guanosine-5--o-(3-thiotriphosphate) and Opioid-Related-Disorders

Chronic exposure to opiates causes the development of tolerance and physical dependence as well as persistent brain neuroplasticity. Despite a wealth of postmortem human studies for opiate addicts, little direct information regarding the functional status of serotonergic and cholinergic receptor-mediated signaling pathways in the human brain of opiate addicts is yet available.. Concentration-response curves for 5-HT and carbachol in individual subjects were analyzed according to a nonlinear regression model, which generated the values of maximum percent increase (%E. Our data demonstrate that the signaling pathways mediated by Gα

Topics: Adult; Analgesics, Opioid; Brain; Dorsolateral Prefrontal Cortex; Female; GTP-Binding Protein alpha Subunits, Gq-G11; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Middle Aged; Neuronal Plasticity; Opiate Alkaloids; Opioid-Related Disorders; Receptor, Muscarinic M1; Receptor, Serotonin, 5-HT2A; Signal Transduction; Young Adult

To examine the relationship between the RAVE (relative activity versus endocytosis) values of opiate agonists and their dependence liability by studying several potent analgesics with special profiles in the development of physical and psychological dependence.. The effects of (-)-cis-(3R,4S,2'R) ohmefentanyl (F9202), (+)-cis-(3R,4S,2'S) ohmefentanyl (F9204), dihydroetorphine (DHE) and morphine on [(35)S]GTP gamma S binding, forskolin-stimulated cAMP accumulation, and receptor internalization were studied in CHO cells stably expressing HA-tagged mu-opioid receptors (CHO-HA-MOR). cAMP overshoot in response to the withdrawal of these compound treatments was also tested.. All four agonists exhibited the same rank order of activity in stimulation of [(35)S]GTP gamma S binding, inhibition of adenylyl cyclase (AC) and induction of receptor internalization: DHE>F9204>F9202>morphine. Based on these findings and the previous in vivo analgesic data obtained from our and other laboratories, the RAVE values of the four agonists were calculated. The rank order of RAVE values was morphine>F9202>F9204>DHE. For the induction of cAMP overshoot, the rank order was F9202>or=morphine>F9204>or=DHE.. Taken in combination with previous findings of these compounds' liability to develop dependence, the present study suggests that the agonist with the highest RAVE value seems to have a relatively greater liability to develop psychological dependence relative to the agonist with the lowest RAVE value. However, the RAVE values of these agonists are not correlated with their probability of developing physical dependence or inducing cAMP overshoot, a cellular hallmark of dependence.

Topics: Analgesics, Opioid; Animals; Cell Membrane; CHO Cells; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Endocytosis; Etorphine; Fentanyl; Guanosine 5'-O-(3-Thiotriphosphate); Morphine; Opioid-Related Disorders; Receptors, Opioid, mu

Analgesics such as morphine cause many side effects including addiction, but kappa-opioid receptor agonist can produce antinociception without morphine-like side effects. With the aim of developing new and potent analgesics with lower abuse potential, we studied the antinociceptive and physical dependent properties of a derivate of ICI-199441, an analogue of (-)U50,488H, named (2-(3,4-dichloro)-phenyl)-N-methyl-N-[(1S)-1-(2-isopropyl)-2-(1-(3-pyrrolinyl))ethyl] acetamides (LPK-26). LPK-26 showed a high affinity to kappa-opioid receptor with the Ki value of 0.64 nM and the low affinities to micro-opioid receptor and delta-opioid receptor with the Ki values of 1170 nM and >10,000 nM, respectively. It stimulated [(35)S]GTPgammaS binding to G-proteins with an EC50 value of 0.0094 nM. In vivo, LPK-26 was more potent than (-)U50,488H and morphine in analgesia, with the ED50 values of 0.049 mg/kg and 0.0084 mg/kg in hot plat and acetic acid writhing tests, respectively. Moreover, LPK-26 failed to induce physical dependence, but it could suppress naloxone-precipitated jumping in mice when given simultaneously with morphine. Taken together, our results show that LPK-26 is a novel selective kappa-opioid receptor agonist with highly potent antinociception effects and low physical dependence potential. It may be valuable for the development of analgesic and drug that can be used to reduce morphine-induced physical dependence.

