Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of agonists at mu and delta opioid receptors were compared by measuring membrane currents under voltage clamp from neurons of the rat nucleus locus coeruleus and guinea pig submucous plexus. In each tissue, the appropriate selective agonist (Tyr-D-Ala-Gly-MePhe-Gly-ol for mu receptors in locus coeruleus or Tyr-D-Pen-Gly-Phe-D-Pen for delta receptors in submucous plexus) increased the conductance of an inwardly rectifying potassium conductance and strongly hyperpolarized the membrane. The properties of the potassium conductance affected by the two opioids could not be distinguished. Experiments with intracellular application of guanosine 5'-[gamma-thio]triphosphate indicated that a guanine nucleotide-binding regulatory protein was involved in the coupling between opioid receptor and potassium channel, but there was no evidence for activation of either cAMP-dependent protein kinase or protein kinase C. It is noted that a number of vertebrate neurotransmitter receptors are coupled to potassium channels. The potassium conductance associated with these channels has properties similar to the conductance activated by mu and delta opioids; this family includes the following receptors: acetylcholine M2, norepinephrine alpha 2, dopamine D2, 5-hydroxytryptamine 5-HT1, adenosine A1, gamma-aminobutyric acid GABAB, and somatostatin. It is suggested that this conductance is a conserved neuronal effector coupled to one of the receptor types that mediates the effects of each of several major transmitters. The mu and delta opioid receptors appear to be unusual in that both utilize this same effector mechanism.
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PMID:Mu and delta receptors belong to a family of receptors that are coupled to potassium channels. 244 52

The mu-opioid receptor agonist stimulation of low-Km GTPase in rat striatal membranes was abolished by islet-activating protein (IAP) treatment, and recovered by Gi reconstitution. When the IAP-treated membranes were phosphorylated with a cAMP-dependent protein kinase, there was no such recovery by Gi. The agonist binding was not affected with respect to Kd, Bmax and sensitivity to guanine nucleotides in the phosphorylated membranes. These findings suggest that phosphorylation of mu-opioid receptors dissociates the agonist change in G-protein activity from the guanine nucleotide-sensitive agonist binding.
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PMID:Phosphorylation of mu-opioid receptors--a putative mechanism of selective uncoupling of receptor--Gi interaction, measured with low-Km GTPase and nucleotide-sensitive agonist binding. 254 27

The molecular basis of opioid receptor mechanisms was studied in reconstitution experiments using purified or membrane-bound opioid receptors and purified GTP-binding proteins (G-proteins). mu-Opioid receptor exclusively purified from rat brains was reconstituted with G-proteins in lipid vesicles. The mu-agonist stimulated the G-protein activity in both G1 or Go-reconstituted vesicles. The stoichiometry revealed that one molecule of mu-receptor is functionally coupled to plural numbers of Gi or Go molecules and that mu-receptor exists in at least two different subtypes, mu i and mu o, separately coupled to Gi and Go, respectively. In addition, when the mu-receptor was phosphorylated by cAMP-dependent protein kinase, the mu-agonist-stimulation of G-protein activity disappeared, while the guanine nucleotide-sensitivity of agonist binding was unchanged. These findings suggest that there are independent domains in the receptor which are related to functional coupling to G-protein and to the agonist-binding modulation by G-protein. kappa-Opioid receptor agonist inhibited the G-protein activity in guinea pig cerebellar membranes. Further experiments revealed that the kappa-opioid receptor is functionally coupled to an inhibition of phospholipase C activity via an inhibition of Gi-activity. Such a receptor-mediated inhibition of G-protein activity may be the first demonstration of a signal transduction mechanism. The delta-opioid receptor agonist showed no effect on G-protein activity in guinea pig striatal and rat cortical membranes, while it stimulated it in NG108-15 cells. In all these membranes, the delta-agonist binding was markedly reduced by GTP gamma S in the presence of MgCl2. These findings suggest that delta-receptors in the brain might be coupled to G-protein without signal transduction.
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PMID:[Molecular pharmacology of opioid receptor mechanisms]. 255 62

