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Growing evidence indicates that targeting nociceptin receptor (NOP) signaling may have therapeutic efficacy in treating alcohol and opioid addiction. However, little is known about the therapeutic value of selective NOP agonists for the treatment of cocaine dependence. Recently, we identified a highly selective, brain-penetrant NOP small molecule agonist (SR-8993), and using this compound, we previously showed that nociceptin receptor activation attenuated consolidation of fear-related memories. Here, we sought to determine whether SR-8993 also affects the rewarding properties of cocaine. Using a conditioned place preference (CPP) procedure, we show that SR-8993 (3 or 10 mg/kg) failed to disrupt acquisition or expression of cocaine CPP (7.5 or 15 mg/kg) in C57BL/6 mice. Additionally, SR-8993 did not affect rate of extinction or reinstatement (yohimbine- and cocaine-induced) of cocaine CPP. These studies indicate that selective activation of NOP may not be sufficient in reducing behavioral responses to cocaine., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cerebral edema is a leading cause of mortality in stroke patients. The purpose of this study was to assess a non-selective opioid receptor agonist, biphalin, in decreasing reducing brain edema formation using both in vitro and in vivo models of stroke. For the in situ model of ischemia, hippocampal slices were exposed to oxygen glucose deprivation (OGD) conditions and we observed that hippocampal water content was increased, compared to normoxia. Treatment with the mu agonist, Tyr-D-Ala', N-CH, -Phe4, Glyol-Enkephalin (DAMGO), delta opioid agonists, D-pen(2), D-phe(5) enkephalin (DPDPE), and kappa agonist, U50 488, all significantly decreased brain slice water gain. Interestingly, the non-selective agonist, biphalin, exhibited a statistically significant (P<0.01) greater effect in decreasing water content in OGD-exposed hippocampal slices, compared with mu, delta, and kappa selective opioid agonists. Moreover, biphalin exhibited anti-edematous effects in a dose responsive manner. The non-selective opioid antagonist, naloxone, returned the water content nearly back to original OGD values for all opioid agonist treatments, supporting that these effects were mediated by an opioid receptor pathway. Furthermore, biphalin significantly decreased edema (53%) and infarct (48%) ratios, and neuronal recovery from stroke, compared with the vehicle-treated groups in a 12h permanent middle cerebral artery occlusion (MCAO) model of focal ischemia. Biphalin also significantly decreased the cell volume increase in primary neuronal cells exposed to OGD condition. These data suggest that opioid receptor activation may provide neuroprotection during stroke and further investigations are needed in the development of novel opioid agonist as efficacious treatments for brain ischemia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This study examines the effects of NOP agonists nociceptin/orphanin FQ (N/OFQ) and Ro 64-6198, NOP antagonists [Nphe(1)]N/OFQ(1-13)-NH(2) Nphe(1) and naloxone benzoylhydrazone (NalBzoH) on spatial memory in NMRI mice and pronociceptin (proNC) knockout (KO) mice using the water maze task. N/OFQ, administered i.c.v. (1, 5 and 10 nmol/mouse) and into hippocampal CA3 (1 nmol/mouse, bilaterally), impaired acquisition and retention in the maze. Impairments were blocked by pre-treatment with Nphe(1) (10 nmol, i.c.v.). Ro 64-6198 (0.1-0.3-1 mg/kg i.p.) also dose-dependently impaired learning. However, pre-treatment with NalBzoH (1 mg/kg, s.c.) failed to modify the effects of Ro 64-6198. Nphe(1) (10 nmol/mouse i.c.v.) and NalBzoH (1 mg/kg, s.c.) by themselves failed to affect maze performance, despite a tendency for enhanced performance. Prepro N/OFQ knockout (ppN/OFQ -/-) showed evidence of improved learning, evident at retention trials and in reversal training. ppN/OFQ -/- mice were not impaired by N/OFQ (10 nmol i.c.v.) in the task, suggesting that changes in postsynaptic NOP receptors may occur in such KO mice. It is concluded that N/OFQ and NOP receptors have an important role in hippocampus-dependent spatial learning and memory, probably by modulation of glutamatergic functions.

Although opioids are known to influence sleep-wake regulation, the neuroanatomic substrate(s) mediating these effects remain unresolved. We hypothesized that the influence of opiates on sleep may be mediated, at least in part, by the ventrolateral preoptic nucleus (VLPO), a key cell group for producing behavioral sleep. By combining in situ hybridization for kappa and mu receptor mRNA with immunostaining of Fos expressed by VLPO cells during sleep we show that >85% of sleep-active VLPO neurons contain mRNA for either or both opioid receptors. Microinfusions of a kappa receptor agonist into the VLPO region increased NREM sleep by 51% during the subjective night, whereas a mu receptor agonist increased wakefulness by 60% during the subjective day. The sleep- and wake-promoting effects of the kappa and mu agonists were blocked by prior administration of their respective antagonist. Combining retrograde tracing from the VLPO with immunohistochemistry for dynorphin (Dyn, the endogenous kappa receptor agonist) or endomorphin 1 (EM1, the endogenous mu receptor agonist) we show that the central lateral parabrachial subnucleus (PBcl) provides Dyn inputs to the VLPO, whereas hypothalamic histaminergic neurons provide EM1 inputs to the VLPO. In summary, results from the present study suggest that central opioid inputs to the VLPO may play a role in sleep-wake regulation and that the VLPO likely mediates the hypnotic response to high levels of opioid analgesics.

We have reported that there is heterologous interaction between the mu, delta or kappa opioid receptors and the receptors for the chemokines CCL5/RANTES or CXCL12/SDF-1 in the regulation of antinociception in rats. CX3CL1/fractalkine, a chemokine that exclusively binds to CX3CR1, has been found to affect morphine analgesia and tolerance in the spinal cord. The purpose of the present study was to see if the interaction between the chemokine CX3CL1/fractalkine receptor and mu, delta or kappa opioid receptors occurs in the periaqueductal grey (PAG) of adult male S-D rats. The cold-water tail-flick (CWT) test was used to measure antinociception. The results showed that intra-PAG injection of 100 ng CX3CL1/fractalkine 30 min before administration of 400 ng DAMGO, 100 ng DPDPE or 20 microg dynorphin significantly reduced the antinociception induced by each of these peptides. These results demonstrate that activation of the CX3CL1 receptor diminishes the effect of mu, delta and kappa opioid agonists on their receptors in the PAG of rats.