Topics: Acetic Acid; Analgesics, Opioid; Animals; Behavior, Animal; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Hot Temperature; Humans; Male; Mice; Motor Activity; Naloxone; Narcotic Antagonists; Opioid-Related Disorders; Pain; Pain Measurement; Pain Threshold; Protein Binding; Pyrroles; Rats; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Time Factors; Transfection

Previous studies established that Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) and (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin) are fully efficacious mu-agonists. Herkinorin (HERK), unlike DAMGO, does not recruit beta-arrestin and promote mu-receptor internalization, even in cells that over express beta-arrestin. We hypothesized that chronic HERK and DAMGO treatment will differentially affect cellular markers of tolerance and dependence. CHO cells expressing the cloned human mu-receptor were treated for 20 h with 10 microM DAMGO, HERK, morphine, or medium. Both DAMGO and HERK acted as full agonists in the [(35)S]GTP-gamma-S binding assay with E(MAX) values of 230% and EC(50) values of 12.8 and 92.5 nM, respectively. In the cAMP assay, DAMGO and HERK had similar E(MAX) values of approximately 80% and EC(50) values of 3.23 and 48.7 nM, respectively. Chronic exposure to both drugs produced moderate tolerance to both drugs ( approximately 2 to 5 fold) in the [(35)S]GTP-gamma-S binding assay. In the cAMP assay, chronic DAMGO produced tolerance to both drugs ( approximately 3 to 4 fold). Chronic HERK eliminated the ability of either drug to inhibit forskolin-stimulated cAMP accumulation. Chronic DAMGO increased, and chronic HERK decreased, forskolin-stimulated cAMP accumulation. Naloxone, after chronic HERK (but not DAMGO) induced a large increase in forskolin-stimulated cAMP accumulation. Viewed collectively with published data, the current data indicate that both internalizing and noninternalizing mu-agonists produce cellular signs of tolerance and dependence.

Topics: Analgesics, Opioid; Animals; Binding, Competitive; Cell Membrane; CHO Cells; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Drug Tolerance; Endocytosis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Furans; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Narcotic Antagonists; Opioid-Related Disorders; Pyrones; Radioligand Assay; Receptors, Opioid, mu

The mu-opioid receptor displays basal signaling activity, which seems to be enhanced by exposure to opioid agonists. This study assesses the in vivo pharmacology of the putative "neutral" antagonist 6beta-naltrexol in comparison to other ligands with varying efficacy, such as naloxone, an inverse agonist in the opioid-dependent state. ICR mice were used to generate full antagonist dose-response curves for naloxone, naltrexone, nalbuphine, and 6beta-naltrexol in blocking acute antinociceptive effects of morphine and precipitating opioid withdrawal in models of physical dependence. 6beta-Naltrexol was roughly equipotent to naloxone and between 4.5- and 10-fold less potent than naltrexone in blocking morphine-induced antinociception and locomotor activity, showing that 6beta-naltrexol enters the central nervous system. In contrast to naloxone and naltrexone, 6beta-naltrexol precipitated only minimal withdrawal at high doses in an acute dependence model and was approximately 77- and 30-fold less potent than naltrexone and naloxone, respectively, in precipitating withdrawal in a chronic dependence model. 6beta-Naltrexol reduced the inverse agonist effects of naloxone in vitro and in vivo, as expected for a neutral antagonist. Therefore, the pharmacological effects of 6beta-naltrexol differ markedly from those of naloxone and naltrexone in the opioid-dependent state. A reduction of withdrawal effects associated with neutral mu-opioid receptor antagonists may offer advantages in treating opioid overdose and addiction.

Topics: Animals; Benzylidene Compounds; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred ICR; Morphine; Motor Activity; Naloxone; Naltrexone; Narcotic Antagonists; Opioid-Related Disorders

