Sensitization of the adenylyl cyclase system in cloned kappa-opioid receptor-transfected cells following sustained agonist treatment: A chimeric study using G protein alpha(i)2/alpha(q) subunits.
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Chronic and/or sustained opioid treatment has been shown to result in development of sensitization of the adenylyl cyclase (AC) system or cAMP overshoot. In this study, we investigated the molecular mechanism responsible for sensitization of the AC system using CHO cells co-expressing cloned kappa-opioid receptor and some chimeric G protein alpha(i2)/alpha(q) subunits. In CHO cells co-expressing the kappa-opioid receptor and pertussis toxin-insensitive chimeric alpha(i2)/alpha(q) subunits with alpha(i2) residues Met244-Asn331, despite pretreatment with pertussis toxin, acute treatment with the kappa-opioid-receptor-selective agonist U69,593 suppressed forskolin-stimulated cAMP accumulation, while sustained treatment with U69,593 (4 h) induced cAMP overshoot over the naive level by the kappa-opioid-receptor-selective antagonist norbinaltorphimine (sensitization of the AC system). On the other hand, in CHO cells co-expressing the kappa-opioid receptor and pertussis toxin-insensitive chimeric alpha(i2)/alpha(q) subunits without alpha(i2) residues Met244-Asn331, pretreatment with pertussis toxin completely blocked these acute and sustained effects of U69,593 on cAMP accumulation. These results suggested that the presence of the specific region of alpha(i2) (Met244-Asn331), which was reported to be responsible for the inhibition of AC, and continuous inhibition of AC by alpha(i2) is necessary for the development of sensitization. (+info)
Protein kinase C-mediated acute tolerance to peripheral mu-opioid analgesia in the bradykinin-nociception test in mice.
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We studied the acute tolerance liability of peripheral opioid analgesia in mice. The analgesia was assessed by the inhibition of bradykinin (BK)-induced nociceptive action by using a newly developed flexor reflex paradigm. Morphine [intraplantarly (i.pl.)] given ipsilaterally to BK showed a dose-dependent reduction of the BK (2 pmol) responses, whereas the administration of 10 nmol of morphine into the contralateral side failed to show any significant analgesic effects. Furthermore, DAMGO ([D-Ala(2),MePhe(4), Gly-ol(5)]-enkephalin), a mu-opioid receptor (MOR) agonist, and U-69593, a kappa-opioid receptor (KOR) agonist, but not DSLET ([D-Ser(2)]Leu-enkephalin-Thr(6)), a delta-opioid receptor agonist, showed similar analgesia on the BK responses. The morphine- or U-69593 [(5alpha,7alpha, 8beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec -8yl] benzeneacetamide]-induced analgesia was markedly attenuated by the intrathecal injection of each antisense oligodeoxynucleotide for the MOR or KOR, respectively, suggesting that these peripheral analgesia are mediated through MORs and KORs located on nociceptor endings, respectively. As BK response was completely recovered to the control level 4 h after morphine (3 nmol i.pl.) or U-69593 (10 nmol i.pl.) administration, these compounds were challenged again to see the inhibition of BK responses. Although morphine analgesia by the second challenge was markedly attenuated, U-69593 analgesia was not. The attenuated morphine analgesia was completely reversed by the pretreatment of calphostin C, Go6976, or HBDDE, a protein kinase C inhibitor, but not by KT-5720, a protein kinase A inhibitor. These results suggest that selective acute tolerance of peripheral morphine analgesia, but not U-69593 analgesia, through MORs and KORs located on polymodal nociceptors, respectively, in the bradykinin-nociception test in mice was mediated through protein kinase C activation. (+info)
G(i1) and G(oA) differentially determine kinetic efficacies of agonists for kappa-opioid receptor.
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We examined the diversity of single receptor function by measuring receptor-G protein coupling in the baculovirus-Sf21 expression system. In comparative studies using Sf21 cell membranes expressing kappa-opioid receptor (KOR) plus Galpha(i1)beta(1)gamma(2) or KOR plus Galpha(oA)beta(1)gamma(2), there was no significant difference between both preparations in the K(i) values of various kappa-opioid ligands for the displacement of [(3)H]U69593 binding. However, a marked difference in the rank order of agonists to stimulate [(35)S]GTPgammaS binding was observed between both preparations. These findings suggest that agonist efficacy is dependent on the population of different G proteins expressed in various tissues. (+info)
Opioid modulation of recurrent excitation in the hippocampal dentate gyrus.