Opioid peptides and glycyl-glutamine (Gly-Gln) have been implicated in the control of ethanol consumption. A recognized beta-endorphin cleavage product, Gly-Gln, inhibits voluntary alcohol consumption when microinjected into the nucleus accumbens (AcbSh) of P rats. To evaluate the site-specific efficacy of Gly-Gln on ethanol consumption following AcbSh application, ethanol preferring (P) rats were allowed to establish individual baseline ethanol/water consumption utilizing a voluntary self-administration paradigm. Subsequent to baseline ethanol consumption being established, bilateral guide cannulae were stereotaxically implanted +1 mm dorsal to the AcbSh for subsequent Gly-Gln (100 nmol/microl) or saline vehicle (1 microl) injections. Alcohol intake, body weight, and water intake were measured at 24 h post-injection intervals. Unilateral Gly-Gln injections reduced ethanol consumption 35.6% (P < 0.05) from pre-established baseline consumption (6.24 +/- 0.64 g/kg to 4.06 +/- 0.28 g/kg). Bilateral Gly-Gln injections further reduced consumption to 51.9% (6.4 +/- 1.0 g/kg to 3.08 +/- 0.65 g/kg at 24 h (P < 0.01) below established baseline values within 24 h without significant changes in body weight or water consumption. Also, the amino acid constituents of the dipeptide had no influence on ethanol consumption behavior; however, Gly-Gln efficacy was shown to be comparable to central beta-endorphin-(1-27) or intraperitoneal (i.p.) naltrexone-induced suppression of ethanol intake. These data indicate that the AcbSh exhibits a site-specific sensitivity to the suppressive actions of Gly-Gln or beta-endorphin-(1-27) injections that modulate voluntary ethanol consumption in P rats. These findings support the broader concept that select forebrain opioid-responsive neural sites may influence the development or expression of alcohol abuse syndromes in animal models or humans.

Cardiovascular effects of activation of opioid receptor like receptors (ORL1 receptors) in the nucleus ambiguus were studied in urethane-anesthetized, adult male Wistar rats. Microinjections of nociceptin (0.31, 0.62, 1.25 and 2.25 mmol/L) into the nucleus ambiguus elicited increases in heart rate (17.5 +/- 4, 33.3 +/- 2.9, 16.5 +/- 1.5 and 13.9 +/- 2.7 beats/min, respectively) which were blocked by an ORL1 receptor antagonist. These results indicate that activation of ORL1 receptors in the nucleus ambiguus elicits tachycardia.

Lithium has been reported to inhibit opioid-induced properties. The present study examined the effect of acute and chronic administration of lithium chloride (LiCl) on morphine's biphasic modulation of susceptibility to pentylenetetrazole (PTZ)-induced clonic seizure in mice. We also examined the possible involvement of nitric oxide (NO) pathway in lithium effect. Both acute (0.1 and 1 mg/kg) and chronic (same doses, 21 consecutive days) administration of LiCl completely inhibited the anticonvulsant and proconvulsant effects of morphine (at doses 1 and 30 mg/kg, respectively). A very low and per se noneffective dose of LiCl (0.05 mg/kg) significantly inhibited both phases of morphine effect when administered concomitant with a noneffective low dose of naloxone (0.1 mg/kg). The NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) at a per se noneffective dose of 0.3 mg/kg potentiated the inhibitory effects of low doses of LiCl (0.01 and 0.05 mg/kg) on both phases of morphine effect. l-arginine, a NO synthase substrate, at a per se noneffective dose of 30 mg/kg reversed the inhibitory effects of lithium (1 mg/kg). Lithium is capable of antagonizing both modulatory effects of morphine on seizure susceptibility even at relatively low doses. These inhibitory effects of lithium may also involve NO synthesis.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP) which is yet to be functionally characterized in dog brain. Ligand binding data reports low NOP density (29 fmol mg(-1) protein) in dog. In this study using dog brain membranes, we have examined the effects of N/OFQ on [leucyl-(3)H]N/OFQ(1-17)OH ([leucyl-(3)H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 microM GTPgammaS. Data from standard [(35)S]GTPgammaS binding and immunoprecipitation (G(alphai1-3)) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHO(hNOP)) cells. N/OFQ displaced [leucyl-(3)H]N/OFQ binding with pK(i) and slope values of 9.62+/-0.07 and 0.38+/-0.05, respectively. Addition of NaCl/GTPgammaS produced a steepening (slope 0.95+/-0.06, n=3) of the curve. N/OFQ stimulated [(35)S]GTPgammaS binding with pEC(50) and E(max) values of 8.21+/-0.17 and 1.17+/-0.01, respectively (in CHO(hNOP), pEC(50) and E(max) values were 8.47+/-0.01 and 7.01+/-0.63). N/OFQ stimulated [(35)S]GTPgammaS binding in dog and CHO(hNOP) cell membranes could be immunoprecipitated with an anti-G(alphai1-3) antibody, indicating coupling to a pertussis toxin (PTx)-sensitive G-protein. N/OFQ actions were competitively antagonized by the selective NOP antagonists, 100 nM J-113397, 1 microM [Nphe(1)]N/OFQ(1-13)NH(2) and 1 microM [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) (partial agonist) yielding pK(B) values of 8.58+/-0.21, 7.06+/-0.59 and 7.32+/-0.41, respectively (in CHO(hNOP), a pK(B) for J-113397 of 8.33+/-0.02 was obtained). Despite relatively low receptor density, we were able to detect functional activity of native dog NOP, with pharmacology consistent with reports for other species.

Nocistatin and nociceptin/orphanin FQ (N/OFQ) are two neuropeptides derived from the same precursor protein, prepronociceptin (ppOFQ), and exhibit different effects on spinal neurotransmission. Nocistatin does not bind to nociceptin/orphanin FQ peptide receptor (NOP), but intrathecal (i.t.) nocistatin has been reported to block the analgesic effect of i.t. N/OFQ. In this study, we investigated the effect of i.t. nocistatin on N/OFQ analgesia to radiant thermal stimuli in chronic constriction injury (CCI) rat. Firstly, to investigate the analgesic effect of N/OFQ, different doses of N/OFQ (3, 10, 30 microg) were intrathecally injected and foot withdrawal latency (FWL) to radiant heat was recorded. It is observed that 3 microg N/OFQ had no effect on FWL, 10 and 30 microg N/OFQ significantly increased FWL of CCI rat. Then, 10 microg N/OFQ, 10 microg nocistatin and a drug cocktail including 10 microg N/OFQ and 10 microg nocistatin were intrathecally injected. The results showed that FWL significantly decreased after using N/OFQ and nocistatin compared with using only N/OFQ, and 10 microg nocistatin had no effect on FWL versus control, suggesting that this dose of nocistatin per se had no effect on the pain threshold of CCI rat, but could block the analgesic effect of N/OFQ. These results indicated that i.t. N/OFQ dose-relatedly depressed thermal hyperalgesia produced by CCI and nocistatin could block N/OFQ analgesia at spinal level in CCI rat.

