In vitro and in vivo pharmacological characterization of the novel neuropeptide S receptor ligands QA1 and PI1.

The pharmacological activity of the novel neuropeptide S (NPS) receptor (NPSR) ligands QA1 and PI1 was investigated. In vitro QA1 and PI1 were tested in calcium mobilization studies performed in HEK293 cells expressing the recombinant mouse (HEK293mNPSR) and human (HEK293hNPSRIle107 and HEK293hNPSRAsn107) NPSR receptors. In vivo the compounds were studied in mouse righting reflex (RR) and locomotor activity (LA) tests. NPS caused a concentration dependent mobilization of intracellular calcium in the three cell lines with high potency (pEC50 8.73-9.14). In inhibition response curve and Schild protocol experiments the effects of NPS were antagonized by QA1 and PI1. QA1 displayed high potency (pKB 9.60-9.82) behaving as a insurmountable antagonist. However in coinjection experiments QA1 produced a rightward swift of the concentration response curve to NPS without modifying its maximal effects; this suggests that QA1 is actually a slow dissociating competitive antagonist. PI1 displayed a competitive type of antagonism and lower values of potencies (pA2 7.74-8.45). In vivo in mice NPS (0.1 nmol, i.c.v.) elicited arousal promoting action in the RR assay and stimulant effects in the LA test. QA1 (30 mgkg(-1)) was able to partially counteract the arousal promoting NPS effects, while PI1 was inactive in the RR test. In the LA test QA1 and PI1 only poorly blocked the NPS stimulant action. The present data demonstrated that QA1 and PI1 act as potent NPSR antagonists in vitro, however their usefulness for in vivo investigations in mice seems limited probably by pharmacokinetic reasons.

The orphanin FQ/nociceptin knockout mouse: a behavioral model for stress responses.

Transgenic mice lacking expression of the OFQ/N precursor protein have provided exciting insights in the physiological functions of this neuropeptide system. While injection of OFQ/N or selective synthetic agonists produces anxiolytic effects in rodents, OFQ/N knockout mice display increased anxiety and impaired adaptation to repeated stress. On the other hand, mice lacking the cognate OFQ/N receptor, ORL1, show improved spatial attention and memory but appear to have normal anxiety and stress behavior. Availability of a selective small molecule OFQ/N antagonist might help clarify this discrepancy.

Involvement of the neuropeptide orphanin FQ/nociceptin in kainate and kindling seizures and epileptogenesis.

PURPOSE: To investigate the role of orphanin FQ/nociceptin (OFQ/N) in epilepsy, we analyzed (a) proOFQ/N (the OFQ/N precursor) and ORL-1 (the OFQ/N receptor) messenger RNA (mRNA) levels in the kainate and in the kindling models of epilepsy in the rat; and (b) seizure expression in proOFQ/N knockout mice. METHODS: Epilepsy models: kainate and kindling. Northern blot analysis, radioactive in situ hybridization. RESULTS: Increased proOFQ/N mRNA levels were found in the thalamus (reticular nucleus) after kainate administration. In contrast, ORL-1 gene expression decreased dramatically in the amygdala, hippocampus, thalamus, and cortex after kainate administration. OFQ/N knockout mice displayed reduced susceptibility to kainate-induced seizures, in that (a) lethality was reduced, (b) latency to generalized seizure onset was significantly prolonged, and (c) behavioral seizure scores were significantly reduced. Furthermore, kindling progression was delayed in OFQ/N-/- mice. CONCLUSIONS: These data indicate that limbic seizures are associated with increased OFQ/N release in multiple brain areas, causing downregulation of ORL-1 receptors and activation of OFQ/N biosynthesis in selected areas, and support the notion that the OFQ/N-ORL-1 system may play a facilitatory role in ictogenesis and in epileptogenesis.

Novel neurotransmitters as natural ligands of orphan G-protein-coupled receptors.

