Investigation of the role of ?arrestin2 in kappa opioid receptor modulation in a mouse model of pruritus.
ABSTRACT: The kappa opioid receptor (KOR) is involved in mediating pruritus; agonists targeting this receptor have been used to treat chronic intractable itch. Conversely, antagonists induce an itch response at the site of injection. As a G protein-coupled receptor (GPCR), the KOR has potential for signaling via G proteins and ?arrestins, however, it is not clear which of these pathways are involved in the KOR modulation of itch. In this study asked whether the actions of KOR in pruritus involve ?arrestins by using ?arrestin2 knockout (?arr2-KO) mice as well as a recently described biased KOR agonist that biases receptor signaling toward G protein pathways over ?arrestin2 recruitment. We find that the KOR antagonists nor-binaltorphimine (NorBNI) and 5'-guanidinonaltrindole (5'GNTI) induce acute pruritus in C57BL/6J mice, with reduced effects in KOR-KO mice. ?Arr2-KO mice display less of a response to KOR antagonist-induced itch compared to wild types, however no genotype differences are observed from chloroquine phosphate (CP)-induced itch, suggesting that the antagonists may utilize a KOR-?arrestin2 dependent mechanism. The KOR agonist U50,488H was equally effective in both WT and ?arr2-KO mice in suppressing CP-induced itch. Furthermore, the G protein biased agonist, Isoquinolinone 2.1 was as effective as U50,488H in suppressing the itch response induced by KOR antagonist NorBNI or CP in C57BL/6J mice. Together these data suggest that the antipruritic effects of KOR agonists may not require ?arrestins.
Project description:Cannabinoid CB(1) receptors (CB(1)Rs) mediate the effects of ?(9)-tetrahydrocannabinol (THC), the psychoactive component in marijuana. Repeated THC administration produces tolerance and dependence, which limit therapeutic development. Moreover, THC produces motor and psychoactive side effects. ?-arrestin2 mediates receptor desensitization, internalization, and signaling, but its role in these CB(1)R effects and receptor regulation is unclear.CB(1)R signaling and behaviors (antinociception, hypothermia, catalepsy) were assessed in ?-arrestin2-knockout (?arr2-KO) and wild-type mice after THC administration. Cannabinoid-stimulated [(35)S]GTP?S and [(3)H]ligand autoradiography were assessed by statistical parametric mapping and region-of-interest analysis.?-arrestin2 deletion increased CB(1)R-mediated G-protein activity in subregions of the cortex but did not affect CB(1)R binding, in vehicle-treated mice. ?arr2-KO mice exhibited enhanced acute THC-mediated antinociception and hypothermia, with no difference in catalepsy. After repeated THC administration, ?arr2-KO mice showed reduced CB(1)R desensitization and/or downregulation in cerebellum, caudal periaqueductal gray, and spinal cord and attenuated tolerance to THC-mediated antinociception. In contrast, greater desensitization was found in hypothalamus, cortex, globus pallidus, and substantia nigra of ?arr2-KO compared with wild-type mice. Enhanced tolerance to THC-induced catalepsy was observed in ?arr2-KO mice.?-arrestin2 regulation of CB(1)R signaling following acute and repeated THC administration was region-specific, and results suggest that multiple, overlapping mechanisms regulate CB(1)Rs. The observations that ?arr2-KO mice display enhanced antinociceptive responses to acute THC and decreased tolerance to the antinociceptive effects of the drug, yet enhanced tolerance to catalepsy, suggest that development of cannabinoid drugs that minimize CB(1)R interactions with ?-arrestin2 might produce improved cannabinoid analgesics with reduced motor suppression.
Project description:Beta-arrestins bind to agonist-activated G-protein-coupled receptors regulating signaling events and initiating endocytosis. In beta-arrestin2-/- (beta arr2-/-) mice, a complex phenotype is observed that includes altered sensitivity to morphine. However, little is known of how beta-arrestin2 affects mu receptor signaling. We investigated the coupling of mu receptors to voltage-gated Ca2+ channels (VGCCs) in beta arr2+/+ and beta arr2-/- dorsal root ganglion neurons. A lack of beta-arrestin2 reduced the maximum inhibition of VGCCs by morphine and DAMGO (D-Ala2-N-Me-Phe4-glycol5-enkephalin) without affecting agonist potency, the onset of receptor desensitization, or the functional contribution of N-type VGCCs. The reduction in inhibition was accompanied by increased naltrexone-sensitive constitutive inhibitory coupling of mu receptors to VGCCs. Agonist-independent mu receptor inhibitory coupling was insensitive to CTAP (Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), a neutral antagonist that inhibited the inverse agonist action of naltrexone. These functional changes were accompanied by diminished constitutive recycling and increased cell-surface mu receptor expression in beta arr2-/- compared with beta arr2+/+ neurons. Such changes could not be explained by the classical role of beta-arrestins in agonist-induced endocytosis. The localization of the nonreceptor tyrosine kinase c-Src appeared disrupted in beta arr2-/- neurons, and there was reduced activation of c-Src by DAMGO. Using the Src inhibitor PP2 [4-amino-5-(4-chlorophenyl)-(t-butyl)pyrazolo[3,4-d]pyrimidine], we demonstrated that defective Src signaling mimics the beta arr2-/- cellular phenotype of reduced mu agonist efficacy, increased constitutive mu receptor activity, and reduced constitutive recycling. We propose that beta-arrestin2 is required to target c-Src to constitutively active mu receptors, resulting in their internalization, providing another dimension to the complex role of beta-arrestin2 and c-Src in G-protein-coupled receptor function.