Immunoreactivity for nociceptin, an endogenous ligand for the ORL1 opioid receptors, has been reported in the nucleus tractus solitarius (nTS). A midline area in the commissural subnucleus (nCom) of nTS is the site of peripheral chemoreceptor projections. This investigation was carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats, to study the cardiovascular effects of the activation of ORL1 receptors in a midline area of the nCom. Microinjections (30 nl) of nociceptin (0.15-0.62 mM) into the nCom elicited depressor and bradycardic responses. Prior microinjections of [N-Phe(1)]-nociceptin-(1-13)-NH(2) (4.5 mM), a specific antagonist for ORL1 opioid receptors, into the nCom blocked the effects of nociceptin (0.31 mM, the maximally effective concentration), but not endomorphin-2 (0.6 mM; an endogenous ligand for micro -opioid receptors). On of other hand, naloxone (0.125 mM; an antagonist for classical opioid receptors) did not block the effects of nociceptin, while it did block the effects of endomorphin-2. The blockade of nociceptin effects by [N-Phe(1)]-nociceptin-(1-13)-NH(2) and endomorphin-2 by naloxone, was not due to some nonspecific effects because the responses to L-Glu (5 mM) remained unaltered after the microinjection of these antagonists. These results indicate that activation of ORL1 receptors in the nCom may play a role in the regulation of cardiovascular function.

The opioid peptide, Orphanin FQ/nociceptin (OFQ/N(1-17))(,) its active fragments, and a related precursor peptide each produce analgesia following microinjection into the amygdala of rats. OFQ/N(1-17)-induced analgesia elicited from the amygdala is blocked by amygdala pretreatment of either general, mu, kappa, or delta-opioid antagonists even though OFQ/N(1-17) binds poorly to these receptor subtypes, and the antagonists bind poorly to the ORL-1/KOR-3 receptor. Agonists at mu and kappa opioid receptors as well as beta-endorphin each produce analgesia elicited from the amygdala that is blocked by opioid antagonist pretreatment in the ventrolateral periaqueductal gray (vlPAG) of rats. The present study examined whether pretreatment of general and selective opioid antagonists in the vlPAG blocked OFQ/N(1-17)-induced analgesia on the tail-flick test elicited from the amygdala, and whether pretreatment of general and selective opioid antagonists in the amygdala blocked OFQ/N(1-17)-induced analgesia elicited from the vlPAG of rats. OFQ/N(1-17)-induced analgesia elicited from the amygdala was significantly and markedly reduced following vlPAG pretreatment with a dose range of either naltrexone, beta-funaltrexamine (beta-FNA, mu), nor-binaltorphamine (NBNI, kappa) or naltrindole (NTI, delta). In contrast, opioid antagonists administered into misplaced mesencephalic control placements ventral and lateral to the vlPAG actually enhanced OFQ/N(1-17)-induced analgesia elicited from the amygdala. OFQ/N(1-17)-induced analgesia elicited from the vlPAG was significantly and markedly reduced following amygdala pretreatment with naltrexone and NBNI, to a lesser degree by NTI, and was unaffected by beta-FNA. Yet, opioid antagonists administered into misplaced amygdala control placements were generally ineffective in altering OFQ/N(1-17)-induced analgesia elicited from the vlPAG. Latencies were transiently increased by general, but not selective opioid antagonist treatment alone in the amygdala, but not the vlPAG. These data indicate reciprocal and regional interactions between the amygdala and vlPAG in the mediation of OFQ/N(1-17) by classic opioid receptor subtype antagonists in rats.

In the present study we investigated long-term interactions between opioid and cannabinoid drugs at several steps along their cellular signal transduction pathways. For this purpose we co-transfected HEK-293 and COS-7 cells with delta-opioid (DOR) and CB1-cannabinoid receptors, and examined the effect of prolonged exposure to either opioid (etorphine) or cannabinoid (DALN) agonists on DOR and CB-1 receptor density and on the ability of subsequent application of the agonists to activate G-proteins (as measured by [35S]GTPgammaS binding) and to inhibit cAMP production. In HEK-293 cells, etorphine induced both homologous and heterologous desensitization, while DALN induced only homologous desensitization. This asymmetric cross-desensitization coincided with asymmetric cross downregulation: etorphine downregulated the binding of the cannabinoid ligand [3H]CP55,940, while DALN failed to reduce the binding of the opioid ligand [3H]diprenorphine. In contrast to the asymmetric desensitization in HEK-293 cells, COS-7 cells presented a two-way cross-desensitization between opioid and cannabinoid agonists, and DALN downregulated the binding of [3H]diprenorphine in these cells. Thus, a complete correlation was found between downregulation and reduction in cell responsiveness ('desensitization'). Moreover, when opioid downregulation in HEK-293 cells was inhibited by either hypertonic sucrose solution or protein kinase inhibitors, desensitization was suppressed to the same extent. These results suggest that, under the present experimental conditions, the reduction in cell responsiveness resulted primarily from downregulation of the receptors.

Physicians have noted since antiquity that their patients complained of less pain and required fewer analgesics at night-time. In humans, the circulating levels of melatonin, a pineal substance with analgesic and hypnotic properties, exhibit a pronounced circadian rhythm with serum levels being high at night and low during day-time. Moreover, pinealectomy abolishes the analgesic effects of melatonin, and naloxone disrupts the day-night rhythm of nociception. In this study, we have attempted to identify and characterize the nature and types of opioid receptor in bovine pinealocyte membranes, using a radioligand binding technique with the selective radioligands [3H]DAMGO, [3H]DPDPE, [3H]U69593 and [3H]orphanin-FQ (OFQ) for identifying mu (mu)-, delta (delta)-, kappa (kappa)- and opioid receptor-like (ORL(1)) receptors, respectively. The saturation experiments on bovine pinealocyte membranes for [3H]DPDPE binding provided B(max) and K(d) values of 553+/-24 fmol/mg protein and 1.3+/-0.6 nM; and for [3H]DAMGO binding provided B(max) and K(d) values of 6.3+/-1.3 fmol/mg protein and 1.2+/-0.4 nM, respectively. On the other hand, the specific radioligands ([3H]U69593 and [3H]OFQ) binding of kappa and ORL(1) receptors were undetectable in bovine pinealocyte membranes. Furthermore, competitive experiments with opioid agonist and antagonist and related compounds confirmed the presence of mu- and delta-opioid binding sites in bovine pinealocyte membranes. These results indicate that neither kappa nor ORL(1) receptors are present on the pinealocytes, and the majority of opioid receptors found in the bovine pineal gland are delta (possibly, both delta(1) and delta(2)) types, with a minority being mu type, and that both are primarily located on the bovine pinealocyte membranes. These opioid receptors, by stimulating the activity of N-acetyltransferase, enhance the synthesis of melatonin.