The "orphan" G-protein-coupled receptors (GPCRs) are cloned GPCRs that bind unknown ligands. Since 1995, nineteen orphan GPCRs have been used as targets to identify and isolate their natural ligands via the application of the "orphan receptor strategy". These ligands are peptides, lipids or biogenic amines, and act as transmitter molecules. One nucleotide-sugar derivative and six peptides or peptide families identified through this strategy are novel and have already enriched our understanding of various brain functions.

Molecular cloning and characterization of a second human cysteinyl leukotriene receptor: discovery of a subtype selective agonist.

The cysteinyl leukotrienes (CysLTs) are potent biological mediators in the pathophysiology of inflammatory diseases, in particular of airway obstruction in asthma. Pharmacological studies have suggested the existence of at least two types of CysLT receptors, designated CysLT(1) and CysLT(2). The CysLT(1) receptor has been cloned recently. Here we report the molecular cloning, expression, localization, and functional characterization of a human G protein-coupled receptor that has the expected characteristics of a CysLT(2) receptor. This new receptor is selectively activated by nanomolar concentrations of CysLTs with a rank order potency of LTC(4) = LTD(4) >> LTE(4). The leukotriene analog BAY u9773, reported to be a dual CysLT(1)/CysLT(2) antagonist, was found to be an antagonist at CysLT(1) sites but acted as a partial agonist at this new receptor. The structurally different CysLT(1) receptor-selective antagonists zafirlukast, montelukast, and MK-571 did not inhibit the agonist-mediated calcium mobilization of CysLT(2) receptors at physiological concentrations. Localization studies indicate highest expression of CysLT(2) receptors in adrenal glands, heart, and placenta; moderate levels in spleen, peripheral blood leukocytes, and lymph nodes; and low levels in the central nervous system and pituitary. The human CysLT(2) receptor gene is located on chromosome 13q14.12-21.1. The new receptor exhibits all characteristics of the thus far poorly defined CysLT(2) receptor. Moreover, we have identified BAY u9773 as a CysLT(2) selective agonist, which could prove to be of immediate use in understanding the functional roles of the CysLT(2) receptor.

The orphanin FQ/nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice.

Like other neuropeptides, orphanin FQ/nociceptin (OFQ/N) is encoded by a larger precursor protein. The cDNA for the OFQ/N precursor has been cloned from human, rat, mouse and bovine tissue demonstrating that this peptidergic system serves important functions that have been conserved during evolution. The structural organization of the precursor protein is similar to opioid peptide precursors, supporting the view of a common origin for the opioid systems and the OFQ/N system. In addition to OFQ/N, the precursor may encode two other biologically active peptides. Anatomic studies have revealed high levels of expression of the OFQ/N messenger RNA in brain structures involved in sensory, emotional and cognitive processing. In particular, high levels of OFQ/N mRNA were detected in the limbic system, underlining the stress attenuating activities that have been described as an important function of OFQ/N. Recently, mutant mice have been generated that lack the precursor protein of OFQ/N to further define the physiological functions of the OFQ/N system. The OFQ/N-deficient mice are characterized by an increased sensitivity to stressful stimuli and a lack of habituation to chronic and repeated stress. This review will summarize recent findings on the molecular biology of the OFQ/N precursor and relate it to possible physiological functions of this newly discovered neuropeptide system.

Functional consequences of reduction in NMDA receptor glycine affinity in mice carrying targeted point mutations in the glycine binding site.

