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.     

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.     

Effects of morphine-3-glucuronide on the antinociceptive activity of peptide and nonpeptide opioid receptor agonists in mice

The effects of morphine-3-glucuronide (M3G), a metabolite of morphine, were determined on the antinociceptive actions, as measured by the tail flick test, of morphine, a μ-opioid receptor agonist, of U-50,488H, a κ-opioid receptor agonist, of [ -Pen², -Pen⁵]enkephalin (DPDPE), a δ₁-opioid receptor agonist, and of [ -Ala²,Glu⁴]deltorphin II (deltorphin II), a δ₂-opioid receptor agonist in mice. Morphine administered ICV (2.5 μg/mouse) or SC (10 mg/kg), U-50,488H (25 mg/kg, IP), DPDPE (15 μg/mouse; ICV), and deltorphin II (15 μg/mouse, ICV) produced antinociception in mice. Intraperitoneal or ICV injections of M3G did not produce any effect on the tail flick latency nor did it affect the antinociception-induced by morphine, U-50,488H, DPDPE, or deltorphin II. Previously M3G has been shown to antagonize the antinociceptive effects of morphine in the rat. It is concluded that in the mouse, M3G neither produces hyperalgesia nor modifies the actions of μ-, κ-, δ₁-, or δ₂-opioid receptor agonists.     

μ-opioid receptor-mediated inhibition of the release of radiolabelled noradrenaline and acetylcholine from rat amygdala slices

Presynaptic modulation by opioids of electrically-evoked neurotransmitter release from superfused rat amygdala slices prelabelled with [³H]noradrenaline (NA) and [¹⁴C]choline was examined. Both [³H]NA and [¹⁴C]acetylcholine release were strongly inhibited by morphine, the mixed δ/μ-receptor agonist [ -Ala², -Leu⁵]enkephalin (DADLE) and the highly selective μ-agonist [ -Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), whereas the highly selective δ-agonist [ -Pen², -Pen⁵]enkephalin and the κ-agonist bremazocine were without effect. The inhibitory effects were potently antagonized by naloxone but not by the selective δ-receptor antagonist fentanylisothiocyanate. When the selective uptake inhibitor desipramine was used to prevent uptake of [³H]NA into noradrenergic nerve terminals, but sparing the uptake into dopaminergic nerve terminals, the electrically evoked release of tritium was strongly inhibited by bremazocine but not by DADLE or DAMGO.

The data indicate, that in the amygdala transmitter release from dopaminergic nerve fibres is inhibited only via activation of κ-receptors, whereas transmitter release from noradrenergic and cholinergic nerve fibers is subjected to inhibition by opioids via activation of μ-receptors only. Regional differences and similarities of modulation of neurotransmitter release by opioids in the rat brain are briefly discussed.     

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.     

Influence of the Hypothalamic Arcuate Nucleus on Intraocular Pressure and the Role of Opioid Peptides

Background

An opioid peptide neuron/humoral feedback regulation might be involved in changes of intraocular pressure (IOP). The aims of this study are to investigate the effects of arcuate nucleus (ARC) and opioid peptides on intraocular pressure (IOP).

Methods

Fifty-four healthy purebred New Zealand white rabbits (108eyes) were randomly divided into 4 groups, including control group, electrical stimulation group, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) group, and [D-Pen 2, D-Pen5]- enkephalin (DPDPE) group. Bilateral IOP was measured after unilateral electrical stimulation of the ARC or unilateral microinjection into the ARC of the selective μ-opioid receptor agonist DAMGO or the selective δ opioid receptor agonist DPDPE, both alone and after pre-administration of either the non-selective opioid receptor antagonist naloxone or saline.

Results

Both electrical stimulation in ARC and micro-injection either <mu> or <delta> opioid receptor agonists, DAMGO or DPDPE, respectively, caused a significant bilateral reduction in IOP (P<0.05) which was more pronounced in the ipsilateral than in the contralateral eye. Pretreatment with naloxone prevented some, but not all IOP reductions.

Conclusion

The ARC takes part in the negative regulation of IOP, an action that may involve opioid neurons.     

