Signaling Complexes of Voltage-Gated Calcium Channels and G Protein-Coupled Receptors

Activation of opioid or opioid-receptor-like (ORL1 a.k.a. NOP or orphanin FQ) receptors mediates analgesia through inhibition of N-type calcium channels in dorsal root ganglion (DRG) neurons (). Unlike the three types of classical μ, δ, and κ opioid receptors, ORL1 mediates an agonist-independent inhibition of N-type calcium channels. This is mediated via the formation of a physical protein complex between the receptor and the channel, which in turn allows the channel to effectively sense a low level of constitutive receptor activity (). Further inhibition of N-type channel activity by activation of other G protein-coupled receptors is thus precluded. ORL1 receptors, however, also undergo agonist-induced internalization into lysosomes, and channels thereby become cointernalized in a complex with ORL1. This then results in removal of N-type channels from the plasma membrane and reduced calcium entry (). Similar signaling complexes between N-type channels and GABA_B receptors have been reported (). Moreover, both L-type and P/Q-type channels appear to be able to associate with certain types of G protein-coupled receptors (). Hence, interactions between receptors and voltage-gated calcium channels may be a widely applicable means to optimize receptor channel coupling.     

Signaling complexes of voltage-gated calcium channels and G protein-coupled receptors

Activation of opioid or opioid-receptor-like (ORL1 a.k.a. NOP or orphanin FQ) receptors mediates analgesia through inhibition of N-type calcium channels in dorsal root ganglion (DRG) neurons (1, 2). Unlike the three types of classical mu, delta, and kappa opioid receptors, ORL1 mediates an agonist-independent inhibition of N-type calcium channels. This is mediated via the formation of a physical protein complex between the receptor and the channel, which in turn allows the channel to effectively sense a low level of constitutive receptor activity (3). Further inhibition of N-type channel activity by activation of other G protein-coupled receptors is thus precluded. ORL1 receptors, however, also undergo agonist-induced internalization into lysosomes, and channels thereby become cointernalized in a complex with ORL1. This then results in removal of N-type channels from the plasma membrane and reduced calcium entry (4). Similar signaling complexes between N-type channels and GABA(B) receptors have been reported (5). Moreover, both L-type and P/Q-type channels appear to be able to associate with certain types of G protein-coupled receptors (6, 7). Hence, interactions between receptors and voltage-gated calcium channels may be a widely applicable means to optimize receptor channel coupling.     

Differential Modulation of ��- and ��-Opioid Receptor Agonists by Endogenous RGS4 Protein in SH-SY5Y Cells

Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins have been reported to modulate opioid receptor signaling. Here we show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express μ- and δ-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by ∼90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of δ-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was increased compared with control cells. This effect was reversed by transient transfection of a stable RGS4 mutant (HA-RGS4C2S). Furthermore, MAPK activation by SNC80 was increased in cells with knockdown of RGS4. In contrast, there was no change in the μ-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the δ-opioid receptor but not the μ-opioid receptor could be precipitated together with the stable RGS4. Using chimeras of the δ- and μ-opioid receptors, the C-tail and third intracellular domain of the δ-opioid receptor were suggested to be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating δ-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4.μ- and δ-opioid receptors are members of the G protein-coupled receptor family and interact with Gα_i/o proteins (1, 2). This results in signaling to a variety of downstream effectors, including adenylyl cyclase and the mitogen-activated protein kinase (MAPK)² cascade. Signaling of opioid receptors is regulated negatively by regulator of G protein signaling (RGS) proteins (3, 4). These are a family of molecules containing a “RGS consensus” domain that bind to Gα subunits and act as GTPase-accelerating proteins to increase the rate of GTP hydrolysis. This results in a decrease in the lifetime of the active Gα-GTP and free Gβγ subunits and limits signaling to downstream effectors (5–8). The mechanisms by which RGS proteins selectively modulate G protein-mediated receptor signal transduction pathways, especially opioid receptor signaling, are beginning to unfold (9–12). The foundation for the function and selectivity of RGS proteins in regulating opioid signaling lies in their ability to interact with opioid receptors and their cognate G proteins. In general, the selectivity or the preference of an RGS protein for a particular receptor is determined by a variety of factors, including tissue-specific expression and precise interaction with the intracellular domains of receptor proteins, G protein subunits, and effectors as well as other pathway-specific components (13).The effects of RGS proteins on opioid receptor signaling have been examined in several systems. The findings are not always consistent, probably due to the different methodologies used. It has been shown that members of the RZ, R4, and R7 subfamilies (7) of RGS proteins play crucial roles not only in terminating acute opioid agonist action but also in opioid receptor desensitization, internalization, recycling, and degradation (3, 14), thereby affecting opioid tolerance and dependence (15–18). Much work has been performed with RGS4, because it is a smaller RGS protein with a structure consisting of the RGS consensus (box) sequence and a small N terminus (19, 20). It also has a wide distribution in the brain, especially in brain regions important for opioid actions, including the striatum, locus coeruleus, dorsal horn of the spinal cord, and cerebral cortex (21). In vitro RGS4 has been shown to reverse δ-opioid receptor agonist-induced inhibition of cAMP synthesis in membranes prepared from NG108-15 cells (6). Overexpression of RGS4 in HEK293 cells also attenuated morphine-, [d-Ala²,N-Me-Phe⁴,Gly-ol⁵]enkephalin (DAMGO)-, and [d-Pen²,d-Pen⁵]enkephalin (DPDPE)-induced inhibition of adenylyl cyclase (22, 23). Co-expression of RGS4 with GIRK1/GIRK2 channels in Xenopus oocytes reduced the basal K⁺ current and accelerated the deactivation of GIRK channels activated by κ-opioid receptor agonist {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (24). Although these previous studies have provided evidence that RGS4 can negatively regulate opioid receptor signaling, they do not confirm a functional role for endogenous RGS4 in endogenous, nontransfected systems.Human neuroblastoma SH-SY5Y cells endogenously express μ- and δ-opioid receptors and a variety of Gα_i/o proteins (25–27). Here we show that RGS4 is abundantly found at both the mRNA and protein levels in these cells. Consequently, we used SH-SY5Y cells to examine the hypothesis that RGS4 negatively modulates opioid receptor signaling under physiological conditions. The endogenously expressed RGS4 level in SH-SY5Y cells was reduced using lentiviral delivery of short hairpin RNA (shRNA) targeting the RGS4 gene. This resulted in changes in δ- but not μ-opioid receptor-mediated signaling to adenylyl cyclase and the MAPK pathway. These findings argue for a selective interaction of RGS4 with the δ-opioid receptor. To test this, we expressed FLAG-tagged μ- and δ-opioid receptors together with a construct for a stable, proteosome-resistant RGS4 protein in HEK293T cells. Co-immunoprecipitation indicated that the δ-opioid but not the μ-opioid receptor was closely associated with RGS4, providing further evidence for a selective interaction between RGS4 and δ-opioid receptor signaling.     

Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

     

Differential Effect of Membrane Cholesterol Removal on ��- and ��-Opioid Receptors: A PARALLEL COMPARISON OF ACUTE AND CHRONIC SIGNALING TO ADENYLYL CYCLASE*

