Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

1. Receptor phosphorylation and down-regulation by protein kinases may be a key event initiating desensitization. The present studies were designed to investigate the effect of a potent protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), on antinociception induced by intrathecal (i.t.) administration of a selective delta-opioid receptor agonist [D-Ala2] deltorphin II in the male ICR mouse and on the specific binding of [3H]-[D-Ser2, Leu5]enkephalin-Thr6 (DSLET), a delta-opioid receptor ligand, in the crude synaptic membrane of the spinal cord. 2. Intrathecal (i.t.) pretreatment with PDBu at low doses, which injected alone did not affect the basal tail-flick latency, dose-dependently attenuated the antinociception induced by i.t. administration of [D-Ala2]deltorphin II. The attenuation of i.t.-administered [D-Ala2] deltorphin II-induced antinociception by PDBu was reversed in a dose-dependent manner by i.t. concomitant pretreatment with a specific PKC inhibitor, calphostin C. 3. In the binding experiment, incubation of the crude synaptic membrane of the spinal cord for 2 h at 25 degrees C with PDBu (0.03 to 10 microM) caused a dose-dependent inhibition of the [3H]-DSLET binding. Scatchard analysis of [3H]-DSLET binding revealed that PDBu at 10 microM displayed a 30.7% reduction in the number of [3H]-DSLET binding sites with no significant change in affinity, compared with the non-treatment control, indicating that the activation of membrane-bound PKC by PDBu causes a decrease in the number of specific delta-opioid agonist binding sites. 4. An i.t. injection of [D-Ala2]deltorphin II produced an acute antinociceptive tolerance to the antinociceptive effect of a subsequent i.t. challenge of [D-Ala2]deltorphin II. Concomitant pretreatment with calphostin C markedly prevented the development of acute tolerance to the i.t.-administered [D-Ala2]deltorphin II-induced antinociception. On the other hand, a highly selective protein kinase A (PKA) inhibitor, KT5720, did not have any effect on the development of acute tolerance to [D-Ala2]deltorphin II antinociception. 5. These findings suggest that a loss of specific delta-agonist binding by the activation of PKC by PDBu is involved in the PDBu-induced antinociceptive unresponsiveness to delta-opioid receptor agonist in the mouse spinal cord. Based on the acute tolerance studies, we propose that PKC, but not PKA, plays an important role in the process of homologous desensitization of the spinal delta-opioid receptor-mediated antinociception.
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PMID:Role of protein kinase C in desensitization of spinal delta-opioid-mediated antinociception in the mouse. 884 50

The effects of activation of the adenylyl cyclase-protein kinase A pathway on the expression of delta-opioid receptor mRNA in the NG108-15 neuroblastoma x glioma cell line has been investigated. Activation of prostaglandin E1 (PGE1) receptors, which are positively coupled to adenylyl cyclase, resulted in a reduction in delta-receptor messenger RNA levels. Direct stimulation of adenylyl cyclase by forskolin or treatment of cells with the cyclic AMP analogue dibutyryl cyclic AMP (db-cAMP) mimicked the effect of PGE1. Down-regulation in receptor protein levels, as measured by loss of radioligand binding sites, was also observed and its extent correlated well with the decrease in the amount of delta-opioid receptor transcripts. D-Ser2-Leu-enkephalin-Thr6 (DSLET) inhibition of adenylyl cyclase activity was also diminished after db-cAMP treatment. Inhibitors of protein kinase A (PKA) partially reversed the PGE1- and db-cAMP-mediated repression of the delta-opioid receptor mRNA levels. The rate of degradation of delta-opioid receptor mRNA in the presence of actinomycin D was not altered in response to db-cAMP, suggesting that mRNA stability is not reduced by PKA action. The regulation of delta-opioid receptor mRNA levels by db-cAMP was not sensitive to the protein synthesis inhibitor cycloheximide, suggesting that de novo protein synthesis is not required in this process.
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PMID:Regulation of delta-opioid receptor mRNA levels by receptor-mediated and direct activation of the adenylyl cyclase-protein kinase A pathway. 900 47

The objective of this study was to characterize the signaling mechanisms of the mu-opioid receptor in its coupling to the cystic fibrosis transmembrane conductance regulator (CFTR) when coexpressed in Xenopus oocytes. Because oocytes do not contain endogenous cAMP-regulated ion channels, the cAMP-modulated CFTR was coexpressed with receptors as a 'reporter' channel. Agonist treatment of oocytes coexpressing mu-opioid receptors, beta2-adrenergic receptors and CFTR produced Cl- currents in a dose-related manner and immunocytochemical analysis confirmed receptor expression. These data suggest that opioid agonists could activate adenylyl cyclase in this system to elevate cAMP levels. Heterotrimeric G protein betagamma-subunits acting on adenylyl cyclase type II would increase cAMP levels. The probable presence of adenylyl cyclase type II and other components of opioid signal transduction such as G(i alpha2), were demonstrated by RT-PCR. However, measurement of cAMP levels in individual oocytes by radioimmunoassay showed that opioid agonist application to oocytes expressing mu-opioid receptors, beta2-adrenergic receptors and CFTR did not increase cAMP levels, whereas application of the beta2-adrenergic agonist, isoproterenol, or IBMX alone did increase cAMP levels. Opioid-induced CFTR activation was not affected by either application of the broad spectrum kinase inhibitor, H7, nor by application of the specific PKA inhibitor, KT5720. Injection of free betagamma-subunits, which could activate the endogenous type II cyclase, was unable to produce measurable currents in oocytes expressing the CFTR. These studies indicate that opioid activation of the CFTR is not mediated through a cAMP/PKA pathway, by either betagamma-subunit activation of an adenylyl cyclase type II or promiscuous coupling to G(s alpha).
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PMID:mu-opioid receptor regulates CFTR coexpressed in Xenopus oocytes in a cAMP independent manner. 903 Jun 98