μ Receptors and opioid cardiovascular effects in the NTS of rat

In order to assess the potential role of mu (μ) and delta (δ) opiate receptors in the central regulation of the cardiovascular and respiratory systems, the cardiovascular and respiratory effects of the relatively selective μ-opioid agonist D-Ala², MePhe⁴, Gly-ol⁵ enkephalin (DAGO) and relatively selective δ-agonist D-Ala²-D-Leu⁵ enkephalin (DADL) were compared following microinjection of these compounds into the nucleus tractus solitarius of pentobarbital-anesthetized rats. Both opioid agonists produced dose dependent increases in systolic and diastolic blood pressure as well as heart rate; but DAGO was nearly ten times more potent in eliciting these changes. Respiratory rate was increased by DADL and by lower doses of DAGO, but was depressed by higher doses of DAGO. Tidal volume was depressed by both peptides. These data support the concept that the cardiovascular pressor responses and tachycardia as well as the respiratory effects of opioids in the rat NTS are mediated by μ receptors.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.     

Heterodimerization of ORL1 and Opioid Receptors and Its Consequences for N-type Calcium Channel Regulation

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, μ-, δ-, and κ-opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The μ-opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of μ-opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.     

Itch-scratch responses induced by opioids through central Mu opioid receptors in mice

We examined scratch-inducing effects of intracisternal, intrathecal and intradermal injections of morphine and some opioid agonists in mice. Intracisternal injection of morphine (3 nmol/animal) and the mu-receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin (DAMGO; 0.2 nmol/animal) elicited scratching of the face, with little effect on scratching of the trunk. Intracisternal injection of the delta-receptor agonist [D-Pen(2,5)]enkephalin (DPDPE) and the kappa-receptor agonist U50488 were without effects. Intrathecal injection of morphine (0.1-3 nmol/animal) produced a dose-dependent increase in body scratching, with little effects on face scratching. Face scratching induced by intrathecal morphine (3 nmol/animal) was almost abolished by subcutaneous pretreatment with naloxone (1 mg/kg). Intradermal injections of morphine (3-100 nmol/site), DAMGO (1-100 nmol/site), DPDPE (10 and 100 nmol/site) and U50488 (10-100 nmol/site) did not elicit scratching of the site of injection. Intradermal injection of histamine (100 nmol/site) induced the scratching in ICR, but not ddY, mice and serotonin (30 and 50 nmol/site) elicited the scratching in either strain of mice. The results suggest that opioids induce scratching, and probably itching, through central mu-opioid receptors in the mouse.     

Delta-Opioid Receptor Analgesia Is Independent of Microglial Activation in a Rat Model of Neuropathic Pain

The analgesic effect of delta-opioid receptor (DOR) ligands in neuropathic pain is not diminished in contrast to other opioid receptor ligands, which lose their effectiveness as analgesics. In this study, we examine whether this effect is related to nerve injury-induced microglial activation. We therefore investigated the influence of minocycline-induced inhibition of microglial activation on the analgesic effects of opioid receptor agonists: morphine, DAMGO, U50,488H, DPDPE, Deltorphin II and SNC80 after chronic constriction injury (CCI) to the sciatic nerve in rats. Pre-emptive and repeated administration of minocycline (30 mg/kg, i.p.) over 7 days significantly reduced allodynia and hyperalgesia as measured on day 7 after CCI. The antiallodynic and antihyperalgesic effects of intrathecally (i.t.) administered morphine (10–20 µg), DAMGO (1–2 µg) and U50,488H (25–50 µg) were significantly potentiated in rats after minocycline, but no such changes were observed after DPDPE (10–20 µg), deltorphin II (1.5–15 µg) and SNC80 (10–20 µg) administration. Additionally, nerve injury-induced down-regulation of all types of opioid receptors in the spinal cord and dorsal root ganglia was not influenced by minocycline, which indicates that the effects of opioid ligands are dependent on other changes, presumably neuroimmune interactions. Our study of rat primary microglial cell culture using qRT-PCR, Western blotting and immunocytochemistry confirmed the presence of mu-opioid receptors (MOR) and kappa-opioid receptors (KOR), further we provide the first evidence for the lack of DOR on microglial cells. In summary, DOR analgesia is different from analgesia induced by MOR and KOR receptors because it does not dependent on injury-induced microglial activation. DOR agonists appear to be the best candidates for new drugs to treat neuropathic pain.     

