The expression of delta opioid receptor mRNA in adult male zebra finches (Taenopygia guttata).

The endogenous opioid system is evolutionarily conserved across reptiles, birds and mammals and is known to modulate varied brain functions such as learning, memory, cognition and reward. To date, most of the behavioral and anatomical studies in songbirds have mainly focused on µ-opioid receptors (ORs). Expression patterns of δ-ORs in zebra finches, a well-studied species of songbird have not yet been reported, possibly due to the high sequence similarity amongst different opioid receptors. In the present study, a specific riboprobe against the δ-OR mRNA was used to perform fluorescence in situ hybridization (FISH) on sections from the male zebra finch brain. We found that δ-OR mRNA was expressed in different parts of the pallium, basal ganglia, cerebellum and the hippocampus. Amongst the song control and auditory nuclei, HVC (abbreviation used as a formal name) and NIf (nucleus interfacialis nidopallii) strongly express δ-OR mRNA and stand out from the surrounding nidopallium. Whereas the expression of δ-OR mRNA is moderate in LMAN (lateral magnocellular nucleus of the anterior nidopallium), it is low in the MSt (medial striatum), Area X, DLM (dorsolateral nucleus of the medial thalamus), RA (robust nucleus of the arcopallium) of the song control circuit and Field L, Ov (nucleus ovoidalis) and MLd (nucleus mesencephalicus lateralis, pars dorsalis) of the auditory pathway. Our results suggest that δ-ORs may be involved in modulating singing, song learning as well as spatial learning in zebra finches.

The Role of Cardiac Opioid Receptors in the Cardioprotective Effect of Continuous Normobaric Hypoxia.

We studied the role of opioid receptor subtypes in improvement of the functional state of the heart during reperfusion after adaptation to continuous normobaric hypoxia. To this end, male Wistar rats were subjected to continuous normobaric hypoxia (12% O2). Then, the hearts were isolated and exposed to total 45-min ischemia followed by 30-min reperfusion. Opioid receptor antagonists were added to the perfusion solution prior to ischemia. It was found that continuous normobaric hypoxia reduced the release of creatine phosphokinase into the effluent, increased myocardial contractile force, and decreased the end-diastolic pressure during reperfusion; these positive effects were related to activation of cardiac δ2- and µ-opioid receptors.

Neuroprotective role of delta opioid receptors in hypoxic preconditioning

Background/aim: The purpose of the present study was to explore the neuroprotective role of delta opioid receptors (DOR) in the rat cortex in hypoxic preconditioning. Materials and methods: Rats were randomly divided into 8 groups: control (C), sham (S), hypoxic preconditioning (PC), severe hypoxia (SH), PC + SH, PC + SH + Saline (PS), PC + SH + DPDPE (DPDPE, selective DOR agonist), PC + SH + NT (NT, Naltrindole, selective DOR antagonist). Drugs were administered intracerebroventrically. Twenty four h after the end of 3 consecutive days of PC (10% O2, 2 h/day), the rats were subjected to severe hypoxia (7% O2 for 3 h). Bcl-2 and cyt-c were measured by western blot, and caspase-3 was observed immunohistochemically. Results: Bcl-2 expressions in the PC group were higher than in control, SH, and PC + SH groups. Even though there were no significant differences between the groups in terms of cyt-c levels, caspase-3 immunoreactivity of cortical neurons and glial cells in the severe hypoxia and NT groups were higher than in the control, sham, and hypoxic preconditioning groups. DPDPE administration diminished caspase-3 immunoreactivity compared with all of the severe hypoxia groups. Conclusions: These results suggest that cortical cells are resistant to apoptosis via increased expression of Bcl-2 and decreased immunoreactivity of caspase-3 in the cortex, and that DOR is involved in neuroprotection induced by hypoxic preconditioning via the caspase-3 pathway in cortical neurons.

Effect of Chronic Continuous Normobaric Hypoxia on Functional State of Cardiac Mitochondria and Tolerance of Isolated Rat Heart to Ischemia and Reperfusion: Role of µ and delta2 Opioid Receptors.

