Agonist-dependent desensitization of the kappa opioid receptor by G protein receptor kinase and beta-arrestin.

We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.     

Serine phosphorylation of insulin receptor substrate 1 by inhibitor kappa B kinase complex

Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor kappaB kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser(307) (equivalent to Ser(312) in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-alpha or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser(312) in response to tumor necrosis factor-alpha was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J(2) as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser(312). Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser(312) (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways.     

Antagonists of the kappa opioid receptor

The research community has increasingly focused on the development of OPRK antagonists as pharmacotherapies for the treatment of depression, anxiety, addictive disorders and other psychiatric conditions produced or exacerbated by stress. Short-acting OPRK antagonists have been recently developed as a potential improvement over long-acting prototypic ligands including nor-BNI and JDTic. Remarkably the short-acting LY2456302 is undergoing phase II clinical trials for the augmentation of the antidepressant therapy in treatment-resistant depression. This Letter reviews relevant chemical and pharmacological advances in the identification and development of OPRK antagonists.     

Long-acting kappa opioid antagonists disrupt receptor signaling and produce noncompetitive effects by activating c-Jun N-terminal kinase

Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective kappa opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14-21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [(3)H]U69,593 to KOR showed that K(d) and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild type mice but not in KOR knock-out mice. Pretreatment of mice with the JNK inhibitor SP600125 before norBNI attenuated the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK.     

Induction of pain facilitation by sustained opioid exposure: relationship to opioid antinociceptive tolerance

Opioid analgesics are frequently used for the long-term management of chronic pain states, including cancer pain. The prolonged use of opioids is associated with a requirement for increasing doses to manage pain at a consistent level, reflecting the phenomenon of analgesic tolerance. It is now becoming clearer that patients receiving long-term opioid therapy can develop unexpected abnormal pain. Such paradoxical opioid-induced pain, as well as tolerance to the antinociceptive actions of opioids, has been reliably measured in animals during the period of continuous opioid delivery. Several recent studies have demonstrated that such pain may be secondary to neuroplastic changes that result, in part, from an activation of descending pain facilitation mechanisms arising from the rostral ventromedial medulla (RVM). One mechanism which may mediate such pain facilitation is through the increased activity of CCK in the RVM. Secondary consequences from descending facilitation may be produced. For example, opioid-induced upregulation of spinal dynorphin levels seem to depend on intact descending pathways from the RVM reflecting spinal neuroplasticity secondary to changes at supraspinal levels. Increased expression of spinal dynorphin reflects a trophic action of sustained opioid exposure which promotes an increased pain state. Spinal dynorphin may promote pain, in part, by enhancing the evoked release of excitatory transmitters from primary afferents. In this regard, opioids also produce trophic actions by increasing CGRP expression in the dorsal root ganglia. Increased pain elicited by opioids is a critical factor in the behavioral manifestation of opioid tolerance as manipulations which block abnormal pain also block antinociceptive tolerance. Manipulations that have blocked enhanced pain and antinociceptive tolerance include reversible and permanent ablation of descending facilitation from the RVM. Thus, opioids elicit systems-level adaptations resulting in pain due to descending facilitation, upregulation of spinal dynorphin and enhanced release of excitatory transmitters from primary afferents. Adaptive changes produced by sustained opioid exposure including trophic effects to enhance pain transmitters suggest the need for careful evaluation of the consequences of long-term opioid administration to patients.     