Orphanin FQ/Nociceptin (OFQ/N), an endogenous peptide found throughout the central nervous system, has been attributed with a wide range of functions, including modulation of motivational and emotional behavior, but most prominently, facilitation of hyperalgesia. It has also been shown that brain OFQ/N is stimulated during locally-induced peripheral inflammation, a condition well known to increase pain sensitivity. However, few studies have addressed whether specific immunological challenge using T-cell dependent and independent stimuli alters OFQ/N gene activation in the brain. Consequently, male C57BL/6J mice were challenged with 5 microg of lipopolysaccharide (LPS) or a T-cell-activating bacterial superantigen, Staphyloccocal enterotoxin A (SEA), and levels of brain OFQ/N precursor, pNOC, mRNA were analyzed by semi-quantitative RT-PCR. In addition, nociceptive thresholds were examined in immunologically challenged mice using the hotplate test. Initial results on a combined region of the brain containing various limbic components, revealed increased levels of pNOC mRNA in response to SEA challenge, but not to LPS. Further analysis of more discrete brain regions revealed increased pNOC mRNA in the hypothalamus and amygdala in response to SEA. Interestingly, challenge with SEA, but not LPS, significantly reduced hindpaw-lick latency in the hot plate test, although this effect was observed only if the hotplate environment was unfamiliar, suggesting an interaction between immunological stimulation and novelty-induced stress. Since SEA induces various cytokines, including TNF-alpha, these results are consistent with a growing literature documenting the effects of cytokines on nociceptive functions, and a possible involvement of the OFQ/nociceptin system., (Copyright 2002 Elsevier Science B.V.)

Orphanin/nociceptin (OFQ/N), a 17-amino-acid peptide, is an endogenous peptide, the receptor for which is similar to mu-, delta- and kappa-opioid receptors ( approximately 65% homology). Reports indicate that OFQ/N can block the antinociception induced by mu-, delta- and kappa-opioid agonists in the rat and in the mouse, indicating that there is a functional interaction between opioid receptors and OFQ/N receptors in the nervous system. It is well known that activation of the mu- and kappa-opioid receptors results in hyperthermia and hypothermia, respectively, in Sprague-Dawley rats. The present studies were designed to examine effects of OFQ/N on body temperature (Tb) and explore whether the mechanism of T(b) change induced by OFQ/N involved the opioid system. The results show that (1) i.c.v. injection of a high dose of OFQ/N (9-18 micro g) produces hypothermia in adult rats; (2) OFQ/N (1.8 micro g, i.c.v., t=+30 s after morphine) can decrease morphine-induced hyperthermia; (3) neither the opioid receptor antagonist, naloxone (10 mg/kg, s.c., t=-15 s before OFQ/N) nor the kappa-opioid receptor antagonist nor-BNI (1 micro g/5 microl, i.c.v., t=-30 s before OFQ/N) reduces the hypothermia induced by i.c.v. injection of OFQ/N at dose of 18 micro g (P>0.05); (4) 60 micro g/5 microl AS oligo (i.c.v. treatment on days 1, 3 and 5) against OFQ/N receptors significantly reduces the hypothermia induced by i.c.v. injection of 9 micro g OFQ/N (P<0.01). These results suggest that the hypothermia induced by i.c.v. injection of a high dose of OFQ/N (9 or 18 micro g) is mediated, at least partially, by its own receptor, independent or downstream of opioid receptors in the rat brain and that OFQ/N probably acts as a physiological antagonist to reduce morphine-induced hyperthermia.

The binding sites of nociceptin (also named orphanin FQ), the endogenous ligand of ORL1 (opiate receptor like 1), were localized in rat brain, using an autoradiographic procedure. High levels of binding were observed in the cingulate, retrosplenial, perirhinal, insular and occipital cortex, anterior and posteromedial cortical amygdaloid nuclei, basolateral amygdaloid nucleus, amygdaloid complex, posterior hippocampus, dorsal endopiriform, central medial thalamic, paraventricular, rhomboid thalamic, suprachiasmatic, ventromedial hypothalamic nuclei, mammillary complex, superficial gray layer of the superior colliculus, locus coeruleus, dorsal raphe nucleus. More moderate labelling was observed in the prefrontal, fronto-parietal, temporal, piriform cortex, dentate gyrus, anterior olfactory nucleus, olfactory tubercle, shell of nucleus accumbens, claustrum, lateral septum, laterodorsal thalamic, medial habenular, subthalamic, reuniens thalamic nuclei, subiculum, periaqueductal grey matter and pons. A lower binding site density was observed in the anterior and medial hippocampus, olfactory bulb, caudate putamen, the core of the nucleus accumbens, medial septum, ventrolateral, ventroposterolateral and mediodorsal thalamic nuclei, lateral and medial geniculate nuclei, hypothalamic area, substantia nigra, ventral tegmentum area and interpedoncular nucleus. A moderate and similar labelling was found in the dorsal and ventral horn of the spinal cord. No labelling was apparent in the corpus callosum. Thus, it appears that the ORL1 receptor is particularly abundant in the cerebral cortex, limbic system of the rat brain and some areas involved in pain perception.

Long-term memory formation for passive-avoidance learning in the day-old chick is known to have two distinct time windows of protein synthesis (F.M. Freeman, S.P.R. Rose, A.B. Scholey, 1995. Two time windows of anisomycin-induced amnesia for passive-avoidance training in the day-old chick. Neurobiol. Learn. Mem. 63, 291-295). The lobus parolfactorius (LPO) is thought to be an important site for the second wave of protein synthesis which occurs 4-5 h after training. Birds received bilateral intracranial injections of agonists and antagonists for the mu-, delta-, kappa-opioid receptors and the opioid receptor-like (ORL(1)) receptor directly into the LPO at 5 h post-training and were tested for recall 24 h later. Also, 100 microM beta-funaltrexamine (beta-FAN), a mu-opioid receptor antagonist, significantly impaired memory formation (P<0.01). The delta-opioid receptor was also involved in memory formation at this time-point since antagonism of this receptor by 1 mM ICI-174,864 caused amnesia (P<0.01) which was reversed by the agonist, DPLPE. The kappa-opioid receptor appeared not to be involved during the second phase of neuronal activity since neither stimulation by dynorphin nor inhibition by nor-BIN caused amnesia for the task. The ORL(1) receptor agonist orphanin FQ also had no effect suggesting that this receptor was not involved at this 5-h time-point. Cytosolic and mitochondrial protein synthesis has been shown to be important in passive-avoidance learning in the day-old chick. Both chloramphenicol (CAP) and anisomycin (ANI), inhibitors of mitochondrial and cytosolic protein synthesis, respectively, caused disruption when injected 5 h post-training into the LPO (P<0.05). Endomorphin-2 (Endo-2), a mu-opioid receptor agonist, reversed both the ANI- and CAP-sensitivity. However, DPLPE, a delta-opioid receptor agonist, only reversed the effect due to CAP. Possible mechanisms for these effects are discussed.