Chronic continuous normobaric hypoxia (CNH) increases cardiac tolerance to ischemia/reperfusion injury in vivo and this effect is mediated via µ and delta2 opioid receptors (ORs) activation. CNH has also been shown to be cardioprotective in isolated rat heart. In this study, we hypothesize that this cardioprotective effect of CNH is mediated by activation of µ and delta2 ORs and preservation of mitochondrial function. Hearts from rats adapted to CNH (12 % oxygen) for 3 weeks were extracted, perfused in the Langendorff mode and subjected to 45 min of global ischemia and 30 min of reperfusion. Intervention groups were pretreated for 10 min with antagonists for different OR types: naloxone (300 nmol/l), the selective delta OR antagonist TIPP(psi) (30 nmol/l), the selective delta1 OR antagonist BNTX (1 nmol/l), the selective delta2 OR antagonist naltriben (1 nmol/l), the selective peptide µ OR antagonist CTAP (100 nmol/l) and the selective delta OR antagonist nor-binaltorphimine (3 nmol/l). Creatine kinase activity in coronary effluent and cardiac contractile function were monitored to assess cardiac injury and functional impairment. Additionally, cardiac tissue was collected to measure ATP and to isolate mitochondria to measure respiration rate and calcium retention capacity. Adaptation to CNH decreased myocardial creatine kinase release during reperfusion and improved the postischemic recovery of contractile function. Additionally, CNH improved mitochondrial state 3 and uncoupled respiration rates, ADP/O, mitochondrial transmembrane potential and calcium retention capacity and myocardial ATP level during reperfusion compared to the normoxic group. These protective effects were completely abolished by naloxone, TIPP(psi), naltriben, CTAP but not BNTX or nor-binaltorphimine. These results suggest that cardioprotection associated with adaptation to CNH is mediated by µ and delta2 opioid receptors activation and preservation of mitochondrial function.

Molecular aspects of delta opioid receptors.

The delta opioid receptor (DOP) belongs to the Class A, rhodopsin-like family of G protein-coupled receptors. Although this receptor has a high level of similarity with the other opioid receptors, it displays unique aspects and functions. Indeed, as opposed to most membrane receptors, DOP is poorly addressed to the plasma membrane. In this chapter, we first review the molecular and cellular mechanisms regulating the expression and the cellular trafficking/sorting of DOP. We then summarize the structural insights of this receptor through the analysis of the existing crystal structures, with a particular focus on the role of the sodium binding site. Finally, we review the current signaling mechanisms mediating receptor function and desensitization.

In vitro and in vivo characterization of the bifunctional µ- and δ- opioid receptors ligand MCRT on mouse gastrointestinal motility.

BACKGROUND: Chimeric opioid MCRT was a novel multi-target ligand based on morphiceptin and PFRTic-NH2, and produced potent analgesia (ED50 = 0.03 nmol/mouse) with less upper gastrointestinal dysmotility. In this study, we sought to perform the tests to evaluate the pharmacological effects of MCRT on distal colon motility and defecation function. Moreover, opioid receptor antagonists and neuropeptide FF (NPFF) receptor antagonists were utilized to explore the mechanisms. METHODS: Isolated mouse colon bioassay and colonic bead expulsion were to characterize MCRT-induced inhibition of colonic motility in vitro and in vivo, respectively. Fecal pellet output was to evaluate the defecation function. RESULTS: (1) In vitro, MCRT increased colonic contraction via µ- and δ- opioid receptors (MOR and DOR). (2) In vivo, MCRT delayed colonic bead expulsion (ED50 = 1.1 nmol/mouse) independent of opioid and NPFF receptors. (3) In vivo, MCRT inhibited fecal number (ED50 = 1.43 nmol/mouse) and dry weight (ED50 = 1.63 nmol/mouse), which was mediated by DOR partially but not MOR. CONCLUSIONS: (1) Data indicated that MCRT was less prone to induce gastrointestinal dysmotility at analgesic doses, and provided a possibility for safer opioid analgesic. (2) Based on the mechanism explorations, we speculated on the existence of such an opioid receptor subtype or MOR/DOR heterodimer, which was involved in the central analgesia and the in vitro colonic contractions but not the central colonic dysmotility.

Stimulation of accumbal GABAB receptors inhibits delta1- and delta2-opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats.