Semisynthetic neoclerodanes as kappa opioid receptor probes

Modification of the furan ring of salvinorin A (1), the main active component of Salvia divinorum, has resulted in novel neoclerodane diterpenes with opioid receptor affinity and activity. Conversion of the furan ring to an aldehyde at the C-12 position (5) has allowed for the synthesis of analogues with new carbon-carbon bonds at that position. Previous methods for forming these bonds, such as Grignard and Stille conditions, have met with limited success. We report a palladium catalyzed Liebeskind-Srogl cross-coupling reaction of a thioester and a boronic acid that occurs at neutral pH and ambient temperature to produce ketone analogs at C-12. To the best of our knowledge, this is the first reported usage of the Liebeskind-Srogl reaction to diversify a natural product scaffold. We also describe a one-step protocol for the conversion of 1 to 12-epi-1 (3) through microwave irradiation. Previously, this synthetically challenging process has required multiple steps. Additionally, we report in this study that alkene 9 and aromatic analogues 12, 19, 23, 25, and 26 were discovered to retain affinity and selectivity at kappa opioid receptors (KOP). Finally, we report that the furan-2-yl analog of 1 (31) has similar affinity to 1. Collectively, these findings suggest that different aromatic groups appended directly to the decalin core may be well tolerated by KOP receptors, and may generate further ligands with affinity and activity at KOP receptors.     

TENA, a selective kappa opioid receptor antagonist

A number of opioid antagonists (TENA, naloxone, Mr 2266, WIN 44441) were evaluated for their selectivity in antagonizing the effect of mu, kappa, and delta agonists in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Among these four antagonists, TENA was the most potent and the only ligand which was selective for kappa receptors. In this regard TENA was approximately 27-times more effective in antagonizing the kappa agonist, U-50488H, relative to the mu agonist, morphine, and it was about 5-times more effective against ethylketazocine (EK) relative to morphine. At the same concentration (20 nM) TENA did not significantly antagonize the delta agonist, [D-Ala2,D-Ala5]enkephalin (DADLE), in the MVD. Also, TENA was more effective than naloxone, EK, or U-50488H in protecting kappa receptors from irreversible blockage by beta-CNA. The results of this study indicate that TENA is the most selective kappa antagonist yet reported.     

Amino acid conjugates as kappa opioid receptor agonists

A novel series of kappa (kappa) opioid receptor agonists were synthesized by incorporating the key structural features of known kappa opioid agonists while replacing the aryl acetamide portion with substituted amino acid conjugates. Compounds 3j (Ki = 6.7 nM), 3k (Ki = 3.6 nM), 3l (Ki = 4.6 nM), 3m (Ki = 0.83 nM) and 3o (Ki = 2 nM) possessed potent affinities for the kappa opioid receptor in vitro with reasonable selectivity over other opioid receptors.     

Arylacetamides as peripherally restricted kappa opioid receptor agonists

Analogues of the kappa (kappa) opioid receptor agonist, ICI 199441, were prepared. Ki values for these analogues at the cloned human kappa opioid receptor ranged from 0.058 to 25 nM. Trifluoromethylaryl derivatives were potent analgesics when administered subcutaneously in the rat and were more peripherally restricted than the parent compound, ICI 199441.     

Protection from cerebral ischemia by U-50,488E, a specific kappa opioid analgesic agent

U-50,488E is a novel analgesic agent with a specific agonist property on the kappa opioid receptor. It is found to protect against the lethal effect of temporary bilateral carotid occlusion (BCO) in Mongolian gerbils and rats of the Fischer strain. Pretreatment with U-50,488E in gerbils before 7 min of BCO reduced the development of behavioral hyperactivity and preserved the hippocampal neurons from ischemic death. This protective effect of U-50,488E resided predominantly in the levo-enantiomer which is also more potent as a kappa analgesic. Two other kappa opioid analgesics, ethylketocyclazocine and bremazocine, shared the effects of U-50,488E in the gerbils. Naloxone and dynorphin 1-13, on the other hand, were without protective effects in the same ischemic model. The ischemic protective effects of U-50,488E may involve the kappa opioid receptor.     

Regulation of opioid receptor trafficking and morphine tolerance by receptor oligomerization

The utility of morphine for the treatment of chronic pain is hindered by the development of tolerance to the analgesic effects of the drug. Morphine is unique among opiates in its ability to activate the mu opioid receptor (MOR) without promoting its desensitization and endocytosis. Here we demonstrate that [D-Ala(2)-MePhe(4)-Gly(5)-ol] enkephalin (DAMGO) can facilitate the ability of morphine to stimulate MOR endocytosis. As a consequence, rats treated chronically with both drugs show reduced analgesic tolerance compared to rats treated with morphine alone. These results demonstrate that endocytosis of the MOR can reduce the development of tolerance, and hence suggest an approach for the development of opiate analogs with enhanced efficacy for the treatment of chronic pain.     

