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Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

ABSTRACT: Kappa opioid receptor (KOR) agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as non-addictive anti-pruritic and analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the beneficial antinociceptive and antipruritic effects of KOR agonists, but attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated signaling in the mouse brain, we observed PKC-dependent modulation of G protein-coupled receptor kinases and Wnt pathways at 5 min; stress signaling, cytoskeleton, mTOR signaling and receptor phosphorylation, including cannabinoid receptor CB1 at 30 min. We further demonstrated that inhibition of CB1 attenuated KOR-mediated CPA. Our results demonstrated the feasibility of in vivo biochemical dissection of signaling pathways that lead to side effects.

SUBMITTER: Liu JJ

PROVIDER: S-EPMC8148297 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

Liu Jeffrey J JJ Chiu Yi-Ting YT Chen Chongguang C Huang Peng P Mann Matthias M Liu-Chen Lee-Yuan LY

Neuropharmacology 20200922

Kappa opioid receptor (KOR) agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as non-addictive anti-pruritic and analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the beneficial antinociceptive and antipruritic effects of KOR agonists, but attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated si ...[more]

PMID: 32976891

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Project description:Kappa Opioid receptors（KORs） belong to family A of G-protein–coupled receptors (GPCRs), and the involvement of their ligands in analgesia and sedation has been extensively studied. KORs, which are expressed in the central and peripheral nervous systems are well known to exert potent anti-nociceptive effects by negatively modulating excitatory neurotransmission. The present study reveals that acute activation of KOR weaken SERT function via protein kinase B/Akt (Akt) and calcium calmodulin dependent kinase II (CaMKII) dependent signaling pathways. Therefore, studying phosphorylation will be helpful to elucidate the cellular mechanisms and downstream effector(s). Studies correlating the dysphoric effects of KOR agonists to the G-protein–independent activation of the p38 MAPK pathway suggest that identification of KOR agonists biased to G-protein–mediated signaling could lead to the development of therapeutics with reduced side effects. Therefore, to better understand the cellular mechanisms and downstream effector(s) by which KOR ligands produce these effects, we employed dimethyl labeling quantitative proteomic approach in this study. In this study, three monoclonal HEK293T cell lines with KOR over-expression were successfully constructed as our experimental models. Then, mass spectrometry-based quantitative approach, based on dimethyl labeling and TiO2 beads-coupled phosphopeptides enrichment, was utilized to analyze the KOR-mediated dynamic phosphorylation network in these three KOR-over-expressed monoclonal cell lines disturbed by LPK-26 or U50,488 treatments. Further Substrate-kinase prediction analysis of 212 up-regulated phosphosites indicated that 18 kinases, such as AKT1 and p70S6K, were involved in the up-regulation of KOR phosphorylation. The up-regulated phosphorylation of AKT1 and p70S6K were further confirmed by western blot, which was consistent with our mass spectrometry results. In summary, quantitative phosphoproteomic study of KOR-over-expressed cells upon agonist stimulation paves the way to systematical investigation of KOR-mediated phosphorylation network dynamics, which will be helpful to research about targeted therapy of KOR related diseases and mechanism study of analgesia addiction.

Project description:Kappa opioid receptor (KOR) agonists are potentially useful as analgesic and anti-pruritic agents, for prevention and treatment of substance use disorders, and for treatment of demyelinating diseases. However, side effects of KOR agonists, including psychotomimesis, dysphoria, and sedation, have caused early termination of clinical trials. Understanding the signaling mechanisms underlying the beneficial therapeutic effects and the adverse side effects may help in the development of KOR agonist compounds. In this review, we summarize the current knowledge in this regard in five sections. First, studies conducted on mutant mouse lines (GRK3-/-, p38alpha MAPK-/-, β-arrestin2-/-, phosphorylation-deficient KOR) are summarized. In addition, the abilities of four distinct KOR agonists, which have analgesic and anti-pruritic effects with different side effect profiles, to cause KOR phosphorylation are discussed. Second, investigations on the KOR agonist nalfurafine, both in vitro and in vivo are reviewed. Nalfurafine was the first KOR full agonist approved for clinical use and in the therapeutic dose range it did not produce significant side effects associated with typical KOR agonists. Third, large-scale high-throughput phosphoproteomic studies without a priori hypotheses are described. These studies have revealed that KOR-mediated side effects are associated with many signaling pathways. Fourth, several novel G protein-biased KOR agonists that have been characterized for in vitro biochemical properties and agonist biases and in vivo behavior effects are described. Lastly, possible mechanisms underlying KOR-mediated CPA, hypolocomotion and motor incoordination are discussed. Overall, it is agreed upon that the analgesic and anti-pruritic effects of KOR agonists are mediated via G protein signaling. However, there is no consensus on the mechanisms underlying their side effects. GRK3, p38 MAPK, β-arrestin2, mTOR pathway, CB1 cannabinoid receptor and protein kinase C have been implicated in one side effect or another. For drug discovery, after initial in vitro characterization, in vivo pharmacological characterizations in various behavior tests are still the most crucial steps and dose separation between beneficial therapeutic effects and adverse side effects are the critical determinant for the compounds to be moved forward for clinical development.

Project description:BACKGROUND:We previously reported that ginsenoside Re (GRe) attenuated against methamphetamine (MA)-induced neurotoxicity via anti-inflammatory and antioxidant potentials. We also demonstrated that dynorphin possesses anti-inflammatory and antioxidant potentials against dopaminergic loss, and that balance between dynorphin and substance P is important for dopaminergic neuroprotection. Thus, we examined whether GRe positively affects interactive modulation between dynorphin and substance P against MA neurotoxicity in mice. METHODS:We examined changes in dynorphin peptide level, prodynorphin mRNA, and substance P mRNA, substance P-immunoreactivity, homeostasis in enzymatic antioxidant system, oxidative parameter, microglial activation, and pro-apoptotic parameter after a neurotoxic dose of MA to clarify the effects of GRe, prodynorphin knockout, pharmacological inhibition of κ-opioid receptor (i.e., nor-binaltorphimine), or neurokinin 1 (NK1) receptor (i.e., L-733,060) against MA insult in mice. RESULTS:GRe attenuated MA-induced decreases in dynorphin level, prodynorphin mRNA expression in the striatum of wild-type (WT) mice. Prodynorphin knockout potentiated MA-induced dopaminergic toxicity in mice. The imbalance of enzymatic antioxidant system, oxidative burdens, microgliosis, and pro-apoptotic changes led to the dopaminergic neurotoxicity. Neuroprotective effects of GRe were more pronounced in prodynorphin knockout than in WT mice. Nor-binaltorphimine, a κ-opioid receptor antagonist, counteracted against protective effects of GRe. In addition, we found that GRe significantly attenuated MA-induced increases in substance P-immunoreactivity and substance P mRNA expression in the substantia nigra. These increases were more evident in prodynorphin knockout than in WT mice. Although, we observed that substance P-immunoreactivity was co-localized in NeuN-immunreactive neurons, GFAP-immunoreactive astrocytes, and Iba-1-immunoreactive microglia. NK1 receptor antagonist L-733,060 or GRe selectively inhibited microgliosis induced by MA. Furthermore, L-733,060 did not show any additive effects against GRe-mediated protective activity (i.e., antioxidant, antimicroglial, and antiapoptotic effects), indicating that NK1 receptor is one of the molecular targets of GRe. CONCLUSIONS:Our results suggest that GRe protects MA-induced dopaminergic neurotoxicity via upregulatgion of dynorphin-mediated κ-opioid receptor and downregulation of substance P-mediated NK1 R.

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Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

Publications

Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

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