Project description:Chronic itch or pruritus is a debilitating disorder that is refractory to conventional anti-histamine treatment. Kappa opioid receptor (KOR) agonists have been used to treat chronic itch, but the underlying mechanism remains elusive. Here, we find that KOR and gastrin-releasing peptide receptor (GRPR) overlap in the spinal cord, and KOR activation attenuated GRPR-mediated histamine-independent acute and chronic itch in mice. Notably, canonical KOR-mediated G?i signaling is not required for desensitizing GRPR function. In vivo and in vitro studies suggest that KOR activation results in the translocation of Ca2+-independent protein kinase C (PKC)? from the cytosol to the plasma membrane, which in turn phosphorylates and inhibits GRPR activity. A blockade of phospholipase C (PLC) in HEK293 cells prevented KOR-agonist-induced PKC? translocation and GRPR phosphorylation, suggesting a role of PLC signaling in KOR-mediated GRPR desensitization. These data suggest that a KOR-PLC-PKC?-GRPR signaling pathway in the spinal cord may underlie KOR-agonists-induced anti-pruritus therapies.
Project description:Beta-arrestins (?-arrestin1 and ?-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, ?-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. ?-arrestin1 and ?-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of ?-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that ?-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. ?-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, ?-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in ?-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of ?-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of ?-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of ?-arrestin2 KO mice is enhanced by MI, which is similar to that in ?-arrestin2 KO mice. In contrast, the inflammation after MI in ?-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that ?-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the ?-arrestin2 gene is knocked out. ?-arrestin2 plays a protective role in MI-induced inflammation.
Project description:Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. ?-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, ?-arrestin2 (?arr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between ?arr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor ?arr2-knockout (?arr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted ?arr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of ?arr2-/- cells. In order to assess the effect of acute loss of ?arr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional ?arr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic ?arr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that ?arr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.
Project description:Although stress has profound effects on motivated behavior, the underlying mechanisms responsible are incompletely understood. In this study we elucidate a functional pathway in mouse brain that encodes the aversive effects of stress and mediates stress-induced reinstatement of cocaine place preference (CPP). Activation of the dynorphin/kappa opioid receptor (KOR) system by either repeated stress or agonist produces conditioned place aversion (CPA). Because KOR inhibition of dopamine release in the mesolimbic pathway has been proposed to mediate the dysphoria underlying this response, we tested dopamine-deficient mice in this study and found that KOR agonist in these mice still produced CPA. However, inactivation of serotonergic KORs by injection of the KOR antagonist norBNI into the dorsal raphe nucleus (DRN), blocked aversive responses to the KOR agonist U50,488 and blocked stress-induced reinstatement of CPP. KOR knockout (KO) mice did not develop CPA to U50,488; however, lentiviral re-expression of KOR in the DRN of KOR KO mice restored place aversion. In contrast, lentiviral expression in DRN of a mutated form of KOR that fails to activate p38 MAPK required for KOR-dependent aversion, did not restore place aversion. DRN serotonergic neurons project broadly throughout the brain, but the inactivation of KOR in the nucleus accumbens (NAc) coupled with viral re-expression in the DRN of KOR KO mice demonstrated that aversion was encoded by a DRN to NAc projection. These results suggest that the adverse effects of stress may converge on the serotonergic system and offers an approach to controlling stress-induced dysphoria and relapse.
Project description:There is growing evidence that kappa opioid receptor (KOR) antagonists could be a useful class of therapeutics for treating depression and anxiety. However, the overwhelming majority of preclinical investigations examining the behavioral effects of KOR antagonists have been in male rodents. Here, we examined the effects of the long-acting KOR antagonist nor-binaltophimine (norBNI) on immobility in the forced swim test in males and females of two different rodent species (C57Bl/6J and California mice). Consistent with previous reports, norBNI (10 mg/kg) decreased immobility in the forced swim test for male C57Bl/6J and California mice. Surprisingly, dose-response studies in female C57Bl/6J and California mice showed that norBNI did not reduce immobility. Pharmacokinetic analyses showed that metabolism and brain concentrations of norBNI were similar in male and female C57Bl/6J. In the nucleus accumbens of male but not female C57Bl/6J, norBNI increased phosphorylation of c-Jun N-terminal kinase (pJNK), a putative mechanism for norBNI action. However, no differences in pJNK were observed in male or female California mice. Together, these results suggest that immobility in the forced swim test is less dependent on endogenous KOR signaling in female rodents and highlight the importance of examining the effects of possible therapeutic agents in both males and females.