The nucleus accumbens contains delta-opioid receptors that may decrease inhibitory neurotransmission. As GABAB receptors inhibit dopamine release, decrease in activation of GABAB receptors may be a mediator of delta-opioid receptor-induced accumbal dopamine efflux. If so, accumbal dopamine efflux induced by delta-opioid receptor activation should be suppressed by stimulating GABAB receptors. As delta-opioid receptors are further subdivided into delta1- and delta2-opioid receptors, we analysed the effects of the GABAB receptor agonist baclofen on delta1- and delta2-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were applied intracerebrally through the dialysis probe. Doses of compounds show total amount administered (mol) during 25-50 min infusions. Baclofen (2.5 and 5.0 nmol), which did not alter basal dopamine levels, inhibited the delta1-opioid receptor agonist DPDPE (5.0 nmol)-induced dopamine efflux. Baclofen (2.5 and 5.0 nmol) also inhibited the delta2-opioid receptor agonist deltorphin II (25.0 nmol)-induced dopamine efflux. A low dose of the GABAB receptor antagonist 2-hydroxysaclofen (100.0 pmol), which failed to alter basal accumbal dopamine levels, counteracted the inhibitory effects of baclofen (5.0 nmol) on DPDPE (5.0 nmol)- and deltorphin II (25.0 nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABAB receptor-mediated inhibition of accumbal dopaminergic activity facilitates activation of delta1- and delta2-opioid receptor-induced increases in accumbal dopamine efflux. This study suggests that activation of delta1- and delta2-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABAB receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux.

Evidence and Function Relevance of Native DOR-MOR Heteromers.

Opioid receptors are the sites of action for morphine and most other clinically used opioid drugs. Abundant evidence now demonstrates that different opioid receptor types can physically associate to form heteromers. Owing to their constituent monomers' involvement in analgesia, mu/delta opioid receptor (M/DOR) heteromers have been a particular focus of attention. Understandings of the physiological relevance and indisputable proof of M/DOR formation in vivo are still evolving. This aspect of the field has been slow to progress in large part by the limitations of most available experimental models; recently however, promising progress is being made. As a result, the long-repeated promise of opioid receptor heteromers as selective therapeutic targets is now being realized.

Allosterism within δ Opioid-κ Opioid Receptor Heteromers in Peripheral Sensory Neurons: Regulation of κ Opioid Agonist Efficacy.

There is abundant evidence for formation of G protein-coupled receptor heteromers in heterologous expression systems, but little is known of the function of heteromers in native systems. Heteromers of δ and κ opioid receptors (DOR-KOR heteromers) have been identified in native systems. We previously reported that activation of DOR-KOR heteromers expressed by rat pain-sensing neurons (nociceptors) produces robust, peripherally mediated antinociception. Moreover, DOR agonist potency and efficacy is regulated by KOR antagonists via allosteric interactions within the DOR-KOR heteromer in a ligand-dependent manner. Here we assessed the reciprocal regulation of KOR agonist function by DOR antagonists in adult rat nociceptors in culture and in a behavioral assay of nociception. Naltrindole enhanced the potency of the KOR agonist 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-phenyl-2-pyrrolidin-1-ylethyl]acetamide (ICI-199441) 10- to 20-fold, but did not alter responses to 2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-ylcyclohexyl]acetamide (U50488). By contrast, the potency of U50488 was enhanced 20-fold by 7-benzylidenenaltrexone. The efficacy of 6'-guanidinonaltrindole (6'-GNTI) to inhibit nociceptors was blocked by small interfering RNA knockdown of DOR or KOR. Replacing 6'-GNTI occupancy of DOR with either naltrindole or 7-benzylidenenaltrexone abolished 6'-GNTI efficacy. Further, peptides derived from DOR transmembrane segment 1 fused to the cell membrane-penetrating HIV transactivator of transcription peptide also blocked 6'-GNTI-mediated responses ex vivo and in vivo, suggesting that 6'-GNTI efficacy in nociceptors is due to its positive allosteric regulation of KOR via occupancy of DOR in a DOR-KOR heteromer. Together, these results provide evidence for the existence of functional DOR-KOR heteromers in rat peripheral sensory neurons and that reciprocal, ligand-dependent allosteric interactions occur between the DOR and KOR protomers.

Delta Opioid Receptors: Learning and Motivation.

Delta opioid receptor (DOR) displays a unique, highly conserved, structure and an original pattern of distribution in the central nervous system, pointing to a distinct and specific functional role among opioid peptide receptors. Over the last 15 years, in vivo pharmacology and genetic models have allowed significant advances in the understanding of this role. In this review, we will focus on the involvement of DOR in modulating different types of hippocampal- and striatal-dependent learning processes as well as motor function, motivation, and reward. Remarkably, DOR seems to play a key role in balancing hippocampal and striatal functions, with major implications for the control of cognitive performance and motor function under healthy and pathological conditions.

Delta Opioid Pharmacology in Relation to Alcohol Behaviors.