Antagonism of kappa opioid mediated effects in the rat by cyclo(Leu-Gly)

The effect of cyclo(Leu-Gly) on U-50,488H- induced pharmacological actions was determined in male Sprague-Dawley rats. Intraperitoneal (i.p.) administration of U-50,488H to rats produced analgesia (tail-flick) and increased urinary output. Cyclo (Leu-Gly) (1-4 mg/kg, s.c.) antagonized the analgesic response to U-50,488H (25 mg/kg; i.p.). A dose of 10 mg/kg (i.p.) of U-50,488H increased the spontaneous urinary output which was antagonized by cyclo (Leu-Gly) (1-4 mg/kg; s.c.). To determine whether cyclo (Leu-Gly) was acting as a kappa-opioid receptor antagonist, the effect of cyclo (Leu-Gly) on the binding of [3H]ethylketocyclazocine (EKC) to membranes of rat cerebral cortex and spinal cord was determined. The IC50 values of cyclo(Leu-Gly) in displacing [3H]EKC from its binding sites in cortex and spinal cord were 1.44 and 0.40 mM, respectively. Chronic administration of U-50,488H (25 mg/kg; i.p., b.i.d.) for 4 days induced tolerance to its analgesic effect. The latter was not affected by cyclo(Leu-Gly) (2 to 8 mg/kg; s.c.) given once a day for 4 days. It is concluded that cyclo(Leu-Gly) antagonizes acute actions of U-50,488H and that such effects of cyclo(Leu-Gly) are not mediated via a direct action on kappa-opioid receptors.     

Dynorphin peptides differentially regulate the human kappa opioid receptor

Dynorphins, endogenous peptides for the kappa opioid receptor, play important roles in many physiological and pathological functions. Here, we examined how prolonged treatment with three major prodynorphin peptides, dynorphin A (1-17) (Dyn A), dynorphin B (1-13) (Dyn B) and alpha-neoendorphin (alpha-Neo), regulated the human kappa opioid receptor (hKOR) stably expressed in Chinese hamster ovary (CHO) cells. Results from receptor binding and [(35)S]GTPgammaS binding assays showed that these peptides were potent full agonists of the hKOR with comparable receptor reserve and intrinsic efficacy to stimulate G proteins. A 4-h incubation with alpha-Neo at a concentration of approximately 600xEC(50) value (from [(35)S]GTPgammaS binding) resulted in receptor down-regulation to a much lower extent than the incubation with Dyn A and Dyn B at comparable concentrations ( approximately 10% vs. approximately 65%). Extending incubation period and increasing concentrations did not significantly affect the difference. The plateau level of alpha-Neo-mediated receptor internalization (30 min) was significantly less than those of Dyn A and Dyn B. Omission of the serum from the incubation medium or addition of peptidase inhibitors into the serum-containing medium enhanced alpha-Neo-, but not Dyn A- or Dyn B-, mediated receptor down-regulation and internalization; however, the degrees of alpha-Neo-induced adaptations were still significantly less than those of Dyn A and Dyn B. Thus, these endogenous peptides differentially regulate KOR after activating the receptor with similar receptor occupancy and intrinsic efficacy. Both stability in the presence of serum and intrinsic capacity to promote receptor adaptation play roles in the observed discrepancy among the dynorphin peptides.     

Novel malonamide derivatives as potent kappa opioid receptor agonists

A novel series of malonamide derivatives was synthesized. These amides were shown to be potent and selective kappa opioid receptor agonists.     