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.     

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.     

Role of Opioid Receptors on Food Choice and Macronutrient Selection in Meat-Type Chick

The present study was designed to examine the role of opioid receptors on food choice and macronutrient selection in neonatal chicks. In this study, 13 experiments designed, experiments 1–3 for effect of specific opioid receptors on appetite and experiments 4–13 on effect of opioid receptors on food choice and macronutrient selection in meat-type chick. In experiment 1, chicken intracerebroventricular (ICV) injected with 125, 250 and 500 pmol of DAMGO (µ-opioid receptor agonist). Experiment 2 was conducted to investigate the effect of DPDPE (δ-opioid receptor agonist) at doses of 20, 40 and 80 nmol. In experiment 3 ICV injection of the U-50488H (κ-opioid receptor agonist, of 10, 20 and 40 nmol) was done. In experiment 4, birds injected with saline and different diets: standard diet without fat, diet containing nutrient energy 20 % higher than standard, diet containing nutrient energy 20 % lower than standard and standard diet containing fat were offered to them to investigate desire of chicken to diets. Experiments 5–7 were similar to experiment 4, except, birds ICV injected with 125, 250 and 500 pmol of DAMGO. In experiments 8–10 chicken received ICV injection of DPDPE (20, 40 and 80 nmol). The experiments 11–13 was similar to previous experiments which birds injected with different doses of U-50488H (10, 20 and 40 nmol), respectively. Then the cumulative food intake measured until 180-min post injection. According to the results, ICV injection of DAMGO diminished food intake while DPDPE and U-50488H increased appetite (P < 0.05). Despite anorexigenic effect, ICV injection of DAMGO increased birds desire to eat fat containing standard diet compared to the standard diet without fat (P < 0.05). These findings suggest endogenous opioids governing preferences for fat rich foods.     

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.     

Inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) on gastrointestinal transit

The inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) (TAPA), a highly selective mu(1)-opioid receptor agonist, on mouse gastrointestinal transit was compared with that of morphine and [D-Ala(2), N-methyl-Phe(4), Gly(5)-ol]-enkephalin (DAMGO). When administered intracerebroventricularly 5 min before the oral injection of charcoal meal, TAPA (10-100 pmol), morphine (0.25-4 nmol), and DAMGO (20-80 pmol) dose-dependently inhibited gastrointestinal transit of charcoal. The inhibitory effect of each mu-opioid receptor agonist was completely antagonized by naloxone, a nonselective opioid receptor antagonist. The inhibitory effects of morphine and DAMGO were significantly antagonized by both beta-funaltrexamine, a selective mu-opioid receptor antagonist, and naloxonazine, a selective mu(1)-opioid receptor antagonist. In contrast, the inhibitory effect of TAPA was not affected at all by beta-funaltrexamine, naloxonazine, nor-binaltorphimine (a selective kappa-opioid receptor antagonist), or naltrindole (a selective delta-opioid receptor antagonist). These results suggest that the inhibitory effect of TAPA on gastrointestinal transit may be mediated through an opioid receptor mechanism different from that of morphine and DAMGO.     

Cardiorespiratory effects of μ and δ opiate agonists following third or fourth ventricular injections

The cardiorespiratory effects of prototype μ (morphine and β-casomorphine 1–4) and δ (D-Ala²-D-Leu⁵Enkephalin—DADLE) opioid ligands were compared following microinjection into third and fourth ventricular spaces in conscious and anesthetized rats. The direction of change in arterial pressure produced by ventricular opioid injections varied according to ligand, site of administration, and state of consciousness of the animal. In general, pentobarbital anesthesia blocked or reversed the pressor response to these opiate agonists; depressor responses became magnified following pentobarbital. Qualitatively, the predominant effect of third ventricular DADLE in anesthetized rats was to produce a depression of arterial pressure and pulse pressure, suggesting an involvement of hypothalamic δ opioid receptors in decreasing sympathetic outflow. By contrast, morphine exerted pronounced bradycardic effects following fourth ventricular administration, suggesting an action at μ opioid receptors which influence vagal parasympathetic activity. Both ligands lowered respiratory rates upon fourth ventricular injection, indicating a possible involvement of either opioid receptor subtype in the depression of brainstem respiratory centers. These depressant effects of opioids upon cardiorespiratory function were readily reversed by naloxone. The qualitative similarity between the cardiovascular effects of third ventricular DADLE administration and various forms of circulatory shock may indicate that both phenomena involve delta opioid receptors at hypothalamic sites.     