Delta opioid receptors (DORs) are heavily involved in alcohol-mediated processes in the brain. In this chapter we provide an overview of studies investigating how alcohol directly impacts DOR pharmacology and of early studies indicating DOR modulation of alcohol behavior. We will offer a brief summary of the different animal species used in alcohol studies investigating DORs followed by a broader overview of the types of alcohol behaviors modulated by DORs. We will highlight a small set of studies investigating the relationship between alcohol and DORs in analgesia. We will then provide an anatomical overview linking DOR expression in specific brain regions to different alcohol behaviors. In this section, we will provide two models that try to explain how endogenous opioids acting at DORs may influence alcohol behaviors. Next, we will provide an overview of studies investigating certain new aspects of DOR pharmacology, including the formation of heteromers and biased signaling. Finally, we provide a short overview of the genetics of the DORs in relation to alcohol use disorders (AUDs) and a short statement on the potential of using DOR-based therapeutics for treatment of AUDs.

Delta Opioid Pharmacology in Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that compromises multiple neurochemical substrates including dopamine, norepinephrine, serotonin, acetylcholine, and glutamate systems. Loss of these transmitter systems initiates a cascade of neurological deficits beginning with motor function and ending with dementia. Current therapies primarily address the motor symptoms of the disease via dopamine replacement therapy. Exogenous dopamine replacement brings about additional challenges since after years of treatment it almost invariably gives rise to dyskinesia as a side effect. Therefore there is a clear unmet clinical need for improved PD therapeutics. Opioid receptors and their respective peptides are expressed throughout the basal ganglia and cortex where monoaminergic denervation strongly contributes to PD pathology. Delta opioid receptors are of particular interest because of their dense localization in basal ganglia and because activating this system is known to enhance locomotor activity under a variety of conditions. This chapter will outline much of the work that has demonstrated the effectiveness of delta opioid receptor activation in models of PD and its neuroprotective properties. It also discusses some of the challenges that must be addressed before moving delta opioid receptor agonists into a clinical setting.

Delta Opioid Receptors and Modulation of Mood and Emotion.

Depression is a pervasive and debilitating mental disorder that is inadequately treated by current pharmacotherapies in a majority of patients. Although opioids have long been known to regulate mood states, the use of opioids to treat depression is rarely discussed. This chapter explores the preclinical and clinical evidence supporting the antidepressant-like effects of opioid ligands, and in particular, delta opioid receptor (DOR) agonists. DOR agonists have been shown to produce antidepressant-like effects in a number of animal models. Some DOR agonists also produce convulsions which has limited their clinical utility. However, DOR agonists that generate antidepressant-like effects without convulsions have recently been developed and these drugs are beginning to be evaluated in humans. Work investigating potential mechanisms of action for the antidepressant-like effects of DOR agonists is also explored. Understanding mechanisms that give rise to DOR-mediated behaviors is critical for the development of DOR drugs with improved safety and clinical utility, and future work should be devoted to elucidating these pathways.

The Delta Opioid Receptor in Pain Control.

Nowadays, the delta opioid receptor (DOPr) represents a promising target for the treatment of chronic pain and emotional disorders. Despite the fact that they produce limited antinociceptive effects in healthy animals and in most acute pain models, DOPr agonists have shown efficacy in various chronic pain models. In this chapter, we review the progresses that have been made over the last decades in understanding the role played by DOPr in the control of pain. More specifically, the distribution of DOPr within the central nervous system and along pain pathways is presented. We also summarize the literature supporting a role for DOPr in acute, tonic, and chronic pain models, as well as the mechanisms regulating its activity under specific conditions. Finally, novel compounds that have make their way to clinical trials are discussed.

The Roles of Opioid Receptors in Cutaneous Wound Healing.

The process of recovery from skin wounding can be protracted and painful, and scarring may lead to weakness of the tissue, unpleasant sensations such as pain or itch, and unfavorable cosmetic outcomes. Moreover, some wounds simply fail to heal and become a chronic burden for the sufferer. Understanding the mechanisms underlying wound healing and the concomitant sensory disorders and how they might be manipulated for therapeutic benefit has attracted much interest in recent years, and here we discuss the latest developments in the field, focusing on the emergent roles of the peripheral opioid receptor (OPr) system.

Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol.

BACKGROUND: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus. METHODS: Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2-6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons. RESULTS: In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol's ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons. CONCLUSIONS: These results suggest that ethanol's neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Lipid Emulsion Inhibits Apoptosis Induced by a Toxic Dose of Verapamil via the Delta-Opioid Receptor in H9c2 Rat Cardiomyoblasts.