According to the lipid raft theory, the plasma membrane contains small domains enriched in cholesterol and sphingolipid, which may serve as platforms to organize membrane proteins. Using methyl-β-cyclodextrin (MβCD) to deplete membrane cholesterol, many G protein-coupled receptors have been shown to depend on putative lipid rafts for proper signaling. Here we examine the hypothesis that treatment of HEK293 cells stably expressing FLAG-tagged μ-opioid receptors (HEK FLAG-μ) or δ-opioid receptors (HEK FLAG-δ) with MβCD will reduce opioid receptor signaling to adenylyl cyclase. The ability of the μ-opioid agonist [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin to acutely inhibit adenylyl cyclase or to cause sensitization of adenylyl cyclase following chronic treatment was attenuated with MβCD. These effects were due to removal of cholesterol, because replenishment of cholesterol restored [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin responses back to control values, and were confirmed in SH-SY5Y cells endogenously expressing μ-opioid receptors. The effects of MβCD may be due to uncoupling of the μ receptor from G proteins but were not because of decreases in receptor number and were not mimicked by cytoskeleton disruption. In contrast to the results in HEK FLAG-μ cells, MβCD treatment of HEK FLAG-δ cells had no effect on acute inhibition or sensitization of adenylyl cyclase by δ-opioid agonists. The differential responses of μ- and δ-opioid agonists to cholesterol depletion suggest that μ-opioid receptors are more dependent on cholesterol for efficient signaling than δ receptors and can be partly explained by localization of μ- but not δ-opioid receptors in cholesterol- and caveolin-enriched membrane domains.Membrane cholesterol can alter the function of integral proteins, such as G protein-coupled receptors, through cholesterol-protein interactions and by changes in membrane viscosity (1). In addition, cholesterol interacts with other lipids found in the bilayer, particularly sphingolipids (2), which allows for tight and organized packing that can precipitate the formation of specialized domains within the plasma membrane (3). These domains have become an area of intense research interest and have been termed lipid or membrane rafts (4). The study of membrane rafts in intact cells is controversial, due in part to the limitations of the current methods used to study rafts (5, 6). Regardless, the membrane environment formed in regions of high cholesterol and sphingolipids may be such that certain proteins have an affinity for these regions, especially proteins with a propensity to interact with cholesterol.Many G protein-coupled receptors and signaling proteins have been found to prefer cholesterol-enriched domains leading to the hypothesis that these domains can organize signaling molecules in the membrane to enhance or inhibit specific signaling events (7). This includes μ- (8, 9), δ- (10, 11), and κ-opioid receptors (12). In addition, Gα_i (12–17), Gα_o (16), and adenylyl cyclase isoforms 3 (18), 5/6 (9, 18, 19), and 8 (20) have been found to associate with cholesterol and/or the cholesterol-binding protein caveolin. Activated opioid receptors couple to Gα_i/o proteins and acutely inhibit the activity of adenylyl cyclase. Longer term exposure to opioid agonists causes sensitization of adenylyl cyclase and a rebound overshoot of cAMP production upon withdrawal of the agonist (21). Consequently, we sought to assess the role of cholesterol depletion on the ability of μ- and δ-opioid receptor agonists to inhibit and cause sensitization of adenylyl cyclase.There are conflicting data for the effect of changes in membrane cholesterol on opioid signaling. For example, an increase in plasma membrane microviscosity by addition of cholesteryl hemisuccinate to SH-SY5Y cell membranes increased μ-opioid receptor coupling to G proteins (22). Conversely, removal of membrane cholesterol from Chinese hamster ovary cells has been shown to either decrease (23) or increase (24) the coupling of μ-opioid receptors to G proteins, as measured by [³⁵S]GTPγS³ binding stimulated by the μ-opioid agonist DAMGO. Furthermore, the effect of cholesterol removal on δ-opioid agonist-stimulated [³⁵S]GTPγS binding varies by cell type (10, 25). In these previous studies, the variety of cell types utilized and the conflicting results make comparisons between opioid receptor types difficult. The objective of this study was to directly compare the role of membrane cholesterol in modulating acute and chronic μ- and δ-opioid signaling in the same cell systems using identical methods, including the following: 1) depletion of cholesterol by the cholesterol-sequestering agent methyl-β-cyclodextrin (MβCD); 2) separation of cholesterol-enriched membranes by sucrose gradient ultracentrifugation; and 3) clustering of lipid raft patches in whole cells with cholera toxin B subunit.In initial experiments using human embryonic kidney (HEK) cells heterologously expressing μ- or δ-opioid receptors, we found that δ-opioid receptors were located in caveolin-poor fractions following 1% Triton X-100 homogenization and sucrose gradient ultracentrifugation. This differs from studies using a detergent-free method to identify lipid raft fractions (10, 11). In contrast, we found that the μ-opioid receptor was found in both caveolin-poor and caveolin-rich fractions, in accordance with previous literature (8, 9). This differential localization of opioid receptors led us to test the hypothesis that, in contrast to the μ-opioid receptor, the δ-opioid receptor would not be dependent on cholesterol for signaling. The results show that μ- but not δ-opioid receptors have a dependence on cholesterol for signaling to adenylyl cyclase and that this effect is much more pronounced following chronic exposure to opioids.     

Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.     

The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats

Previous studies suggested that electroacupuncture (EA) can suppress opioid dependence by the release of endogenous opioid peptides. To explore the site of action and the receptors involved, we tried to inject highly specific agonists for μ-, δ- and κ-opioid receptors into the CNS to test whether it can suppress morphine-induced conditioned place preference (CPP) in the rat. Male Sprague–Dawley rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP model. This CPP can be prevented by (a) i.p. injection of 3 mg/kg dose of morphine, (b) intracerebroventricular (i.c.v.) injection of micrograms doses of the selective μ-opioid receptor agonist DAMGO, δ-agonist DPDPE or κ-agonist U-50,488H or (c) microinjection of DAMGO, DPDPE or U50488H into the shell of the nucleus accumbens (NAc). The results suggest that the release of endogenous μ-, δ- and κ-opioid agonists in the NAc shell may play a role for EA suppression of opiate addiction.     

Effect of Chronic Administration of Morphine, U-50, 488H and [D-Pen, D-Pen]enkephalin on the Concentration of cGMP in Brain Regions and Spinal Cord of the Mouse

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of morphine, U-50,488H and [ -Pen², -Pen⁵]enkephalin on the concentration of cGMP in brain regions and spinal cord of the mouse. Peptides 18(10) 1629–1634, 1997.—The effects of chronic administration and subsequent withdrawal of μ-, κ- and δ-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at μ-, κ- and δ-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla(76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of μ- and κ- opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to μ- and κ- but not to δ-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by μ-, κ- and δ-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to μ-, κ- and δ-opioid receptor agonists.     

μ-Opioid Receptor Antibody Reveals Tissue-Dependent Specific Staining and Increased Neuronal μ-Receptor Immunoreactivity at the Injured Nerve Trunk in Mice

Neuropathic pain is a debilitating chronic disease often resulting from damage to peripheral nerves. Activation of opioid receptors on peripheral sensory neurons can attenuate pain without central nervous system side effects. Here we aimed to analyze the distribution of neuronal μ-opioid receptors, the most relevant opioid receptors in the control of clinical pain, along the peripheral neuronal pathways in neuropathy. Hence, following a chronic constriction injury of the sciatic nerve in mice, we used immunohistochemistry to quantify the μ-receptor protein expression in the dorsal root ganglia (DRG), directly at the injured nerve trunk, and at its peripheral endings in the hind paw skin. We also thoroughly examined the μ-receptor antibody staining specificity. We found that the antibody specifically labeled μ-receptors in human embryonic kidney 293 cells as well as in neuronal processes of the sciatic nerve and hind paw skin dermis, but surprisingly not in the DRG, as judged by the use of μ/δ/κ-opioid receptor knockout mice. Therefore, a reliable quantitative analysis of μ-receptor expression in the DRG was not possible. However, we demonstrate that the μ-receptor immunoreactivity was strongly enhanced proximally to the injury at the nerve trunk, but was unaltered in paws, on days 2 and 14 following injury. Thus, μ-opioid receptors at the site of axonal damage might be a promising target for the control of painful neuropathies. Furthermore, our findings suggest a rigorous tissue-dependent characterization of antibodies'' specificity, preferably using knockout animals.     