Potential anxiolytic-like properties of intracerebroventricular (i.c. v.) infusion of orphanin FQ (OFQ), a recently discovered neuropeptide, were investigated in the mouse defense test battery, a well-validated anxiolytic screening test. In this model, Swiss mice are directly confronted with a natural threat (a rat) as well as situations associated with this threat. Primary measures taken during and after rat confrontation were flight, risk assessment, defensive attack and escape attempts. Unlike the anxiolytic drug diazepam (3-10 microgram/5 microliter, i.c.v.), which affected all defensive responses, OFQ (0.3-3 nM/5 microliter) only clearly reduced defensive upright postures and biting reactions. Subjects displayed these latter defensive behaviors upon forced contact with the threat stimulus, a situation which is considered to be highly stressful. These results suggest that the OFQ system may not be primarily involved in anxiety-related responses including cognitive aspects (i. e., risk assessment), while it may play a role in the adaptative responses to unavoidable or extreme stress stimuli., (Copyright 1999 Published by Elsevier Science B.V.)

Orphanin FQ (also known as nociceptin) is a 17-amino-acid peptide which acts as a potent endogenous agonist of the orphan opioid receptor-like (ORL1) receptor. Endomorphin-1, a 4-amino-acid peptide discovered recently, is a potent and selective endogenous agonist for the mu-opiate receptor. In the present study, the effect of OFQ or/and endomorphin-1 on the response to noxious thermal stimuli was observed using the tail-flick test in rats. Intracerebroventricular (i.c.v.) administration of OFQ (1, 5 microg) could shorten tail-flick latency; In contrast, intrathecal (i.t.) administration of OFQ (1, 2 or 10 microg) could increase the latency; i.c.v. (1, 2, 5 microg) or i.t. (0.2, 2, 5 microg) administration of endomorphin-1 dose-dependently increased the latency, indicating an analgesic effect. Furthermore, OFQ (0.1-5 microg) when intraventricularly injected together with endomorphin-1 (5 microg), could dose-dependently reverse the analgesia induced by the latter. On the contrary, OFQ (1 microg) intrathecally injected together with endomorphin-1 (0.2 microg) could further increase the tail-flick latency. The results showed that OFQ at the supraspinal level produces hyperalgesia and is antagonistic to endomorphin-1, while at the spinal level it produces analgesia and is synergic with endomorphin-1. Different interaction mechanism between OFQ and endomorphin-1 in the brain and the spinal cord is thus suggested., (Copyright 1999 Elsevier Science B.V.)

Nociceptin/orphanin FQ (NOC/oFQ) is a recently discovered endogenous ligand for the opioid like receptor, ORL-1. In the piglet, cGMP activates the ATP sensitive (K(ATP)) while cAMP activates both the K(ATP) and the calcium sensitive (K(ca)) K(+) channel to elicit vasodilation. The present study was designed to characterize the role of cGMP, cAMP, K(ATP), and K(ca) channel activation in NOC/oFQ-induced pial artery dilation in newborn pigs equipped with a closed cranial window. NOC/oFQ (10(-8), 10(-6) M) induced pial arteriole dilation was decreased by the protein kinase A inhibitor Rp 8-Br cAMPs (16+/-1 and 30+/-1 vs. 5+/-1 and 10+/-1%). NOC/oFQ dilation was associated with elevated CSF cAMP (1037+/-58 vs. 1919+/-209 fmol/ml for control and 10(-6) M NOC/oFQ). Glibenclamide and iberiotoxin, K(ATP) and K(ca) channel antagonists, attenuated NOC/oFQ induced dilation (15+/-1 and 28+/-1 vs. 10+/-1 and 19+/-1% before and after iberiotoxin). In contrast, the nitric oxide synthase inhibitor, L-NNA, and the protein kinase G inhibitor, Rp 8-Br cGMPs had no effect on NOC/oFQ dilation while such dilation was not associated with a change in CSF cGMP. The putative ORL-1 receptor antagonist [F/G] NOC/oFQ (1-13)-NH(2) blocked NOC/oFQ dilation while responses were unchanged after naloxone (17+/-1 and 30+/-2 vs. 3+/-1 and 5+/-1%, before and after [F/G] NOC/oFQ (1-13)-NH(2)). Dilation to other opioids (e.g., methionine enkephalin) was unchanged by [F/G] NOC/oFQ (1-13)-NH(2). These data show that NOC/oFQ elicits pial artery dilation, at least in part, via cAMP, K(ATP), and K(ca) channel dependent mechanisms. These data suggest that such a mechanism involves the sequential release of cAMP and subsequent K(ATP) and K(ca) channel activation., (Copyright 1999 Elsevier Science B.V.)

Nociceptin (orphanin FQ), the newly discovered endogenous ligand for the novel opioid receptor-like 1 receptor, has been initially found to participate in pain modulation. In this study, centrally mediated cardiovascular actions of this peptide were investigated in the alpha-chloralose/urethane-anesthetized rats. We found that bilateral injection of nociceptin (10 nmol) into the rostral ventrolateral medulla (RVLM), wherein injection of excitatory amino acid dl-homocysteic acid (3 nmol) induced typical pressor responses, significantly reduced arterial blood pressure and heart rate by -32% and -15%, respectively. Reduction of blood pressure and heart rate in response to intra-RVLM injection of nociceptin was dose-dependent with a threshold dose being 3 nmol. Pretreatment with the selective nociceptin receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 (10 nmol), into the RVLM abolished the nociceptin-induced cardiovascular inhibition. In contrast, non-selective opioid receptor antagonist, naloxone (10 nmol), did not modify the hypotension and bradycardia induced by nociceptin, even though naloxone at the same dose prevented reduction of blood pressure and heart rate induced by intra-RVLM injection of methionine-enkephalin (3 nmol). Both [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 and naloxone injection alone had no significant effect on baseline blood pressure and heart rate. These data suggest that the newly discovered opioid-like neuropeptide nociceptin in the CNS exert powerful influence on hemodynamic activity by affecting the RVLM neurons. This influence is inhibitory in nature, which may not be active in normal physiological conditions. Moreover, the cardiovascular effects of nociceptin were mediated by activation of specific nociceptin receptors rather than typical naloxone-sensitive opioid receptors., (Copyright 1999 Elsevier Science B.V.)