Project description:Kappa opioid receptor (KOR) agonists produce analgesic and anti-pruritic effects, but their clinical application was limited by dysphoria and hallucinations. Nalfurafine, a clinically used KOR agonist, does not cause dysphoria or hallucinations at therapeutic doses in humans. We found that in CD-1 mice nalfurafine produced analgesic and anti-scratch effects dose-dependently, like the prototypic KOR agonist U50,488H. In contrast, unlike U50,488H, nalfurafine caused no aversion, anhedonia, or sedation or and a low level of motor incoordination at the effective analgesia and anti-scratch doses. Thus, we established a mouse model that recapitulated important aspects of the clinical observations. We then employed a phosphoproteomics approach to investigate mechanisms underlying differential KOR-mediated effects. A large-scale mass spectrometry (MS)-based analysis on brains revealed that nalfurafine perturbed phosphoproteomes differently from U50,488H in a brain-region specific manner after 30-min treatment. In particular, U50,488H and nalfurafine imparted phosphorylation changes to proteins found in different cellular components or signaling pathways in different brain regions. Notably, we observed that U50,488H, but not nalfurafine, activated the mammalian target of rapamycin (mTOR) pathway in the striatum and cortex. Inhibition of the mTOR pathway by rapamycin abolished U50,488H-induced aversion, without affecting analgesic, anti-scratch, and sedative effects and motor incoordination. The results indicate that the mTOR pathway is involved in KOR agonist-induced aversion. This is the first demonstration that phosphoproteomics can be applied to agonist-specific signaling of G protein-coupled receptors (GPCRs) in mouse brains to unravel pharmacologically important pathways. Furthermore, this is one of the first two reports that the mTOR pathway mediates aversion caused by KOR activation.
Project description:Patients with cholestatic disease exhibit pruritus and analgesia, but the mechanisms underlying these symptoms are unknown. We report that bile acids, which are elevated in the circulation and tissues during cholestasis, cause itch and analgesia by activating the GPCR TGR5. TGR5 was detected in peptidergic neurons of mouse dorsal root ganglia and spinal cord that transmit itch and pain, and in dermal macrophages that contain opioids. Bile acids and a TGR5-selective agonist induced hyperexcitability of dorsal root ganglia neurons and stimulated the release of the itch and analgesia transmitters gastrin-releasing peptide and leucine-enkephalin. Intradermal injection of bile acids and a TGR5-selective agonist stimulated scratching behavior by gastrin-releasing peptide- and opioid-dependent mechanisms in mice. Scratching was attenuated in Tgr5-KO mice but exacerbated in Tgr5-Tg mice (overexpressing mouse TGR5), which exhibited spontaneous pruritus. Intraplantar and intrathecal injection of bile acids caused analgesia to mechanical stimulation of the paw by an opioid-dependent mechanism. Both peripheral and central mechanisms of analgesia were absent from Tgr5-KO mice. Thus, bile acids activate TGR5 on sensory nerves, stimulating the release of neuropeptides in the spinal cord that transmit itch and analgesia. These mechanisms could contribute to pruritus and painless jaundice that occur during cholestatic liver diseases.
Project description:Psychotomimetic and prodepressive effect by kappa opioid receptor (KOR) activation in rodents and human is widely known. Significantly, recent clinical investigations demonstrated the salutary effects of KOR antagonists in patients with treatment resistant depression, indicating essential role of KOR signaling in refractory depression. This study was undertaken to reveal the molecular determinant of KOR mediated depression and antidepressant response of KOR antagonist. We observed that chronic KOR activation by U50488, a selective KOR agonist, significantly increased depression like symptoms (behavioral despair, anhedonia and sociability) in C57BL/6J mice, which were blocked by KOR antagonist norBNI and antidepressant imipramine, but not by fluoxetine or citalopram. Further, chronic KOR activation increased phosphorylation of NR2B subunit of NMDA at tyrosine 1472 (pNR2B NMDA) in the hippocampus, but not in the cortex. Similar to behavioral effects norBNI and imipramine, but not SSRIs, blocked NR2B phosphorylation. Moreover, KOR induced depression like behaviors were reversed by NR2B selective inhibitor Ro 25-6981. Mechanistic studies in primary cultured neurons and brain tissues using genetic and pharmacological approaches revealed that stimulation of KOR modulates several molecular correlates of depression. Thus, these findings elucidate molecular mechanism of KOR signaling in treatment resistant depression like behaviors in mice.