Effects of steroids on the brain opioid system

The experiments reported here add further evidence in support of the view that sex steroids may influence the binding characteristics of brain opioid receptors. In particular, it has been shown that: (a) the number of μ-opioid receptors varies in the hypothalamus of regularly cycling female rats according to the different phases of the estrous cycle, which are characterized by fluctuations of circulating levels of sex steroids; (b) the number of μ-opioid receptors decreases in the hypothalamus and in the corpus striatum when ovariectomized rats are submitted to treatments with estradiol and progesterone able to induce a “positive” feedback effect on LH release. A treatment with estrogen alone able to induce a “negative” feedback effect on LH release brings about an increase of the number of μ-opioid receptors in the thalamus and in the hippocampus; (c) in addition to the μ-receptors, receptors of the delta type may also be involved in the control of gonadotropin secretion; recent results here presented indicate that a line of immortalized hypothalamic cells (GT1 cells), which synthesize and secrete LHRH, present δ opioid receptors on their membranes; these are apparently involved in the control of LHRH release from these cells.     

Opiate-Induced Suppression of Rat Hypoglossal Motoneuron Activity and Its Reversal by Ampakine Therapy

Background

Hypoglossal (XII) motoneurons innervate tongue muscles and are vital for maintaining upper-airway patency during inspiration. Depression of XII nerve activity by opioid analgesics is a significant clinical problem, but underlying mechanisms are poorly understood. Currently there are no suitable pharmacological approaches to counter opiate-induced suppression of XII nerve activity while maintaining analgesia. Ampakines accentuate α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor responses. The AMPA family of glutamate receptors mediate excitatory transmission to XII motoneurons. Therefore the objectives were to determine whether the depressant actions of μ-opioid receptor activation on inspiratory activity includes a direct inhibitory action at the inspiratory premotoneuron to XII motoneuron synapse, and to identify underlying mechanism(s). We then examined whether ampakines counteract opioid-induced depression of XII motoneuron activity.

Methodology/Principal Findings

A medullary slice preparation from neonatal rat that produces inspiratory-related output in vitro was used. Measurements of inspiratory burst amplitude and frequency were made from XII nerve roots. Whole-cell patch recordings from XII motoneurons were used to measure membrane currents and synaptic events. Application of the μ-opioid receptor agonist, DAMGO, to the XII nucleus depressed the output of inspiratory XII motoneurons via presynaptic inhibition of excitatory glutamatergic transmission. Ampakines (CX614 and CX717) alleviated DAMGO-induced depression of XII MN activity through postsynaptic actions on XII motoneurons.

Conclusions/Significance

The inspiratory-depressant actions of opioid analgesics include presynaptic inhibition of XII motoneuron output. Ampakines counteract μ-opioid receptor-mediated depression of XII motoneuron inspiratory activity. These results suggest that ampakines may be beneficial in countering opiate-induced suppression of XII motoneuron activity and resultant impairment of airway patency.     

Src Phosphorylation of ��-Receptor Is Responsible for the Receptor Switching from an Inhibitory to a Stimulatory Signal