The effects of nociceptin (orphanin FQ) on medial vestibular nucleus (MVN) neurons in vitro, and on vestibulo-ocular reflex (VOR) function in vivo, were investigated in order to determine the role of 'opioid-like orphan' (ORL1) receptors in modulating vestibular reflex function in the rat. Nociceptin (100 nM-1 microM) potently inhibited the spontaneous discharge of the majority (86%) of MVN neurons tested in the rat dorsal brainstem slice preparation in vitro. This inhibition was dose-dependent and persisted after blockade of synaptic transmission in low Ca2+/Co2+ medium. The inhibitory effects were insensitive to the opioid antagonist naloxone, but were effectively antagonised by the selective ORL1 receptor antagonist, [Phe1Psi(CH2-NH)Gly2]Nociceptin(1-13)NH2. The majority of MVN neurons ( approximately 70%) were inhibited by both nociceptin and the delta-opioid receptor agonist, [D-ala2, D-leu5]-enkephalin (DADLE), while a minority of cells (approximately 30%) were selectively responsive either to DADLE or to nociceptin, but not both. Co-application of nociceptin and DADLE to neurons that were responsive to both agonists, resulted in an inhibitory response that was the same as or less than the inhibition evoked by either agonist alone. Intracellular whole-cell patch clamp recordings from identified Type A and Type B MVN cells showed that both these cell types are responsive to nociceptin, which induced membrane hyperpolarisation and decrease in input resistance consistent with its known effects on membrane K currents in other cell types. In alert rats, i.c.v. injection of nociceptin caused a significant decrease in the gain of the hVOR and resulted in a prolongation of post-rotatory nystagmus in darkness. The decrease in VOR gain and the increase in the VOR time-constant was significant even at low doses of nociceptin which did not cause other observable behavioural effects. These findings demonstrate that endogenously released nociceptin may have a hitherto unexplored role in the functional modulation of the neural pathways that mediate vestibular reflexes in vivo., (Copyright 1999 Elsevier Science B.V.)

We evaluated the opioid antinociceptive mechanism of the calcium channel blockers verapamil and flunarizine in groups of mice with the hotplate test. Both produced a naloxone-sensitive dose-dependent analgesia. The antinociceptive effect of both was reversed by beta-FNA, (mu1 and mu2 antagonists), and both enhanced the antinociceptive activity of morphine, implying a role for mu receptors. Furthermore, since the analgesic effect of flunarizine, but not verapamil, was reversed by naloxonazine (mu1 antagonist), we suggest that the mu1 subtype is involved in flunarizine analgesia, but not in verapamil analgesia. Studies with the selective delta opioid agonist DPDPE and the selective antagonists naltrindole indicated that the antinociceptive activity of verapamil is also mediated by delta receptor agonistic activity (primarily following i.c.v. administration); flunarizine, by contrast, exhibited antagonistic activity at this receptor. Verapamil amplified the antinociceptive activity of kappa1 (U50,488H) and kappa3 (nalorphine) agonists, but its known analgesic activity was inhibited only partially by the kappa1 antagonist Nor-BNI, indicating partial involvement of kappa1 receptor. Flunarizine, however, demonstrated antagonistic activity at both kappa1 and kappa3 receptors, with more prominent inhibitory activity at the latter one. These findings suggest that verapamil and flunarizine elicit analgesia at both the spinal and supraspinal levels. Verapamil's analgesia was explained by agonistic activity at the mu, delta and may also be kappa3 receptor subtypes. Flunarizine exhibited a mixed agonistic-antagonistic opioid activity as shown by its agonistic activity at the mu1 receptor and antagonistic activity at delta, kappa1 and kappa3 receptor subtypes., (Copyright 1999 Elsevier Science B.V.)

The biotransformation of nociceptin/orphanin FQ (NOFQ) by enzyme activity isolated from U1690 human lung carcinoma and SH-SY5Y human neuroblastoma cell lines, and from rat brain cortex cells in primary culture was investigated. The identification and quantification of the cleavage products were performed using electrospray ionization mass spectrometry linked to size-exclusion chromatography. The effect of chronic morphine treatment of the cells (5 days) on NOFQ biotransformation was also studied. It was found that major products generated from NOFQ were the amino-terminal peptides N1-9 and N1-13. The pattern of NOFQ biotransformation was quite similar for all three cell cultures. However, different proportions of the formed peptides were noted. The cleavage was inhibited by EDTA, PMSF, Hg2+, Cu2+ and Zn2+. Dynorphin A2-13 inhibited NOFQ cleavage in a manner suggesting competition of the two peptides for the same enzyme. Chronic morphine treatment of the cell cultures resulted in a substantial increase in the enzyme activity, leading to higher levels of the major fragments and accumulation of N1-12 and the shorter peptides N1-5, N1-6. Since the effect of morphine treatment of the cells was blocked by naloxone, it is likely that it was receptor specific. Taken together, the findings suggest that a metallosensitive endopeptidase, the activity of which is increased by chronic morphine treatment of the cells, is responsible for the biotransformation of NOFQ with fragments N1-9 and N1-13 being the major products., (Copyright 1999 Elsevier Science B.V.)

Intracerebroventricular administration of Orphanin FQ (5.5, 55 or 550 pmol) caused a dose-related increase in prolactin secretion in both male and female rats and stimulated GH secretion in males. The magnitude of the prolactin secretory response was greater in females than in males. These effects of OFQ on prolactin and growth hormone release are the same as the stimulatory effects of the endogenous opioid peptides., (Copyright 1998 Elsevier Science B.V.)

N18TG2 neuroblastoma cells co-express delta-opioid and CB1-cannabinoid receptors. Both receptors are negatively coupled to adenylyl cyclase through pertussis toxin-sensitive GTP-binding proteins. In the present study, we confirmed the independent activity of opioid and cannabinoid agonists, and investigated chronic interactions between the two signal transduction pathways in these cells. Opioid and cannabinoid agonists stimulated [35S]guanosine-5'-O-(3-thiotriphosphate) binding to N18TG2 membranes. When the opioid agonist etorphine and the cannabinoid agonist desacetyllevonantradol (DALN) were applied together, the stimulation was similar to the arithmetic sum of the two separate effects. This additivity existed even after partial ablation of the G-proteins reservoir with a low concentration of pertussis toxin, indicating that opioid and cannabinoid receptors activate different pools of G-proteins in N18TG2 cells. Chronic treatment of the cells with either opioid or cannabinoid agonists induced desensitization to the respective drug. In addition, asymmetric cross-desensitization was found: while long-term exposure to DALN induced homologous desensitization, and did not reduce the effect of etorphine, long-term exposure to etorphine attenuated the cannabinoid activation of G-proteins. Chronic exposure to either DALN or etorphine not only induced desensitization, but also elevated the basal activity of G-proteins in the exposed cells. The combination of the two drugs did not yield an additive activation, suggesting that chronic exposure of N18TG2 cultures to cannabinoid and opioid agonists modified a common responding element within the cells. This work presents the N18TG2 neuroblastoma as a suitable experimental model to study the molecular mechanism(s) underlying chronic interactions between opioid and cannabinoid drugs., (Copyright 1998 Elsevier Science B.V.)