The duration of the calcium component of the action potential (APD) of dorsal root ganglion (DRG) neurons in mouse spinal cord-ganglion explants has been shown to be dually modulated via excitatory and inhibitory opioid receptors. In order to determine if opioid-induced APD prolongation is modulated by receptors that are positively coupled to the adenylate cyclase (AC)/cyclic AMP second messenger system, whole-cell recordings were made from mouse DRG neurons grown in dissociated cell cultures. Tests for opioid responsivity were carried out after intracellular dialysis of an inhibitor of cAMP-dependent protein kinase (PKI). In control recordings, both DADLE-induced APD prolongation as well as shortening were prevented by co-perfusion with the opioid antagonist, diprenorphine (10 nM). Intracellular dialysis of PKI in these neurons completely blocked opioid-induced APD prolongation but did not attenuate APD shortening generally elicited by higher opioid concentrations. Bath perfusion of 10 nM DADLE elicited APD prolongation in 59% of the DRG neurons (n = 34) tested with control solution in the recording pipette, whereas none showed APD prolongation when the pipette contained PKI (n = 18). In control tests with 1 microM DADLE, the APD was prolonged in 37% of the cells and shortened in 26% (n = 19); in contrast, a matched group of PKI-treated cells showed no APD prolongation, whereas 42% showed APD shortening (n = 26). The results support the hypothesis that opioid-induced APD prolongation in DRG neurons is mediated by opioid receptor subtypes that are positively coupled via Gs to AC/cAMP-dependent voltage-sensitive ionic conductances.
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PMID:Inhibitor of cyclic AMP-dependent protein kinase blocks opioid-induced prolongation of the action potential of mouse sensory ganglion neurons in dissociated cell cultures. 284 53

A mu opioid receptor and a G protein-activated K+ channel were coexpressed in Xenopus oocytes. Stimulation of the mu opioid receptor induced an inwardly rectifying current that was blocked by opioid receptor antagonist naloxone, indicating that the mu opioid receptor is functionally coupled to the K+ channel. The coupling is mediated by G proteins, since pertussis toxin treatment reduced the K+ current and injection of GTP gamma S (guanosine 5'-O-(thiotriphosphate)) enhanced it. Repeated stimulation of the mu receptor leads to desensitization, as the K+ current from the second stimulation was reduced to 70% of that from the first one. Both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) regulate this process, but in opposite direction. Activation of PKC by treatment of the oocyte with phorbol ester potentiated the desensitization of the mu receptor-induced current. However, incubation of the cell with a membrane-permeable cAMP analog, 8-chlorophenylthio-cAMP, completely abolished the desensitization. The cAMP effect appears to be mediated by PKA, since injection of a PKA catalytic subunit showed the same effect as cAMP incubation. These results suggest that PKA and PKC differentially regulate the mu opioid receptor coupling to the G protein-activated K+ channel.
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PMID:Differential regulation by cAMP-dependent protein kinase and protein kinase C of the mu opioid receptor coupling to a G protein-activated K+ channel. 813 98

We describe a mild and convenient labeling method for obtaining opioid radioligands which exhibit high specific activity together with a high affinity for the delta-opioid receptor. We chemically synthesized and tested the affinity of enkephalin- and deltorphin-like peptides that contain a phosphorylation site at their C-terminus. The peptide YdAGFLTPRRASLGC (peptide B), labeled to 700 Ci/mmol in the presence of cAMP-dependent protein kinase and [gamma-32P]ATP, bound to the receptor with high affinity (Kd = 3.62 +/- 0.29 nM). This peptide was also chemically coupled to bovine serum albumin and provided a multivalent opioid protein (B-BSA) with interesting properties: compared with peptide B, B-BSA was a better substrate for the kinase (100% 32P incorporation, sp act > or = 7000 Ci/mmol when labeled) and a better ligand for the receptor (Kd = 0.20 +/- 0.02 nM). The concept of peptide extension by a short phosphorylatable sequence should be more generally applicable to other small peptidic hormones or neurotransmitters and provide useful probes for biochemical studies and expression cloning of membrane receptors.
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PMID:32P-labeled opioid peptides with high affinity for the delta-opioid receptor. 829