17beta-Estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic (beta-endorphin) neurons. In the current study, we used intracellular recordings from guinea pig hypothalamic slices to characterize the receptor and intracellular effector system mediating the rapid effects of E2. E2 acted stereospecifically with physiologically relevant concentration dependence (EC50 = 8 nM) to cause a 4-fold reduction in the potency of a mu-opioid agonist to activate an inwardly rectifying K+ conductance. Using Schild analysis to estimate the affinity of the mu-opioid receptor for an antagonist (naloxone), we found that estrogen did not compete for the mu-opioid receptor or alter the affinity of the mu receptor. Both the nonsteroidal estrogen diethylstilbestrol and the "pure" antiestrogen ICI 164,384 blocked the actions of E2, the latter with a subnanomolar affinity. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a nongenomic mechanism of action by E2. The actions of E2 were mimicked by the protein kinase A (PKA) activators forskolin and cAMP, Sp-isomer triethylammonium salt. Furthermore, the selective PKA antagonists cAMP, Rp-isomer triethylammonium salt and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. Thus, estrogen binds to a specific receptor that activates PKA to rapidly uncouple the mu-opioid receptor from its K+ channel. Because we have previously shown that gamma-aminobutyric acidB receptors are also uncoupled by estrogen, this mechanism of action has the potential to alter synaptic transmission via G protein-coupled receptors throughout the brain.
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PMID:Modulation of G protein-coupled receptors by an estrogen receptor that activates protein kinase A. 910 25

Opioid peptide receptor (OPR) agonists are co-released with the beta-adrenergic receptor (beta-AR) agonist norepinephrine (NE) from nerve terminals in the heart during sympathetic stimulation. Whereas recent studies indicate that OPR and beta-AR coexist on the surface of cardiac myocytes, whether significant "cross talk" occurs between OPR and beta-AR signaling cascades within heart cells is unknown. In the present study we demonstrate a marked effect of delta-OPR stimulation to modulate beta-adrenergic responses in single isolated rat ventricular myocytes. Nanomolar concentrations (10(-8) M) of the OPR agonist leucine enkephalin (LE), a naturally occurring delta-opioid peptide, inhibited NE-induced increases in sarcolemmal L-type Ca2+ current, cytosolic Ca2+ transient, and contraction. The antiadrenergic effect of LE was pertussis toxin sensitive and abolished by naloxone, an opioid receptor antagonist. In contrast, LE was unable to inhibit the positive inotropic effects induced by equipotent concentrations of 8-(4 chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, a cell-permeant adenosine 3',5'-cyclic monophosphate analog, or by the non-receptor-induced increase in contraction by elevated bathing Ca2+ concentration. These results indicate that an interaction of the OPR and beta-AR systems occurs proximal to activation of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase of the beta-AR intracellular signaling pathway. This modulation of beta-adrenergic effects by OPR activation at the myocyte level may have important implications in the regulation of cardiac Ca2+ metabolism and contractility, particularly during the myocardial response to stress.
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PMID:Opioid peptide receptor stimulation reverses beta-adrenergic effects in rat heart cells. 912 41

We have used a sensitive solution hybridization assay with a riboprobe transcribed from the coding sequence of the delta-opioid receptor gene (DOR) to study the up-regulation of the DOR mRNA by ethanol in NG108-15 cells. Exposure of the cells to compounds that increase cAMP levels (forskolin, forskolin + IBMX, or dibutyryl cAMP) resulted in the attenuation of ethanol-induced up-regulation of DOR mRNA. The inactive analogue of forskolin, 1,9-dideoxy forskolin had no effect. Northern blot analysis of RNA extracts from ethanol-, forskolin- or ethanol + forskolin-treated cells showed proportional changes in each of the multiple DOR mRNA bands, so that no difference was observed in the fraction of the total hybridization signal produced by each band of the DOR mRNA. In the absence of ethanol, forskolin or dibutyryl cAMP reduced the basal levels of DOR mRNA. The cAMP analogue (Rp)-cAMPS, a protein kinase A (PKA) inhibitor, increased DOR mRNA levels. However, the combination of (Rp)-cAMPS and ethanol did not further increase DOR mRNA levels compared to ethanol or (Rp)-cAMPS alone. Signaling through cAMP and PKA down-regulates DOR mRNA levels. The ethanol-induced increase in DOR mRNA levels in NG108-15 cells appears to be mediated via a reduction of PKA.
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PMID:Activation of protein kinase A prevents the ethanol-induced up-regulation of delta-opioid receptor mRNA in NG108-15 cells. 922

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


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