Tyrosine kinase mediated phosphorylation of the hexamerin receptor in the rice moth Corcyra cephalonica by ecdysteroids

Hexamerins are multifunctional insect storage proteins utilized during metamorphosis of holometabolous insects. These proteins are stage specifically taken up by the fat body cells from the haemolymph due to receptor-mediated endocytosis. The hexamerin receptor and the concomitant hexamerin sequestration in the rice moth Corcyra cephalonica is controlled by the steroid hormone 20-hydroxy-ecdysone (20E). However, the mechanism of receptor activation for hexamerin uptake is not yet clear. We report here that 20E stimulates the phosphorylation of 120 kDa hexamerin binding protein which has been demonstrated to represent the receptor. Phosphorylation of the receptor is suggested to be essential for receptor activation and occurs prior to the hexamerin uptake. The 20E stimulated phosphorylation is mediated partly by a tyrosine kinase as phosphotyrosine antibodies cross-react with the receptor and its phosphorylation is blocked partly by genistein. Back phosphorylation study provides additional evidence for 20E regulation of hexamerin receptor phosphorylation in intact fat body. The receptor phosphorylation is developmentally regulated. This is the first report demonstrating that (i) the uptake of hexamerin is dependent on the phosphorylation of hexamerin receptor and (ii) the phosphorylation is catalyzed partly by a tyrosine kinase which is activated by 20E through a non-genomic action.     

Regulation of insulin receptor kinase by multisite phosphorylation

The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse-phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta-catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMDA受体拮抗剂对阿片类药物耐受和依赖的阻断作用

本文综述阻断ＮＭＤＡ受体离子通道复合药物对阿惩耐受和成瘾发生的影响。行为药理学研究显示,非竞争性ＮＭＤＡ受体拮抗剂、竞争性ＮＭＤＡ受体拮抗剂和甘氨酸受占拮抗剂能抑制阿片耐受和戒断反应,其药理学特性明显不同于其他类型抗阿片耐受和成瘾的药物,阐述了ＮＭＤＡ受体拮抗剂治疗阿片类芗耐受和领事的系列化机制。并指出ＮＭＤＡ受体拮抗剂具有神经毒性。     

Mechanisms of mu opioid receptor/G-protein desensitization in brain by chronic heroin administration

Previous studies have shown that chronic opiate treatment decreases mu opioid-stimulated [35S]GTPgammaS binding in specific brain regions. To extend these findings, the present study investigated DAMGO-stimulated [35S]GTPgammaS binding in membrane homogenates and coronal sections from rats non-contingently administered heroin. Rats were administered saline or increasing doses of heroin i.v. hourly up to 288 mg/kg/day over 40 days. In brain sections, chronic heroin administration decreased DAMGO-stimulated [35S]GTPgammaS binding in medial thalamus and amygdala, with no effect in cingulate cortex or nucleus accumbens. Chronic heroin administration also reduced [35S]GTPgammaS binding stimulated by the principal metabolite of heroin, 6-monoacetylmorphine. In contrast, no significant changes in mu opioid receptor binding were observed in amygdala or thalamus using [3H]DAMGO autoradiography. In membranes from amygdala and thalamus, chronic heroin treatment decreased the maximal effect of DAMGO in stimulating [35S]GTPgammaS binding, with no effect on DAMGO potency. GTPgammaS saturation analysis showed that chronic heroin treatment decreased the Bmax, and increased the K(D), of DAMGO-stimulated [35S]GTPgammaS binding. These data suggest potential mechanisms by which chronic agonist treatment produces opioid receptor/G-protein desensitization in brain.     

Involvement of dynorphin and the kappa opioid receptor in feeding

Dynorphin-(1–17) produces a highly specific increase in food ingestion. Similar enhancement of food ingestion is found with dynorphin fragments (1–10), (1–11), (1–13) and (3–13) but not with (1–8) and (1–9). Dynorphin B (rimorphin) also enhances food intake. The highly specific kappa agonist U-50,488 also enhances food intake as do a number of other kappa-opiate receptor agonists. These studies provided further support for the role of a highly specific dynorphin-kappa opioid receptor in the modulation of feeding.     

Morphine induces terminal micro-opioid receptor desensitization by sustained phosphorylation of serine-375

Morphine is a poor inducer of micro-opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxy-terminal residue 375 (Ser(375)). Ser(375) phosphorylation was sufficient and required for morphine-induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO-desensitized receptors were rapidly dephosphorylated. In contrast, morphine-desensitized receptors remained at the plasma membrane in a Ser(375)-phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser(375).