Opiates produce analgesia by activating mu opioid receptor-linked inhibitory G protein signaling cascades and related ion channel interactions that suppress cellular activities by hyperpolarization. After chronic opiate exposure, an excitatory effect emerges contributing to analgesic tolerance and opioid-induced hyperalgesia. Ultra-low-dose opioid antagonist co-treatment blocks the excitatory effects of opiates in vitro, as well as opioid analgesic tolerance and dependence, as was demonstrated here with ultra-low-dose naloxone combined with morphine. While the molecular mechanism for the excitatory effects of opiates is unclear, a switch in the G protein coupling profile of the mu opioid receptor and adenylyl cyclase activation by Gbetagamma have both been suggested. Using CNS regions from rats chronically treated with vehicle, morphine, morphine+ultra-low-dose naloxone or ultra-low-dose naloxone alone, we examined whether altered mu opioid receptor coupling to G proteins or adenylyl cyclase activation by Gbetagamma occurs after chronic opioid treatment. In morphine-naïve rats, mu opioid receptors coupled to Go in striatum and to both Gi and Go in periaqueductal gray and spinal cord. Although chronic morphine decreased Gi/o coupling by mu opioid receptors, a pronounced coupling to Gs emerged coincident with a Gbetagamma interaction with adenylyl cyclase types II and IV. Co-treatment with ultra-low-dose naloxone attenuated both the chronic morphine-induced Gs coupling and the Gbetagamma signaling to adenylyl cyclase, while increasing Gi/o coupling toward or beyond vehicle control levels. These findings provide a molecular mechanism underpinning opioid tolerance and dependence and their attenuation by ultra-low-dose opioid antagonists.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Blotting, Western; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Protein alpha Subunits; GTP-Binding Protein beta Subunits; GTP-Binding Protein gamma Subunits; Guanosine 5'-O-(3-Thiotriphosphate); Hot Temperature; Immunoprecipitation; Isoenzymes; Male; Naloxone; Narcotic Antagonists; Opioid-Related Disorders; Pain Threshold; Protein Binding; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, G-Protein-Coupled; Receptors, Opioid, mu; Signal Transduction; Substance Withdrawal Syndrome

Narcotic analgesics cause addiction by poorly understood mechanisms, involving mu opoid receptor (MOR). Previous cell culture studies have demonstrated significant basal, spontaneous MOR signaling activity, but its relevance to narcotic addiction remained unclear. In this study, we tested basal MOR-signaling activity in brain tissue from untreated and morphine-pretreated mice, in comparison to antagonist-induced withdrawal in morphine-dependent mice. Using guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding and adenylyl cyclase activity assay in brain homogenates, we demonstrated that morphine pretreatment of mice enhanced basal MOR signaling in mouse brain homogenates and, moreover, caused persistent changes in the effects of naloxone and naltrexone, antagonists that elicit severe withdrawal in dependent subjects. Naloxone and naltrexone suppressed basal [(35)S]GTP gamma S binding (acting as "inverse agonists") only after morphine pretreatment, but not in drug-naive animals. Moreover, naloxone and naltrexone stimulated adenylyl cyclase activity in striatum homogenates only after morphine pretreatment, by reversing the inhibitory effects of basal MOR activity. After cessation of morphine treatment, the time course of inverse naloxone effects on basal MOR signaling was similar to the time course of naltrexone-stimulated narcotic withdrawal over several days. The neutral antagonist 6 beta-naltrexol blocked MOR activation without affecting basal signaling (G protein coupling and adenylyl cyclase regulation) and also elicited substantially less severe withdrawal. These results demonstrate long-lasting regulation of basal MOR signaling as a potential factor in narcotic dependence.

Topics: Adenylyl Cyclases; Animals; Brain; Disease Models, Animal; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred ICR; Morphine; Motor Activity; Naltrexone; Narcotic Antagonists; Opioid-Related Disorders; Receptors, Opioid, mu; Signal Transduction; Substance Withdrawal Syndrome; Sulfur Radioisotopes

We used a previously reported model of morphine sensitization that elicited a complex behavioral syndrome involving stereotyped and non stereotyped activity. To identify the mechanism of these long-lasting processes, we checked the density of mu opioid receptors, receptor-G-protein coupling and the cyclic AMP (cAMP) cascade. In morphine-sensitized animals mu opioid receptor autoradiography revealed a significant increase in the caudate putamen (30% versus controls), nucleus accumbens shell (16%), prefrontal and frontal cortex (26%), medial thalamus (43%), hypothalamus (200%) and central gray (89%). Concerning morphine's activation of G proteins in the brain, investigated in the guanylyl 5'-[gamma-(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding assay, a significant increase in net [(35)S]GTPgammaS binding was seen in the caudate putamen (39%) and hypothalamus (27%). In the caudate putamen this was due to an increase in the amount of activated G proteins, and in the hypothalamus to a greater affinity of G proteins for guanosine triphosphate (GTP). The main second messenger system linked to the opioid receptor is the cAMP pathway. In the striatum basal cAMP levels were significantly elevated in sensitized animals (70% versus controls) and [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) significantly inhibited forskolin-stimulated cAMP production in control (30%) but not in sensitized rats. In the hypothalamus no significant changes were observed in basal cAMP levels and DAMGO inhibition. These cellular events induced by morphine pre-exposure could underlie the neuroadaptive processes involved in morphine sensitization.