The goals of this study were to investigate the effects of lipid emulsion (LE) on apoptosis induced by a toxic dose of verapamil in H9c2 cells and to elucidate the associated cellular mechanism. The effects of LE alone and combined with an inhibitor on the decreases in cell counts and viability induced by verapamil and diltiazem were examined using the MTT assay. The effects of verapamil alone, combined LE and verapamil treatment, and combined inhibitor, LE and verapamil treatment on cleaved caspase-3, caspase-8 and Bax expression, were examined using Western blotting. The effects of verapamil alone and combined with LE on the number of TUNEL-positive H9c2 cells were also examined. LE attenuated the decreases in cell counts and viability induced by verapamil and diltiazem. However, the magnitude of the LE-mediated attenuation of decreased cell viability was enhanced by verapamil compared with diltiazem treatment. Naloxone, naltrindole hydrochloride, LY294002 and MK-2206 inhibited the LE-mediated attenuation of increased cleaved caspase-3 and caspase-8 expression induced by verapamil. LE attenuated the increase in the number of TUNEL-positive cell induced by verapamil. These results suggest that LE attenuates apoptosis induced by verapamil via activation of the delta-opioid receptor, phosphoinositide 3-kinase and Akt.

The Delta-Opioid Receptor Agonist D-Ala2-D-Leu2-Enkephalin Enhances Hypoxic Depression of Excitatory Synaptic Transmission and Improves Recovery of Rat Cortical Neurons from Hypoxia in vitro.

We investigated the influence of the delta-opioid receptor-preferring agonist D-Ala2-D-Leu2-enkephalin (DADLE) in vitro during long- and short-term hypoxia on the single cortical neuron membrane currents, the postsynaptic currents (PSCs), and the postsynaptic potentials (PSPs) in rats. Rat cortical pyramidal neurons showed 2 distinct and prognostically relevant responses to hypoxia. Type A neurons that responded to hypoxia by an inward current, followed by a steady outward current, were shown to recover during subsequent reoxygenation. In contrast, type B neurons that responded by a steady inward current, indicative of gradual anoxic depolarisation, suffered irreversible membrane dysfunction and did not recover completely during reoxygenation. Pre-treatment with 1 µmol/l DADLE attenuated the hypoxic inward current and favored complete recovery of holding current and input resistance during reoxygenation, even when neurons were challenged by a second exposure to hypoxia. DADLE enhanced the inhibitory effect of hypoxia on PSPs and PSCs. We assume that this neuroprotective effect is transmitted by the additive effects of DADLE on the hypoxic PSP/PSC suppression, thereby inhibiting presynaptic glutamate release.

Mixed MOP/DOP agonist biphalin elicits anti-transit effect in mouse models mimicking diarrhea-predominant irritable bowel syndrome symptoms.

BACKGROUND: Opioid receptors play a crucial role in the maintenance of homeostasis in the gastrointestinal (GI) tract. The aim of this study was to characterize the effect of biphalin, a mixed MOP/DOP agonist, on mouse intestinal contractility in vitro and GI motility in vivo and in animal models mimicking symptoms of diarrhea-predominant irritable bowel syndrome (IBS-D). METHODS: The effect of biphalin on muscle contractility in vitro was characterized in the ileum and colon. The anti-transit activity of biphalin in vivo was assessed in the following tests: whole gastrointestinal transit, colonic bead expulsion, fecal pellet output and castor oil-induced diarrhea, alone and in the presence of naloxone, and MOP and DOP antagonists. RESULTS: In vitro, biphalin (10(-10)-10(-6)M) inhibited colonic and ileal smooth muscle contractions in a concentration-dependent, opioid antagonist-reversible manner. In vivo, biphalin at the dose of 5mg/kg ip prolonged the whole GI transit and inhibited colonic bead expulsion. Biphalin reversed hypermotility and exerted anti-diarrheal effect in mouse models mimicking IBS-D symptoms. CONCLUSION: Biphalin is an interesting template for novel opioid-based agents to be used in therapy of functional GI diseases.

Voltage-Gated R-Type Calcium Channel Inhibition via Human µ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gßγ Protein Subunits.

Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by µ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human µ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of µ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via µ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein ßγ (Gßγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[ß-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gßγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gßγ subunits in a voltage-independent manner.