Recent studies have revealed that in G protein-coupled receptor signalings switching between G protein- and β-arrestin (βArr)-dependent pathways occurs. In the case of opioid receptors, the signal is switched from the initial inhibition of adenylyl cyclase (AC) to an increase in AC activity (AC activation) during prolonged agonist treatment. The mechanism of such AC activation has been suggested to involve the switching of G proteins activated by the receptor, phosphorylation of signaling molecules, or receptor-dependent recruitment of cellular proteins. Using protein kinase inhibitors, dominant negative mutant studies and mouse embryonic fibroblast cells isolated from Src kinase knock-out mice, we demonstrated that μ-opioid receptor (OPRM1)-mediated AC activation requires direct association and activation of Src kinase by lipid raft-located OPRM1. Such Src activation was independent of βArr as indicated by the ability of OPRM1 to activate Src and AC after prolonged agonist treatment in mouse embryonic fibroblast cells lacking both βArr-1 and -2. Instead the switching of OPRM1 signals was dependent on the heterotrimeric G protein, specifically G_i2 α-subunit. Among the Src kinase substrates, OPRM1 was phosphorylated at Tyr³³⁶ within NPXXY motif by Src during AC activation. Mutation of this Tyr residue, together with mutation of Tyr¹⁶⁶ within the DRY motif to Phe, resulted in the complete blunting of AC activation. Thus, the recruitment and activation of Src kinase by OPRM1 during chronic agonist treatment, which eventually results in the receptor tyrosine phosphorylation, is the key for switching the opioid receptor signals from its initial AC inhibition to subsequent AC activation.Classical G protein-coupled receptor (GPCR)² signaling involves the activation of specific heterotrimeric G proteins and the subsequent dissociation of α- and βγ-subunits. These G protein subunits serve as the activators and/or inhibitors of several effector systems, including adenylyl cyclases, phospholipases, and ion channels (1). However, recent studies have shown that GPCR signaling deviates from such a classical linear model. For example, in kidney and colonic epithelial cells, protease-activated receptor 1 can transduce its signals through either Gα_i/o or Gα_q subunits via inhibition of small GTPase RhoA or activation of RhoD. Thus, RhoA and RhoD act as molecular switches between the negative and positive signaling activity of protease-activated receptor 1 (2). Another example is the ability of β₂-adrenergic receptor to switch from G_s-dependent pathways to non-classical signaling pathways by coupling to pertussis toxin-sensitive G_i proteins in a cAMP-dependent protein kinase/protein kinase C phosphorylation-dependent manner. In this case, the phosphorylation-induced switch in G protein coupling provides the receptor access to alternative signaling pathways. For β₂-adrenergic receptors, this leads to a G_i-dependent activation of MAP kinase (3, 4). Furthermore the involvement of protein scaffolds, such as β-arrestins in the MAP kinase cascade, could also alter the GPCR signaling (5–8). Hence the formation of “signaling units” or “receptosomes” would influence the GPCR signaling process and destination.For opioid receptors, which are members of the rhodopsin GPCR subfamily receptors, signal switching is also observed. Normally opioid receptors inhibit AC activity, activate the MAP kinases and Kir3 K⁺ channels, inhibit the voltage-dependent Ca²⁺ channels, and regulate other effectors such as phospholipase C (9). However, during prolonged agonist treatment, not only is there a blunting of these cellular responses but also a compensatory increase in intracellular cAMP level, which is particularly significant upon the removal of the agonist or the addition of an antagonist such as naloxone (10–12). This compensatory adenylyl cyclase activation phenomenon has been postulated to be responsible for the development of drug tolerance and dependence (13). The observed change from receptor-mediated AC inhibition to receptor-mediated AC activation reflects possible receptor signal switching. Although the exact mechanism for such signal changes has yet to be elucidated, activation of specific protein kinases and subsequent phosphorylation of AC isoforms (14, 15) and other signaling molecules (16) have been suggested to be the key for observed AC activation. Among all the protein kinases studied, involvement of protein kinase C, MAP kinase, and Raf-1 has been implicated in the activation of AC (17–19). Alternative mechanisms, such as agonist-induced receptor internalization and the increase in the constitutive activities of the receptor, also have been suggested to play a role in increased AC activity after prolonged opioid agonist treatment (20). Earlier studies also implicated the switching of the opioid receptor from G_i/G_o to G_s coupling during chronic agonist treatment (21). Regardless of the mechanism, the exact molecular events that lead to the switching of opioid receptor from an inhibitory response to a stimulatory response remain elusive.Src kinases, which are members of the nonreceptor tyrosine kinase family, have been implicated in GPCR function because several Src family members such as cSrc, Fyn, and Yes have been reported to be activated by several GPCRs, including β₂- (22) and β₃ (23)-adrenergic, M₂- (24) and M₃ (25)-muscarinic, and bradykinin receptors (26). The GPCRs that are capable of activating Src predominantly couple to G_i/o family G proteins (27). Src kinases appear to associate with, and be activated by, GPCRs themselves either through direct interaction with intracellular receptor domains or by binding to GPCR-associated proteins, such as G protein subunits or β-arrestins (27). Src kinase has been reported to be activated by κ- (28) and δ (29)-opioid receptors and regulate the c-Jun kinase and MAP kinase activities. Src kinase within the nucleus accumbens has been implicated in the rewarding effect and hyperlocomotion induced by morphine in mice (30). However, it is not clear whether the Src kinase is activated and involved in the signal transduction in AC activation after chronic opioid agonist administration.Previously we reported that the lipid raft location of the receptor and the Gα_i2 proteins are two prerequisites for the observed increase in AC activity during prolonged agonist treatment (31, 32). Because various protein kinases including Src kinases and G proteins have been shown to be enriched in lipid rafts (33), the roles of these cellular proteins in the eventual switching of opioid receptor signals from inhibition to stimulation of AC activity were examined in the current studies. We were able to demonstrate that the association with and subsequent activation of Src kinase by the μ-opioid receptor (OPRM1), which leads to eventual tyrosine phosphorylation of OPRM1, are the cellular events required for the switching of opioid receptor signaling upon chronic agonist treatment.     