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kappa opioid receptor activation inhibits granule cell-mediated excitatory neurotransmission in the hippocampal formation via a decrease in glutamate release from both perforant path and mossy fiber terminals. We now report a third, anatomically and pharmacologically distinct site of such kappa opioid inhibition within the hippocampus. Granule cell population responses to selective stimulation of an excitatory hilar pathway were decreased by the kappa(1) opioid receptor agonist U69,593, an effect blocked by the kappa(1) antagonist norbinaltorphimine. U69,593 also inhibited hilar path induced long-term potentiation (LTP) of granule cell responses. LTP in this pathway was also blocked by the NMDA receptor antagonist d-2-amino-5-phosphonovalerate, unlike granule cell mossy fiber LTP in CA3. The kappa opioid peptide dynorphin is present in hilar mossy fiber collaterals. Ultrastructural analysis of these collaterals demonstrated dynorphin-containing vesicles in asymmetric synapses formed between axon terminals and granule cell dendrites, suggesting direct granule cell-granule cell connections. Evoked release of endogenous dynorphin within the hilus was effective in reducing hilar excitation of granule cells, although this release, in contrast to the release of dynorphin in the dentate molecular layer, was not dependent on L-type calcium channels. No hilar path excitation was observed in the absence of bicuculline, suggesting a strong GABA(A)-mediated inhibition of this pathway. However, hilar path activity could be seen after LTP, with or without bicuculline. Thus, kappa opioids can inhibit granule cell recurrent excitation, likely via effects on excitatory mossy fiber collaterals. Such collaterals are thought to be important in mediating temporal lobe epilepsy. (+info)
Inhibition by unsaturated fatty acids of type II secretory phospholipase A2 synthesis in guinea-pig alveolar macrophages evidence for the eicosanoid-independent pathway.
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The effect of arachidonic acid (C20:4) on the production of secretory type II phospholipase A2 (sPLA2-II) by guinea-pig alveolar macrophages was investigated. We show that incubation of these cells with 1-30 microM of arachidonic acid inhibits the synthesis of sPLA2-II in a concentration-dependent manner with an IC50 of approximately 7.5 microM. The inhibition by low concentrations (5 microM) of arachidonic acid was partially reduced by pretreatment of alveolar macrophages with cyclooxygenase or cytochrome P450 inhibitors (aspirin and 1-aminobenzotriazole, respectively), but not by lipoxygenase inhibitor, BW A4C. However, these inhibitors failed to interfere with the effect of high concentrations (30 microM) of arachidonic acid, suggesting that the latter may act on the expression of sPLA2-II, at least in part, independently of eicosanoid generation. Indeed, a similar inhibitory effect on sPLA2-II activity and mRNA expression was observed with other unsaturated fatty acids such as eicosapentaenoic (C20:5) and oleic (C18:1) acids, but not with the saturated fatty acid, palmitic acid (C16:0). In addition, arachidonic acid partially reduced the secretion of tumor necrosis factor alpha, an important intermediate in the induction of sPLA2-II synthesis by guinea-pig alveolar macrophages. However, addition of recombinant tumor necrosis factor alpha failed to reverse the inhibitory effect of arachidonic acid on sPLA2-II expression, suggesting that this process occurs downstream of tumor necrosis factor alpha secretion. We conclude that the expression of sPLA2-II in alveolar macrophages is down-regulated at the transcriptional level by arachidonic acid either directly or via its cyclooxygenase and cytochrome P450-derived metabolites. (+info)
kappa-Opioid inhibits catecholamine biosynthesis in PC12 rat pheochromocytoma cell.