We have used site-directed mutagenesis in conjunction with homologous recombination to generate two mouse lines carrying point mutations in the glycine binding site of the NMDAR1 subunit (Grin1). Glycine concentration-response curves from acutely dissociated hippocampal neurons revealed a 5- and 86-fold reduction in receptor glycine affinity in mice carrying Grin1(D481N) and Grin1(K483Q) mutations, respectively, whereas receptor glutamate affinity remained unaffected. Homozygous mutant Grin1(D481N) animals are viable and fertile and appear to develop normally. However, homozygous mutant Grin1(K483Q) animals are significantly lighter at birth, do not feed, and die within a few days. No gross abnormalities in CNS anatomy were detected in either Grin1(D481N) or Grin1(K483Q) mice. Interestingly, in situ hybridization and Western blot analysis revealed changes in the expression levels of NMDA receptor subunits in Grin1(D481N) mice relative to wild type that may represent a compensatory response to the reduction in receptor glycine affinity. Grin1(D481N) mice exhibited deficits in hippocampal theta burst-induced long-term potentiation (LTP) and spatial learning and also a reduction in sensitivity to NMDA-induced seizures relative to wild-type controls, consistent with a reduced activation of NMDA receptors. Mutant mice exhibited normal prepulse inhibition but showed increased startle reactivity. Preliminary analysis indicated that the mice exhibit a decreased natural aversion to an exposed environment. The lethal phenotype of Grin1(K483Q) animals confirms the critical role of NMDA receptor activation in neonatal survival. A milder reduction in receptor glycine affinity results in an impairment of LTP and spatial learning and alterations in anxiety-related behavior, providing further evidence for the role of NMDA receptor activation in these processes.

Orphan receptors, novel neuropeptides and reverse pharmaceutical research.

By the beginning of the next millennium, the search for the natural ligands of the orphan G-protein-coupled receptors will lead to the discovery of so many new neuropeptides that it may well double their present number. This bounty of new tools will direct us to new insights in brain function and to better understanding of brain disorders. It is expected that the novel neuropeptides will have a particular impact on molecular psychiatry. In view of their potential, the novel neuropeptides should also become the focus of drug discovery programs. It is hoped that these programs will be initiated at an early stage, when understanding of novel neuropeptide function has not necessarily been reached, to allow for the design of neuropeptide chemical surrogates that are crucial to the study of the novel neuropeptide system and may serendipitously develop into highly successful drugs.

Targeted disruption of the orphanin FQ/nociceptin gene increases stress susceptibility and impairs stress adaptation in mice.

The neuropeptide orphanin FQ (also known as nociceptin; OFQ/N) has been implicated in modulating stress-related behavior. OFQ/N was demonstrated to reverse stress-induced analgesia and possess anxiolytic-like activity after central administration. To further study physiological functions of OFQ/N, we have generated OFQ/N-deficient mice by targeted disruption of the OFQ/N gene. Homozygous mice display increased anxiety-like behavior when exposed to a novel and threatening environment. OFQ/N-null mice show elevated basal pain threshold but develop normal stress-induced analgesia. Interestingly, these mice show impaired adaptation to repeated stress when compared with wild-type mice, whereas their performance in spatial learning remained unaffected. Basal and poststress plasma corticosterone levels were found to be elevated in OFQ/N-deficient animals. Thus, OFQ/N appears to be crucially involved in the neurobiological regulation of stress-coping behavior and fear.

Orphan receptors and the concept of reverse physiology: discovery of the novel neuropeptide orphanin FQ/nociceptin.

The cloning of numerous orphan members from the supergene family of G protein-coupled receptors implies the existence of many as yet undiscovered neurotransmitters and neuropeptides. Recently, new technologies were developed to isolate natural ligands for orphan receptors, using the receptor as a biological sensor during the purification process. This manuscript will present the concept and technology of an approach which starts from a cloned receptor to ultimately describe the physiological functions of the transmitter system. This strategy inverts the classical order of biomedical research and was thus termed "reverse physiology". The first natural ligand isolated by this strategy is a peptide with significant similarity to the opioid peptides and has been named orphanin FQ or nociceptin (OFQ/NOC). Evidence for characterizing OFQ/NOC as a genuine neuropeptide will be reviewed. OFQ/NOC is biosynthetically derived from a larger precursor protein which may encode additional bioactive peptides. Since its discovery, a large number of studies have described numerous physiological functions of OFQ/NOC. Because of its relation to the opioid system, much attention has been focused on the involvement of OFQ/NOC in nociception, sometimes with controversial results. However, the pharmacological profile of the OFQ/NOC system suggests a clear separation from the opioids. The discovery of OFQ/NOC and the subsequent analyses of its physiological functions is an example which has already been followed by the identification of two other novel neuropeptides. The orphan receptor strategy holds a lot of promises for the postgenomic era, helping to fill the vast amount of sequence data with life.

Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding.

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor-like (ORL1) receptor, exhibit structural features suggestive of the micro, kappa, and delta opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with (125)I-[(14)Tyr]-OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes.

Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat.

Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor-like receptor (ORL-1). It and its precursor, prepro-OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro-OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes.

Reverse physiology: discovery of the novel neuropeptide, orphanin FQ/nociceptin.

The search for novel neurotransmitters and neuropeptides has been recently revolutionized by the development of a purification strategy based on orphan G protein-coupled receptors, cloned receptors for which no natural ligands are known. This strategy uses the orphan receptor as bait to identify its natural ligand. This article will review the discovery of the first natural ligand isolated following this strategy. This ligand is a peptide that shares some striking sequence similarity to the opioid peptides and has been named Orphanin FQ or Nociceptin (OFQ/NOC). The discovery of OFQ/NOC will be described as one example of the use of orphan receptors in identifying novel neurotransmitters and neuropeptides, an example that has already been followed in the identification of other novel neuropeptides. After reviewing the conceptual and technological basis of the strategy and its successful first application, we discuss the criteria used to validate OFQ/NOC as the natural ligand of the orphan receptor and as a genuine neuropeptide. We also discuss the importance and implications of discovering OFQ/NOC mode of synthesis, which is synthesized as expected in the form of a larger polypeptide precursor, which in turn raises the question of the existence of other OFQ/NOC-related peptides. We then present an overview of the numerous studies that have blossomed after the OFQ/NOC discovery and describe the numerous physiological roles that have already been attributed to OFQ/NOC, and in particular the controversy regarding its involvement in pain perception. Because of the similarities between the OFQ/NOC and opioid systems, we also discuss overlaps between these systems and present evidence favoring a pharmacological separation between these systems. We finish by outlining the power of the orphan receptor strategy and by discussing some of its pitfalls.

Creating a functional opioid alkaloid binding site in the orphanin FQ receptor through site-directed mutagenesis.

Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a "gain of function" approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [3H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and (-)-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the delta receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.

Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities.

Strict pharmacological selectivity in families of structurally related ligands and receptors may result from a key process in evolution aiming at increasing diversity in neurotransmission. An intriguing example of such exclusive specificity can be found in the newly discovered orphanin FQ (OFQ) system when it is compared with the opioid system. Both OFQ and its receptor share a high degree of sequence similarity to the opioid peptides and their corresponding receptors, respectively. However, OFQ does not activate opioid receptors, nor do the opioid peptides elicit biological activity at the OFQ receptor. We have therefore investigated the basis for the inherent selectivity of the primary structures of OFQ and dynorphin A, its closest counterpart. A series of truncated and/or chimeric peptides led to the conclusion that both peptides contain domains which establish their pharmacological selectivity. In the OFQ molecule we could delineate a domain that prevents its ability to activate the kappa-opioid receptor by apparently repelling its binding. In both peptides the selectivity-generating domains are composed of single residues in key positions together with short stretches of amino acids which do not overlap. To prove this concept, we designed a universal agonist and found it active at both the OFQ receptor and the kappa-opioid receptor. Our observations suggest that a coordinated mechanism of evolution has separated the orphanin FQ system from the opioid system.

Interaction of [3H]orphanin FQ and 125I-Tyr14-orphanin FQ with the orphanin FQ receptor: kinetics and modulation by cations and guanine nucleotides.