Involvement of opioid receptors in the oxytocin-induced antinociception in the central nervous system of rats

Recent studies showed that oxytocin and opioid peptides play important roles in pain modulation at different levels in the central nervous system. The present study was performed to explore whether opioid system is involved in the oxytocin-induced antinociception in the brain of rats. The results showed that: (1) intracerebroventricular injection of oxytocin induced dose-dependent increases in hindpaw withdrawal latencies (HWL) to noxious thermal and mechanical stimulation in rats. (2) The antinociceptive effect of oxytocin was attenuated dose-dependently by intracerebroventricular injection of naloxone, indicating an involvement of opioid system in the oxytocin-induced antinociception. (3) It is interesting that the antinociceptive effect of oxytocin was attenuated by subsequent intracerebroventricular injection of the μ-opioid antagonist β-funaltrexamine (β-FNA) and the κ-opioid antagonist nor-binaltorphimine (nor-BNI), but not the δ-opioid antagonist naltrindole. The results indicate that oxytocin plays an antinociceptive role in the brain of rats; μ- and κ-opioid receptors, not δ-receptors, are involved in the oxytocin-induced antinociception in the central nervous system of rats.     

Binding of [H][D-Ala, MePhe, Gly-ol] Enkephalin, [H][D-Pen, D-Pen]Enkephalin, and [H]U-69,593 to Airway and Pulmonary Tissues of Normal and Sensitized Rats

Bhargava, H. N., V. M. Villar, J. Cortijo and E. J. Morcillo. Binding of [³H][D-Ala², MePhe⁴, Gly-ol⁵]enkephalin, [³H][D-Pen², D-Pen⁵]enkephalin, and [³H]U-69,593 to airway and pulmonary tissues of normal and sensitized rats. Peptides 18(10) 1603–1608, 1997.—The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [³H]labeled ligands of μ-([D-Ala², MePhe⁴, Gly-ol⁵]enkephalin; DAMGO), δ ([D-Pen², D-Pen⁵]enkephalin; DPDPE), and κ-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (unsensitized) and actively IgE-sensitized rats acutely challenged with the specific antigen. [³H]DAMGO, [³H]DPDPE and [³H]U-69,593 bound to membranes of normal and sensitized tissues at a saturable, single high-affinity site. The rank order of receptor densities in normal tissues was δ- ≥ κ- ≥ μ-, with lung parenchyma exhibiting the greatest binding capacity for δ- and μ- receptors compared to the other regions examined. The K_d values showed small differences between ligands and regions tested. The μ- and δ-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. By contrast, κ-opioid receptor density was augmented in sensitized lung parenchyma but an increase in K_d values was also observed. These differential changes in the density and affinity of opioid receptor types may be related to alterations in endogenous opioid peptides during the process of sensitization.     