The heptadecapeptide orphanin FQ or nociceptin (OFQ/N), the endogenous ligand for the orphan opioid receptor, has a complex pharmacology in mice, eliciting either an anti-opioid/hyperalgesic action or analgesia depending upon the dose and testing paradigm. Unlike mice, orphanin FQ/nociceptin fails to elicit hyperalgesia in the rat following intracerebroventricular injection. Both OFQ/N and a truncated version, OFQ/N(1-11), produce a robust analgesic response. OFQ/N analgesia is readily antagonized by the opioid antagonists naloxone or diprenorphine, despite their very poor affinity for the cloned orphan opioid receptor. Antisense studies revealed that probes targeting the second and third coding exon of the orphan clone significantly attenuate OFQ/N analgesia, while the exon 1 probe was inactive. These results indicate that OFQ/N elicits a naloxone-sensitive analgesia in rats similar to that previously reported in mice., (Copyright 1998 Elsevier Science B.V.)

Nitric oxide (NO) is involved in neurohormonal secretion from median eminence neuroendocrine nerve terminals. We report that stimulation of NO release from median eminence fragments including vascular tissues occurs by mu3 receptor activation by morphine, or by cannabinoid type 1 receptor activation by anandamide. The released levels of NO are lower after anandamide than after morphine stimulation. These processes can be blocked by L-NAME, a specific nitric oxide synthase inhibitor, by naloxone for the morphine-stimulated NO release, or SR 141716A, a specific CB1 receptor inhibitor, for the anandamide-stimulated NO release. Furthermore, morphine and anandamide, by this NO dependent process, influences neurohormonal release from median eminence nerve terminals within 10 min. Via this NO dependent process, morphine stimulates both GnRH and CRF release, whereas anandamide selectively stimulates GnRH release. These observations together with previous data suggest that morphine and the anandamide-stimulated NO originates from the vascular endothelium of the portal plexus. These results indicate that endothelial cells of the median eminence may be involved in the release of neurohormones., (Copyright 1998 Elsevier Science B.V.)

Nociceptin (NOC), an endogenous ligand for the orphan receptor ORL1, has recently been recognized as a neuropeptide. We used brain microdialysis and on-line high performance liquid chromatography to examine the effect of NOC on the basal outflow of dopamine (DA) and its metabolite in the freely moving rat striatum in vivo. The percent change of DA release induced by NOC at concentrations of 10-6 and 10-5 M were 383% and 398%, respectively. This effect of NOC was attenuated by naloxone, suggesting that NOC activates classic (micro, delta, kappa) receptors in a very little way. These data indicates that NOC may act as a neuropeptide which enhances DA release from the striatum of rat brain via an opioid receptor., (Copyright 1998 Elsevier Science B.V.)

The activity of the recently isolated endogenous opioid-like peptide orphanin FQ (OFQ) was measured in two classical bioassays of opioid action. OFQ potently and concentration-dependently suppressed the electrically stimulated contractions of the guinea pig ileum (GPI) and the mouse vas deferens (MVD) with EC50 values of 1.82 +/- 0.16 and 2.97 +/- 0.01 nM, and Emax values of 56 +/- 3% and 96 +/- 4%, respectively. This effect of OFQ, in both the GPI and MVD, was insensitive to the opioid receptor antagonist naloxone. OFQ competed with [3H]diprenorphine binding to mu-, delta- or kappa-opioid receptors stably expressed in Chinese hamster ovary cell lines with IC50 values of 2.1 +/- 0.4, 2.2 +/- 0.3, 0.75 +/- 0.3 microM, respectively. Low affinity for the classical opioid receptors together with the inability of naloxone to antagonize its effect suggest that the inhibitory action of OFQ is mediated via a distinct OFQ receptor in the GPI and MVD. Consequently, the MVD could serve as a valuable bioassay of potential OFQ receptor antagonists.

Whole-cell patch-clamp recordings were used to study Ba2+ currents through voltage-dependent Ca2+ channels in dorsal root ganglion x mouse neuroblastoma hybrid (F-11) cells. Opioid agonists selective for either mu (Tyr-D-Ala-Gly-Mephe-Gly-ol; DAMGO) or delta (Tyr-D-Pen-Gly-Phe-D-Pen-OH; DPDPE) receptors inhibited high-threshold Ba2+ currents. The inhibition was reversible, naloxone-sensitive, and dose-dependent. The inhibitory effects of both DAMGO and DPDPE were blocked by pretreatment of the cells with pertussis toxin (PTX) as well as by brief exposure to the sulfhydryl alkylating agent, N-ethylmaleimide (NEM). The N-type Ca2+ channel antagonist omega-conotoxin GVIA (omega-CTX GVIA) irreversibly inhibited high threshold Ba2+ currents by 66% and blocked the inhibitory effect of DAMGO or DPDPE. In contrast, the L-type Ca2+ channel blocker nifedipine inhibited high threshold Ba2+ currents by 15% and failed to block the inhibitory effect of DAMGO or DPDPE. These results demonstrate that mu and delta opioid receptors are negatively coupled to N-type Ca2+ channels via PTX- and NEM-sensitive GTP-binding proteins in F-11 cells.

Opioid receptors have been demonstrated by light microscopic techniques in fine cutaneous nerves in naive animals. The present study extends these findings by showing that 29 and 38% of unmyelinated cutaneous sensory axons can be immunostained for mu- or delta-opioid receptors respectively. Local cutaneous injection of DAMGO, a mu-opioid ligand, ameliorates the nociceptive behaviors caused by local cutaneous injection of glutamate, a purely nociceptive chemical stimulus showing that the mu-receptors are functional. By contrast the delta-opioid ligand [2-D-penicillamine, 5-D-penicillamine]enkephalin (DPDPE) had no effect on these behaviors. These findings indicate a wider function for opioid receptors in naive animals than previously envisioned.