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It was reported that nicotine-induced dopamine release in the rat pheochromocytoma cell line, PC12 cells, was inhibited by kappa-opioid. However, it is not known whether inhibition of catecholamine biosynthesis is involved in the inhibitory mechanisms of kappa-opioids in PC12 cells. U-69593 (a kappa-opioid agonist: >/=100 nM) significantly inhibited the nicotine-induced increase of tyrosine hydroxylase (TH, a rate-limiting enzyme in biosynthesis of catecholamine) enzyme activity and TH mRNA levels. These inhibitory effects were completely reversed by naloxone and nor-binaltorphimine dihydrochloride (nor-BNI), a specific kappa-antagonist, whereas pertussis toxin (PTX) only partially reversed this inhibitory effect. Also, U-69593 (>/=100 nM) significantly inhibited the nicotine-induced increase of cAMP production. This inhibitory effect was completely reversed by naloxone and nor-BNI, whilst only partially reversed by PTX. Moreover, U-69593 (>/=100 nM) significantly inhibited the nicotine-induced increase of both the TH protein level and intracellular catecholamine levels. These results indicate that the anti-cholinergic actions of kappa-opioid can be explained partially by its inhibition of both TH enzyme activity and TH synthesis, through suppression of the cAMP/protein kinase A pathway. It would also appear that the PTX-sensitive G-protein mediates the inhibitory effect of this pathway, at least in part. (+info)
Tyrosine phosphorylation of the kappa -opioid receptor regulates agonist efficacy.
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To explore the role of highly conserved tyrosine residues in the putative cytoplasmic domains of the seven-transmembrane G protein-coupled opioid receptors, we expressed the rat kappa-opioid receptor (KOR) in Xenopus oocytes and then activated the intrinsic insulin receptor tyrosine kinase. KOR activation by the agonist produced a strong increase in potassium current through coexpressed G protein-gated inwardly rectifying potassium channels (K(IR)3). Brief pretreatment with insulin caused a 60% potentiation of the KOR-activated response. The insulin-induced increase in kappa-opioid response was blocked by the tyrosine kinase inhibitor genistein. In contrast, insulin had no effect on the basal activity of K(IR)3, suggesting that KOR is the target of the tyrosine kinase cascade. Mutation of tyrosine residues to phenylalanines in either the first or second intracellular loop of KOR to produce KOR(Y87F) and KOR(Y157F) had no effect on either the potency or maximal effect of. However, neither KOR(Y87F)- nor KOR(Y157F)-mediated responses were potentiated by insulin treatment. Insulin pretreatment shifted the dose-response curve for activation of KOR by increasing the maximal response without changing the EC(50) value for. These results suggest that insulin increases the efficacy of KOR activation by phosphorylating two tyrosine residues in the first and second intracellular loops of the receptor. Thus, tyrosine phosphorylation may provide an important mechanism for modulation of G protein-coupled receptor signaling. (+info)
Kappa-opioid receptor activation modifies dopamine uptake in the nucleus accumbens and opposes the effects of cocaine.
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Coadministration of kappa-opioid receptor agonists (kappa-agonists) with cocaine prevents alterations in dialysate dopamine (DA) concentration in the nucleus accumbens (Acb) that occur during abstinence from repeated cocaine treatment. Quantitative microdialysis was used to determine the mechanism producing these effects. Rats were injected with cocaine (20 mg/kg, i.p.), or saline, and the selective kappa-agonist U-69593 (0.32 mg/kg, s.c.), or vehicle, once daily for 5 d. Extracellular DA concentration (DA(ext)) and extraction fraction (E(d)), an indirect measure of DA uptake, were determined 3 d later. Repeated cocaine treatment increased E(d), whereas repeated U-69593 treatment decreased E(d), relative to controls. Coadministration of both drugs yielded intermediate E(d) values not different from controls. In vitro DA uptake assays confirmed that repeated U-69593 treatment produces a dose-related, region-specific decrease in DA uptake and showed that acute U-69593 administration increases DA uptake in a nor-binaltorphimine reversible manner. Repeated U-69593 also led to a decrease in [(125)I]RTI-55 binding to the DA transporter (DAT), but did not decrease total DAT protein. These results demonstrate that kappa-opioid receptor activation modulates DA uptake in the Acb in a manner opposite to that of cocaine: repeated U-69593 administration decreases the basal rate of DA uptake, and acute U-69593 administration transiently increases DA uptake. kappa-agonist treatment also alters DAT function. The action of kappa-agonists on DA uptake or DAT binding, or both, may be the mechanism(s) mediating the previously reported "cocaine-antagonist" effect of kappa-opioid receptor agonists. (+info)