Topics: Animals; Behavior, Animal; Binding, Competitive; Brain; Brain Chemistry; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Morphine; Neurons; Opioid-Related Disorders; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Signal Transduction

In cellular models, chronic exposure to mu-opioid agonists converts antagonists into inverse agonists at mu-receptors. Such adaptations could contribute to the development of tolerance and/or dependence. To determine whether delta-receptors respond similarly, or whether this adaptation is unique for mu-receptors, this study examined the effects of prolonged agonist exposure on the intrinsic activity of several delta-opioid ligands in GH(3) cells expressing delta-receptors. In opioid naive cells, delta-receptors were constitutively active, and a series of delta-ligands displayed a range of intrinsic activities for G protein activation. Chronic treatment with the full delta-agonist [D-Pen(2,5)]-enkephalin reduced the acute ability of [D-Pen(2,5)]-enkephalin to stimulate and the full inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174864) to inhibit G protein activation. In contrast, although naloxone and naltriben exhibited weak partial agonism in opioid naive cells, both ligands acted as full inverse agonists to produce concentration-dependent inhibition of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding after prolonged exposure to [D-Pen(2,5)]-enkephalin or to the partial agonist morphine. This effect was reversed by a neutral delta-antagonist (N,N-bisallyl)-Tyr-Gly-Gly-psi-(CH(2)S)-Phe-Leu-OH (ICI-154129). Finally, as is also characteristic of inverse agonists, naloxone and naltriben demonstrated higher affinities for uncoupled delta-receptors in cells chronically treated with [D-Pen(2,5)]-enkephalin, relative to opioid naive cells. Therefore, this relatively novel adaptation is shared by both mu- and delta-opioid receptors and therefore may serve as an important common mechanism involved the development of tolerance and/or dependence.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Cell Line; Cell Membrane; Drug Tolerance; Enkephalin, D-Penicillamine (2,5)-; Guanosine 5'-O-(3-Thiotriphosphate); Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Narcotics; Opioid-Related Disorders; Receptors, Opioid, delta; Receptors, Opioid, mu

Repeated opioid administration has been associated in human brain with unaltered density of mu-opioid receptors (agonist radioligand binding sites and immunodetected receptor protein). These receptors are coupled to Gi/Go-proteins, which are increased in brain of heroin addicts. To assess the activity of G-proteins and their coupling to receptors after chronic opioid abuse, [35S]GTPgammaS binding was quantified in postmortem prefrontal cortices of 15 opioid-dependent subjects and 15 matched controls. The stimulation of [35S]GTPgammaS binding by the mu-opioid receptor agonist DAMGO or the alpha2-adrenoceptor agonist UK14304 was used as a functional measure of the status of the receptor-G-protein coupling. [35S]GTPgammaS binding basal values were similar in opioid addicts (819+/-83 fmol mg-1 of protein) and controls (918+/-106 fmol mg(-1) of protein). In opioid addicts, [35S]GTPgammaS binding stimulation by DAMGO showed a maximal effect (62+/-8%) and a potency (EC50 = 1.09+/-0.26 microM) that did not differ from the maximal effect (60+/-12%) and potency (EC50 = 2.01+/-0.58 microM) in controls. In opioid addicts, [35S]GTPgammaS binding stimulation by UK14304 was not different in maximal effect (28+/-3%) from controls (32+/-8%), but the potency of the agonist was decreased (EC50 = 4.36+/-1.81 microM) when compared with controls (EC50 = 0.41+/-0.15 microM). The results provide a direct evidence of an apparent normal functional activity of brain mu-opioid receptors (Gi/Go-protein coupling) during the opioid dependence process in humans. The data also demonstrate a functional uncoupling of alpha2-adrenoceptors from G-proteins, which indicates a heterologous desensitization of these receptors. This finding could represent an adaptive mechanism against the decreased noradrenergic activity induced by the chronic presence of opioid drugs.

Topics: Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-Agonists; Adult; Autopsy; Brain; Brimonidine Tartrate; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Opioid-Related Disorders; Postmortem Changes; Quinoxalines; Receptors, Opioid, mu; Reference Values; Sulfur Radioisotopes