This is a renewal application of our grant DA00564-28 which was committed to study the neurochemical mechanisms of narcotic tolerance since its inception. Though under intensive investigation, the molecular mechanism of opioid tolerance and dependence has yet to be determined. A general concept of homeostasis has been the focus of much discussion, i.e. drug exposure disrupts the balance of the system, thus tolerance and dependence are cellular adaptations to regain the homeostasis. There are many theories and experimental data in support of different hypotheses such as transcriptional regulation leading to macromolecule synthesis or the alteration in the neurotransmission. In this proposal, we test the hypothesis that opioid tolerance stems from the decrease in the receptor signaling during chronic treatment, or receptor desensitization. The uncoupling of the receptor from the effectors might not be the case in the long-term effects of the drug, but it is the most efficient manner to regain the system's homeostasis. During the past funding period, by generating the mu opioid receptor knockout animals, we conclusively demonstrated that the morphine in vivo activities are absolutely dependent on the concentration of the mu opioid receptor. In parallel studies, we identified the agonist-induced phosphorylation sites of the mu opioid receptor in the in vitro cell lines models. Agonists, such as morphine, induced the phosphorylation of Thr370 and Ser375 residues within the carboxyl domain of the mu-receptor. A basal phosphorylation at the Ser363 was also observed. Now, we are in the position to address the functional roles of the phosphorylation of these critical Ser/Thr residues. We will investigate the roles of these residues in the rapid and slow desensitization of the mu-opioid receptor. We will investigate the existence of these residues in the agonist-induced internalization of the receptor. We will investigate the existence of possible cell line specificity in the phosphorylation of these residues by using various clonal cell lines of primary in vitro neuronal cultures transiently transfected with the adenovirus containing the wild type or mutant receptors. We will determine the identity of the kinases that might be involved in the phosphorylation of these residues. Finally, we will generate mutant receptor in vivo by the gene targeting approach to address the question of the receptor phosphorylation on opioid tolerance development in vivo. Hence, we anticipate that, by carrying out both the in vitro and in vivo studies, we will be able to demonstrate opioid tolerance is a consequence of receptor signaling decrease, or receptor desensitization.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
2R01DA000564-29
Application #
6330780
Study Section
Special Emphasis Panel (ZRG1-MDCN-5 (01))
Program Officer
Koustova, Elena
Project Start
1979-01-01
Project End
2006-03-31
Budget Start
2001-06-01
Budget End
2002-03-31
Support Year
29
Fiscal Year
2001
Total Cost
$456,159
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Pharmacology
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee et al. (2017) Phosphorylation of poly(rC) binding protein 1 (PCBP1) contributes to stabilization of mu opioid receptor (MOR) mRNA via interaction with AU-rich element RNA-binding protein 1 (AUF1) and poly A binding protein (PABP). Gene 598:113-130
Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee et al. (2015) Analysis of epigenetic mechanisms regulating opioid receptor gene transcription. Methods Mol Biol 1230:39-51
Wagley, Yadav; Hwang, Cheol Kyu; Lin, Hong-Yiou et al. (2013) Inhibition of c-Jun NH2-terminal kinase stimulates mu opioid receptor expression via p38 MAPK-mediated nuclear NF-?B activation in neuronal and non-neuronal cells. Biochim Biophys Acta 1833:1476-88
Law, Ping-Yee; Reggio, Patricia H; Loh, Horace H (2013) Opioid receptors: toward separation of analgesic from undesirable effects. Trends Biochem Sci 38:275-82
Wu, Qifang; Hwang, Cheol Kyu; Zheng, Hui et al. (2013) MicroRNA 339 down-regulates ?-opioid receptor at the post-transcriptional level in response to opioid treatment. FASEB J 27:522-35
Song, Kyu Young; Choi, Hack Sun; Law, Ping-Yee et al. (2013) Vimentin interacts with the 5'-untranslated region of mouse mu opioid receptor (MOR) and is required for post-transcriptional regulation. RNA Biol 10:256-66
Miller, Eric C; Zhang, Lei; Dummer, Benjamin W et al. (2012) Differential modulation of drug-induced structural and functional plasticity of dendritic spines. Mol Pharmacol 82:333-43
Song, Kyu Young; Choi, Hack Sun; Law, Ping-Yee et al. (2012) Post-transcriptional regulation of mu-opioid receptor: role of the RNA-binding proteins heterogeneous nuclear ribonucleoprotein H1 and F. Cell Mol Life Sci 69:599-610
Lin, Hong-Yiou; Law, Ping-Yee; Loh, Horace H (2012) Activation of protein kinase C (PKC)? or PKC? as an approach to increase morphine tolerance in respiratory depression and lethal overdose. J Pharmacol Exp Ther 341:115-25
Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee et al. (2012) MicroRNAs in opioid pharmacology. J Neuroimmune Pharmacol 7:808-19

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