Effects of a cAMP-dependent protein kinase and protein kinase C inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) and a cAMP- and cGMP-dependent protein kinase inhibitor, H-8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide), on the behavioral signs of naloxone (an opioid receptor antagonist)-precipitated withdrawal syndrome and effects of H-7 on the change of protein kinase C activity in the pons/medulla region induced by morphine (a mu-opioid receptor agonist) or butorphanol (a mu/delta/kappa mixed opioid receptor agonist) were investigated in this study. Rats were intracerebroventricularly (i.c.v.) infused with morphine (26 nmol/microliters/h) or butorphanol (26 nmol/microliters/h) through osmotic minipumps for 3 days. In some groups, either saline or drug-treated groups were concomitantly infused with H-7 (1 and 10 nmol/microliters/h) or H-8 (10 nmol/microliters/h). The expression of physical dependence produced by morphine or butorphanol, as evaluated by naloxone (5 mg/kg i.p.)-precipitated withdrawal signs, was reduced by concomitant infusion of H-7 or H-8. In the same condition, morphine and butorphanol chronic treatment enhanced (28.1% and 26.3% enhancement over the saline-treated group, respectively) cytosolic protein kinase C activity in the pons/medulla, but not in the membrane fraction. Furthermore, concomitant infusion of H-7 inhibited the enhancement of protein kinase C activity. These results indicate that various types of protein kinases may play an important role in the development and/or expression of physical dependence on opioids. Among them, the enhancement of cytosolic protein kinase C activity in the pons/medulla region seems to be one of the major underlying mechanisms in opioid physical dependence.
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PMID:Possible involvement of protein kinases in physical dependence on opioids: studies using protein kinase inhibitors, H-7 and H-8. 854 12

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.
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PMID:Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid. 924 1

To investigate the role of glutamate in the locus coeruleus (LC) during opioid withdrawal, rats were continuously infused with morphine (a mu-opioid receptor agonist, 26 nmol/microl/h) or butorphanol (a mu/delta/kappa-mixed opioid receptor agonist, 26 nmol/microl/h) intracerebroventricularly (i.c.v.) via osmotic minipumps for 3 days. A direct LC injection of glutamate (1 or 10 nmol/5 microl) or naloxone (an opioid receptor antagonist, 24 nmol/5 microl) induced withdrawal signs in morphine- or butorphanol-dependent animals. However, these agents failed to precipitate any withdrawal signs in saline-treated control animals. On the other hand, the expression of withdrawal signs precipitated by the administration of glutamate or naloxone in opioid-dependent animals was completely blocked by concomitant infusion with 1 or 10 nmol/microl/h of an inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase and protein kinase C, H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine]. In animals that had been infused with opioids in the same manner, i.c.v. injection of naloxone (48 nmol/5 microl) precipitated withdrawal signs and increased extracellular fluid levels of glutamate in the LC of morphine- or butorphanol-dependent rats measured by in vivo microdialysis method. However, concomitant infusion with H-7 inhibited the increases of glutamate levels in the LC. These results strongly suggest that an expeditious release of glutamate in the LC region plays an important role in the expression of physical dependence on opioids. Furthermore, the action on glutamate release might be increased by the enhancement of cAMP-dependent protein kinase and/or protein kinase C activity.
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PMID:The role of glutamate in the locus coeruleus during opioid withdrawal and effects of H-7, a protein kinase inhibitor, on the action of glutamate in rats. 957 May 13

Reversible phosphorylation events can alter many critical cellular functions and has been hypothesized to play a role in the development of tolerance to chronically administered agonists. The technique described here was developed to assess the phosphorylation state of mu-opioid receptor protein isolated from rodent brain tissue following 4 days of continual, subcutaneously released morphine. We have adapted and combined two techniques: back-phosphorylation reactions, which make use of tissue from animal models, and phosphorylation followed by immunoprecipitation, an approach used in cell culture models. mu-receptor protein is precipitated using a receptor-specific antibody, and the protein phosphorylation state is preserved by the use of phosphatase inhibitors. Phosphorylation sites that are dephosphorylated on the isolated protein are then radiolabelled by the addition of purified cAMP-dependent protein kinase (PKA) catalytic subunit and [32P]ATP. The samples are separated by gel electrophoresis, and the resulting bands are quantified using a phosphoimager. The amount of 32P incorporated into the protein is assumed to be inversely related to the degree of phosphorylation prior to the reaction (i.e., the in vivo state of the protein). These estimates of micro-opioid receptor phosphorylation can then be correlated with a behavioral endpoint such as antinociception, to better understand the role of phosphorylation events in tolerance development.
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PMID:A method for the determination of activated receptor phosphorylation state following in vivo drug treatment. 1059 53


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