The heptadecapeptide orphanin FQ (OFQ) has been identified as the endogenous ligand for a G protein-coupled receptor (OFQ-R), which, despite its high degree of sequence similarity to opioid receptors, fails to bind opioid ligands. We developed two radioligands for the OFQ-R: a tritiated native OFQ peptide ([3H]OFQ) and a radioiodinated form in which Leu14 was substituted by tyrosine (125I-Tyr14-OFQ). Their binding properties were examined in human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells heterologously expressing the OFQ-R at different levels (HEK 293 expressed 40-fold more OFQ-R than did CHO). Both ligands exhibited rapid, monophasic association kinetics in each cell line. Dissociation of both ligands from OFQ-R expressed in HEK 293 cells was biphasic, whereas dissociation of 125I-Tyr14-OFQ from OFQ-R expressed in CHO cells was monophasic and slow. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically. In CHO cells, 125I-Tyr14-OFQ detected a single affinity state with an intermediate Kd value of 54 pM. Optimal binding of the radioligands required 1-5 mM MgCl2, whereas millimolar concentrations of ZnCl2, CaCl2, MnCl2, and NaCl reduced specific binding of both ligands. A nonhydrolyzable GTP analog [guanosine-5'-(beta,gamma-imido)triphosphate] reduced the affinity of both OFQ ligands to their receptor without significant changes in the total binding capacity, indicating functional interactions between the OFQ-R and G proteins. In rat brain membranes, specific, saturable binding of 125I-Tyr14-OFQ was demonstrated to be pharmacologically identical to the heterologously expressed OFQ-R. Taken together, these results indicate that 125I-Tyr14-OFQ and [3H]OFQ exhibit virtually identical characteristics and are suitable for the pharmacological analysis of the OFQ-R.

Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress.

Orphanin FQ (OFQ, Nociceptin) is a recently discovered 17-amino acid neuropeptide that is structurally related to the opioid peptides but does not bind opioid receptors. OFQ has been proposed to act as an anti-opioid peptide, but its widespread sites of action in the brain suggest that it may have more general functions. Here we show that OFQ plays an important role in higher brain functions because it can act as an anxiolytic to attenuate the behavioral inhibition of animals acutely exposed to stressful/anxiogenic environmental conditions. OFQ anxiolytic-like effects were consistent across several behavioral paradigms generating different types of anxiety states in animals (light-dark preference, elevated plus-maze, exploratory behavior of an unfamiliar environment, pharmacological anxiogenesis, operant conflict) and were observed at low nonsedating doses (0.1-3 nmol, intracerebroventricular). Like conventional anxiolytics, OFQ interfered with regular sensorimotor function at high doses (>3 nmol). Our results show that an important role of OFQ is to act as an endogenous regulator of acute anxiety responses. OFQ, probably in concert with other major neuropeptides, exerts a modulatory role on the central integration of stressful stimuli and, thereby, may modulate anxiety states generated by acute stress.

Moving from the orphanin FQ receptor to an opioid receptor using four point mutations.

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons.

Orphanin FQ (OFQ) has recently been reported to be an endogenous ligand for the opioid-like LC132 receptor. The effect of OFQ on high voltage-gated calcium channels (VGCCs) was examined in freshly dissociated rat pyramidal neurons using the whole-cell configuration of the patch-clamp technique. High-threshold Ba2+ currents were reversibly inhibited by OFQ. The depression of the currents was associated with a slowed rate of activation and a change in the activation I-V relationship at step potentials higher than +30 mV. In concentration-response experiments, a mean (+/-SEM) pEC50 value of 7.0 +/- 0.07 and a Hill coefficient of 1.5 +/- 0.08 (n = 5) were obtained. The near-maximum inhibition of the Ba2+ currents by OFQ (1 microM) amounted to 31 +/- 2.2% of control (n = 15). Opioid receptors could not account for the effects of OFQ on VGCCs, because naloxone, a broad spectrum mu-, delta-, and kappa-receptor antagonist, did not reduce the effectiveness of OFQ. When GTP-gamma-S was included in the pipette, the depression of the currents by OFQ was irreversible, whereas currents from neurons preincubated with pertussis toxin were not inhibited by OFQ, consistent with the involvement of a PTX-sensitive G-protein. When selective blockers of VGCCs were used, it was demonstrated that all subtypes of VGCCs were affected by OFQ. In conclusion, the effect of OFQ on VGCCs expressed in hippocampal CA3 and CA1 neurons may play an important role in the regulation of hippocampal cell excitability and neurotransmitter release.