Microinjections of excitatory amino acids made into the ventrolateral midbrain periaqueductal gray of the rat have revealed that neurons in this region integrate a reaction characterised by quiescence, hyporeactivity, hypotension and bradycardia. Microinjections of both excitatory amino acids and opioids into the ventrolateral periaqueductal gray have shown also that it is a key central site mediating analgesia. The effects of injections of opioids into the ventrolateral periaqueductal gray on arterial pressure and heart rate or behaviour are unknown. In this study we first mapped in the rat the extent of the ventrolateral periaqueductal gray hypotensive region as revealed by microinjections of excitatory amino acids. We found that ventrolateral periaqueductal gray depressor region extended more rostrally than previously thought into the tegmentum ventrolateral to the periaqueductal gray. Subsequently we studied for the first time, the effects of microinjections of mu-, delta-, and kappa-opioid agonists made into the ventrolateral periaqueductal grey depressor region. In contrast to the effects of excitatory amino acid injections, microinjections of the mu-opioid agonist ([D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin) evoked hypertension and tachycardia at approximately 50% of sites. Similar to excitatory amino acid injections, microinjections of both the delta-opioid agonist ([D-Pen2,D-Pen5]enkephalin), and the kappa-opioid agonist ((5,7,8)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-y l]-benzeneacetamide) evoked either a hypotension and bradycardia, or had no effect. These results indicate that different opiate receptor subtypes are present on a distinct population of ventrolateral periaqueductal gray neurons, or at different ventrolateral periaqueductal gray synaptic locations (pre- or post-synaptic).

Nociceptin is a 17 amino acid peptide and acts as a potent endogenous agonist of the opioid receptor-like1 (ORL1) receptor. Nociceptin is reported to depress glutamatergic transmission and to block the spinal facilitation which is thought to be mediated by the activation of N-methyl-D-aspartate (NMDA) receptor. Paw carageenan injection induces thermal hyperalgesia which has been reported to be maintained by the NMDA receptor dependent spinal facilitation. In the present study, we investigated the effect of intrathecally administered nociceptin on the level of thermal hyperalgesia induced by carageenan injection in the rat. Nociceptin depresses the level of thermal hyperalgesia in a dose-dependent manner. These data suggest that spinal ORL1 receptor activation depresses the spinal facilitation state induced by paw carageenan injection.

Nociceptin (Orphanin FQ) is a newly discovered endogenous heptadecapeptide substrate for the opioid-receptor-like 1 receptor, a G protein coupled receptor that bears striking amino acid sequence homology to opiate receptors. In rats, intrathecal (i.t.) administration of nociceptin is without effect on basal thresholds for responsiveness to electric food shock. However, during either late gestation or its hormonal simulation, when nociceptive thresholds are elevated by approximately 70%, i.t. nociceptin substantially attenuates jump thresholds in a dose-dependent fashion. This hypoalgesic effect of nociceptin is not limited to attenuating the gestational or sex steroid-induced increment in pain thresholds. Following highest i.t. dose of nociceptin employed (20 nmol), the gestational or sex steroid-induced increment in jump thresholds is not only abolished but a significant hyperalgesia is observed. These results underscore the importance of the hormonal milieu to nociceptin hypoalgesic sensitivity. The potential contribution of spinal nociceptive pathways that utilize nociceptin to the etiology of extraordinary painful pregnancy and labor should not be ignored.

The effects of the mu opioid receptor agonists, morphine and Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAGO), the delta opioid receptor agonist, Tyr-D-Pen-Gly-Phe-D-penicillamine (DPDPE) and the kappa-opioid receptor agonist, dynorphin A-(1-13) on the whole-cell K+ currents (IK) of cultured mouse DRG neurons and neuroblastoma X DRG neuron hybrid F11 cells were studied. These opioid ligands all elicited dual effects. Low concentrations (< nM) usually elicited a transient increase in IK (within 1 min), followed by a sustained decrease in IK. In contrast, microM concentrations rapidly elicited a sustained increase in IK. After brief treatment with cholera toxin subunit B (CTX-B), the usual sustained decrease in IK evoked by < nM opioid agonists no longer occurred. Low concentrations then elicited only a sustained increase in IK. On the other hand, after chronic treatment with pertussis toxin (PTX), the usual microM opioid-induced increases in IK no longer occurred and more than half of the cells responded with a sustained decrease of IK to microM as well as nM opioids. The results suggest that mu, delta and kappa opioid receptors are each coupled to K+ channels through CTX-B- and PTX-sensitive transduction systems. Both systems have similar threshold concentrations to opioids. Activation of the CTX-B-sensitive opioid receptor/transduction system resulted in a decrease in K+ conductance of the cell which is generally associated with an increase in neuronal excitability. Activation of the other system resulted in an increase in K+ conductance which will, in general, decrease neuronal excitability. The net change in the IK depends upon which effect predominates. The dominance at different opioid concentrations may depend on the relative efficacies of the coupling of these two systems to K+ channels.

The possible modulations by morphine and various opioids of the spinal release of cholecystokinin-like material (CCKLM) evoked by 30 mM K+ was studied in vitro, using slices of the dorsal part of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid. Addition of the mu agonist, DAGO (0.1-10 microM), to the perfusing fluid produced a concentration-dependent decrease in the peptide release, which could be prevented by the preferential mu antagonist, naloxone. Complex modulations were induced by the delta agonist, DTLET, as this drug inhibited CCKLM release when added at 10 nM-3 microM to the perfusing fluid, but enhanced it at 10 microM. Both effects were preventable by the delta antagonists naltrindole and ICI 154129, suggesting that delta receptors, possibly of different subtypes, mediated the inhibition and stimulation by DTLET. Morphine also exerted a biphasic effect, as the alkaloid decreased CCKLM release at 0.01-0.1 microM and enhanced it at 10 microM. Morphine-induced inhibition was preventable by naloxone, whereas its stimulatory effect could be blocked by naltrindole and ICI 154129. Although inactive on its own on CCKLM release, the selective kappa 1 agonist U 50488H (1 microM) prevented the inhibitory effects of both DAGO (10 microM) and morphine (0.1 microM), suggesting the existence of interactions between kappa 1 and mu receptors within the dorsal zone of the rat spinal cord. These data indicate that low concentrations of morphine exert an inhibitory influence on spinal CCKergic neurons that depends on the stimulation of mu opioid receptors. The excitatory influence of 10 microM morphine likely results from the simultaneous stimulation of mu, delta and kappa receptors, as the inhibitory effect of mu receptor stimulation can be masked by that of kappa 1 receptors, allowing only the expression of a delta-dependent excitatory effect similar to that induced by 10 microM DTLET.