Arrestin is required for agonist-induced trafficking of voltage-dependent calcium channels

Many metabotropic receptors in the nervous system act through signaling pathways that result in the inhibition of voltage-dependent calcium channels. Our previous findings showed that activation of seven-transmembrane receptors results in the internalization of calcium channels. This internalization takes place within a few seconds, raising the question of whether the endocytic machinery is in close proximity to the calcium channel to cause such rapid internalization. Here we show that voltage-dependent calcium channels are pre-associated with arrestin, a protein known to play a role in receptor trafficking. Upon GABAB receptor activation, receptors are recruited to the arrestin-channel complex and internalized. beta-Arrestin 1 selectively binds to the SNARE-binding region of the calcium channel. Peptides containing the arrestin-binding site of the channel disrupt agonist-induced channel internalization. Taken together these data suggest a novel neuronal role for arrestin.     

μ-opioid and 5-HT1A receptors heterodimerize and show signalling crosstalk via G protein and MAP-kinase pathways

μ-opioid receptors have been shown to form heterodimers with several G protein coupled receptors involved in pain regulation such as α(2A)-adrenergic and neurokinin 1 receptors. Because the 5-HT(1A) receptor is also involved in pain control, we investigated whether it can interact with the μ-opioid receptor in cell lines. Using epitope-tagged μ-opioid and 5-HT(1A) receptors, we show that both receptors can co-immunoprecipate when expressed in the same cells. This physical interaction was corroborated by a Bioluminescence Resonance Energy Transfer signal between the μ-opioid receptor fused to Renilla luciferase and the 5-HT(1A) receptor fused to the Green Fluorescent Protein. Consistent with the presence of functional heterodimers, the μ-opioid receptor activated a Gα(o) protein covalently fused to the 5-HT(1A) receptor in membrane preparations as well as a Gα(15) protein fused to the 5-HT(1A) receptor in living cells. We demonstrate that both receptors can coexerce control of the ERK1/2 pathway: for example, μ-opioid receptor-induced ERK1/2 phosphorylation was selectively desensitized by 5-HT(1A) receptor activation. Although 5-HT(1A) and μ-opioid receptors were capable to internalize in response to their own activation, they were ineffective to induce the co-internalization of their partners. Thus, we show a functional heterodimerization of μ-opioid and 5-HT(1A) receptors in cell lines, a complex that might play a role in the control of pain in vivo. These results also support the potential therapeutic action of 5-HT(1A) agonists against nociceptive processes.     

Lipo-Endomorphin-1 Derivatives with Systemic Activity against Neuropathic Pain without Producing Constipation

To enhance the drug-like properties of the endogenous opioid peptide endomorphin-1 (1 = Tyr-Pro-Trp-Phe-NH₂), the N-terminus of the peptide was modified with 2-aminodecanoic acid, resulting in compound 3. Tyr in compound 1 was replaced with 2,6-dimethyltyrosine yielding compound 2. Derivative 2 was also substituted with 2-aminodecanoic acid producing compound, 4. Lipoamino acid-modified derivatives showed improved metabolic stability and membrane permeability while maintaining high μ-opioid (MOP) receptor binding affinity and acting as a potent agonist. In vivo studies showed dose-dependent antinociceptive activity following intravenous (i.v.) administration of compounds 3 and 4 in a chronic constriction injury (CCI)-rat model of neuropathic pain with ED₅₀ values of 1.22 (±0.93) and 0.99 (±0.89) µmol/kg, respectively. Pre-treatment of animals with naloxone hydrochloride significantly attenuated the anti-neuropathic effects of compound 3, confirming the key role of opioid receptors in mediating antinociception. In contrast to morphine, no significant constipation was produced following i.v. administration of compound 3 at 16 µmol/kg. Furthermore, following chronic administration of equi-potent doses of compound 3 and morphine to rats, there was less antinociceptive tolerance for compound 3 compared with morphine.