vivo. Ovariectomies were performed on female Sprague-Dawley rats, and their plasma luteinizing hormone (LH) levels were measured after icv injection of OFQ with or without [Nphe1]NC(1-13)NH2, a competitive antagonist of opioid receptor-like1 receptor (ORL1 receptor). Reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate the expression of the ORL1 receptor in rat pituitary. GnRH release from hypothalamus slices was inhibited 90 min after the administration of 2 mmol/L and 20 mmol/L OFQ (P<0.05). Accordingly, GnRH release from hypothalamus POA was also significantly reduced by the injection of 0.2 mmol/L and 2 mmol/L OFQ. Plasma LH levels were also decreased significantly 2 h after icv injection of 20 nmol OFQ in OVX rats (P<0.05) and this effect could be abolished by pretreatment with 20 nmol [Nphe1]NC(1-13)NH2, that is, NC13. More interestingly, plasma LH levels in OVX rats increased markedly 2 h after icv injection of 100 nmol and 200 nmol NC13. RT-PCR analysis further revealed that the ORL1 receptor was not expressed in the pituitary of OVX rats. Central administration of nociceptin/orphanin FQ might inhibit the release of hypothalamic GnRH and decrease the plasma LH levels through ORL1 receptors in OVX rats.

Effects and mechanisms of supraspinal administration of rat/mouse hemokinin-1, a mammalian tachykinin peptide, on nociception in mice

Rat/mouse hemokinin 1 (r/m HK-1) is a novel tachykinin peptide whose biological functions are not fully understood. This work was designed to observe the effects of r/m HK-1 in pain modulation at supraspinal level in mice using tail-flick test. Intracerebroventricular (i.c.v.) administration of r/m HK-1 (0.1, 0.3, 1, 3 nmol/mouse) dose-dependently induced potent analgesic effect (ED 50 = 0.2877 nmol/mouse). When r/m HK-1 co-injected (i.c.v.) with SR140333 (a selective NK 1 receptor antagonist), SR140333 could fully antagonize the analgesic effect of r/m HK-1. The maximal analgesic effect of r/m HK-1 (3 nmol/mouse) could also be reversed by naloxone (i.p., 2 mg/kg). However, i.c.v. low dose administration of r/m HK-1 (10, 3, 1 pmol/mouse) induced hyperalgesia with a “U” shape curve, which means that the maximal hyperalgesic effect appeared at 3 pmol/mouse, and this effect of r/m HK-1 could also be fully blocked by SR140333. Interestingly, [Nphe 1]NC(1–13)NH 2, a selective opioid receptor like-1 (ORL-1) receptor antagonist, could fully reverse the maximal hyperalgesic effect of r/m HK-1 (3 pmol/mouse). In addition, when r/m HK-1 co-injected (i.c.v.) with SR48968 (a selective NK 2 receptor antagonist), SR48968 could hardly affect the nociceptive effects of r/m HK-1 either at nanomole concentration or at picomole concentration. These findings suggested that r/m HK-1 might play an important role in pain modulation at supraspinal level in mice and these effects were first elicited through the activation of NK 1 receptor, subsequently, whether activation of the classical opioid receptor or the ORL1 receptor depending on the dose of i.c.v. administration of r/m HK-1.

Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in guinea-pig vessels, but not in rat and mouse aorta

Cannabinoids exert complex effects on blood pressure related to their interference with cardiovascular centres in the central nervous system and to their direct influence on vascular muscle, vascular endothelium and heart. In view of the relative lack of information on the occurrence of CB1 receptors on the vascular postganglionic sympathetic nerve fibres, the aim of the present study was to examine whether cannabinoid receptor ligands affect the electrically evoked tritium overflow in superfused vessels (tissue pieces) from the guinea-pig, the rat and the mouse preincubated with 3H-noradrenaline. The cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]1,4-benzoxazinyl](1-naphthalenyl) methanone) inhibited the evoked tritium overflow in the guinea-pig aorta, but not in that of the rat or mouse. The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB1 receptor antagonist rimonabant, yielding an apparent pA2 value of 7.9. The most pronounced (near-maximum) inhibition obtained at the highest WIN 55,212-2 concentration applied (3.2 microM) amounted to 40%. WIN 55,212-2 also inhibited the evoked overflow in guinea-pig pulmonary artery, basilar artery and portal vein, again in a manner sensitive to antagonism by rimonabant. The latter did not affect the evoked overflow by itself in the four vessels, but did increase the electrically evoked tritium overflow from superfused guinea-pig hippocampal slices preincubated with 3H-choline and from superfused guinea-pig retina discs preincubated with 3H-noradrenaline (labelling dopaminergic cells in this tissue). The inhibitory effect of 3.2 microM WIN 55,212-2 on the evoked overflow from the guinea-pig aorta was comparable in size to that obtained with agonists at the histamine H3, kappa opioid (KOP) and ORL1 (NOP) receptor (1 or 10 microM, producing the respective near-maximum effects) whereas prostaglandin E2 1 microM caused a higher near-maximum inhibition of 70%. Prostaglandin E2 also induced an inhibition by 65 and 80% in the rat and mouse aorta respectively, indicating that the present conditions are basically suitable for detecting presynaptic receptor-mediated inhibition of noradrenaline release. The results show that the postganglionic sympathetic nerve fibres in the guinea-pig aorta, but not in the rat or mouse aorta, are endowed with presynaptic inhibitory cannabinoid CB1 receptors; such receptors also occur in guinea-pig pulmonary artery, basilar artery and portal vein. These CB1 receptors are not subject to an endogenous tone and the extent of inhibition obtainable via these receptors is within the same range as that of several other presynaptic heteroreceptors, but markedly lower than that obtainable via receptors for prostaglandin E2.