Differential Regulation of D1 Dopamine Receptor- and of A2a Adenosine Receptor-Stimulated Adenylyl Cyclase by μ-, δ1-, and δ2-Opioid Agonists in Rat Caudate Putamen

Abstract: Inhibition and stimulation of adenylyl cyclase by opioid and D₁ dopamine or A_2a adenosine agonists, respectively, were characterized in the caudate putamen of rats. D₁ dopamine receptors have been reported to be localized preferentially on striatonigral neurons and A_2a adenosine receptors on striatopallidal neurons. The aim of the present study was to evaluate the effects of μ-[Tyr-d -Ala-Gly-(N-Me)Phe-Gly-ol (DAMGO)], δ₁-[Tyr-d -Pen-Gly-Phe-d -Pen (DPDPE)], and δ₂- ([d -Ala²]deltorphin-II [DT-II]) opioid agonists on the D₁ dopamine receptor- and A_2a adenosine receptor-stimulated adenylyl cyclase in membranes from rat caudate putamen. The results show that DAMGO, DPDPE, and DT-II inhibit forskolin-stimulated adenylyl cyclase [selectively antagonized by d -Phe-Cys-Tyr-d -Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; μ antagonist), 7-benzylidenenaltrexone (BNTX; δ₁ antagonist), and naltriben (NTB; δ₂ antagonist), respectively], but only μ- and δ₂-opioid agonists inhibit D₁ dopamine-stimulated adenylyl cyclase (antagonized by CTOP and NTB, respectively). Furthermore, DT-II and DPDPE inhibit A_2a adenosine-stimulated adenylyl cyclase (antagonized by NTB and BNTX, respectively), whereas DAMGO did not inhibit A_2a adenosine-stimulated adenylyl cyclase activity. These results suggest that μ-, δ₁-, and δ₂-opioid receptors display differential localization and provide neurochemical evidence suggesting the differential location of the δ₁ and δ₂ subtypes. μ-Opioid receptors may be preferentially expressed by striatonigral neurons, δ₁- by striatopallidal neurons, and δ₂- by these two striatal efferent neuron populations.     

An examination of the relationship between μ-opioid antinociceptive efficacy and G-protein coupling using pertussis and cholera toxins

The hypothesis that μ-opioid agonists having low antinociceptive efficacy might be more susceptible to interference with G-protein coupling than μ-opioid agonists having higher antinocicep-tive efficacy was tested. Supraspinal antinociceptive efficacy for the three μ-opioid agonists morphine, [D-Ala², NMePhe⁴, Gly⁵-ol]-enkephalin (DAMGO) and sufentanil in the mouse 55 °C warm-water tail-flick test was evaluated 18–24 h after intracerebroventricular (i.c.v.) administration of β-funaltrexamine (β-FNA). The β-FNA pretreatment (0.2–2.0 nmol) attenuated antinociception in the order morphine > DAMGO > sufentanil, consistent with previous reports of their relative antinociceptive efficacy. The association of efficacy with G-protein coupling was then assessed by determining sensitivity to i.c.v. (0.1–3.0 μg) pertussis toxin (PTX) or cholera toxin (CTX). The effect of PTX on equiantinociceptive doses was in the inverse order of agonist efficacy. CTX augmented sufentanil-induced antinociception. Morphine- and DAMGO-induced antinociception were unaffected by CTX. These data suggest that: (i) highly efficacious μ agonists (viz., sufentanil) couple more efficiently to PTX-sensitive inhibitory G_i-proteins than do agonists of lower efficacy (viz., morphine, DAMGO) and (ii) highly efficacious μ agonists have greater capacity to utilize CTX-sensitive stimulatory G_s-proteins than do μ-agonists with lower efficacy.