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The leaves of Mitragyna speciosa (kratom), a plant native to Southeast Asia, are increasingly used as a pain reliever and for attenuation of opioid withdrawal symptoms. Using the tools of natural products chemistry, chemical synthesis, and pharmacology, we provide a detailed in vitro and in vivo pharmacological characterization of the alkaloids in kratom. We report that metabolism of kratom’s major alkaloid, mitragynine, in mice leads to formation of (a) a potent mu opioid receptor agonist antinociceptive agent, 7-hydroxymitragynine, through a CYP3A-mediated pathway, which exhibits reinforcing properties, inhibition of gastrointestinal (GI) transit and reduced hyperlocomotion, (b) a multifunctional mu agonist/delta-kappa antagonist, mitragynine pseudoindoxyl, through a CYP3A-mediated skeletal rearrangement, displaying reduced hyperlocomotion, inhibition of GI transit and reinforcing properties, and (c) a potentially toxic metabolite, 3-dehydromitragynine, through a non-CYP oxidation pathway. Our results indicate that the oxidative metabolism of the mitragynine template beyond 7-hydroxymitragynine may have implications in its overall pharmacology in vivo.

Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, β-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, β-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, β-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of β-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of β-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.

In this work, we studied a series of carfentanyl amide-based opioid derivatives targeting the mu opioid receptor (μOR) and the delta opioid receptor (δOR) heteromer as a credible novel target in pain management therapy. We identified a lead compound named MP135 that exhibits high G-protein activity at μ–δ heteromers compared to the homomeric δOR or μOR and low β-arrestin2 recruitment activity at all three. Furthermore, MP135 exhibits distinct signaling profile, as compared to the previously identified agonist targeting μ–δ heteromers, CYM51010. Pharmacological characterization of MP135 supports the utility of this compound as a molecule that could be developed as an antinociceptive agent similar to morphine in rodents. In vivo characterization reveals that MP135 maintains untoward side effects such as respiratory depression and reward behavior; together, these results suggest that optimization of MP135 is necessary for the development of therapeutics that suppress the classical side effects associated with conventional clinical opioids.

This report describes the unique pharmacological profile of FBNTI, a potent DOR antagonist that acts as a MOR agonist via an allosteric mechanism. Binding of FBNTI to opioid receptors expressed in HEK 293 cells revealed a 190-fold greater affinity for DOR (Ki = 0.84 nM) over MOR (Ki = 160 nM). In mice, intrathecal FBNTI produced potent antinociception (ED50 = 46.9 pmol/mouse), which was antagonized by selective MOR antagonists (CTOP, β-FNA). Autoantagonism of the MOR agonism by FBNTI was observed above the ED75 dose, suggesting antagonism of activated MOR. That FBNTI is devoid of agonism in DOR knockout mice is consistent with allosteric activation of the MOR protomer via FBNTI bound to within a MOR-DOR heteromer. This proposed mechanism is supported by calcium mobilization assays, which indicate that FBNTI selectively activates the MOR-DOR heteromer and functionally antagonizes the MOR protomer at >ED75. The unprecedented mode of MOR activation by FBNTI may be responsible for the lack of tolerance after intrathecal (i.t.) administration. FBNTI was highly effective upon topical administration to the ipsolateral hind paw in the Hargreaves assay (EC50 = 0.17 ± 0.08 μM) and without significant contralateral activity, suggesting a lack of systemic exposure.

Tianeptine is an atypical antidepressant used in Europe to treat patients who respond poorly to selective serotonin reuptake inhibitors (SSRIs). The recent discovery that tianeptine is a mu opioid receptor (MOR) agonist has provided a potential avenue for expanding our understanding of antidepressant treatment beyond the monoamine hypothesis. Thus, our studies aim to understand the neural circuits underlying tianeptine's antidepressant effects. We show that tianeptine induces rapid antidepressant-like effects in mice after as little as one week of treatment. Critically, we also demonstrate that tianeptine's mechanism of action is distinct from fluoxetine in two important aspects: (1) tianeptine requires MORs for its chronic antidepressant-like effect, while fluoxetine does not, and (2) unlike fluoxetine, tianeptine does not promote hippocampal neurogenesis. Using cell-type specific MOR knockouts we further show that MOR expression on GABAergic cells-specifically somatostatin-positive neurons-is necessary for the acute and chronic antidepressant-like responses to tianeptine. Using central infusion of tianeptine, we also implicate the ventral hippocampus as a potential site of antidepressant action. Moreover, we show a dissociation between the antidepressant-like phenotype and other opioid-like phenotypes resulting from acute tianeptine administration such as analgesia, conditioned place preference, and hyperlocomotion. Taken together, these results suggest a novel entry point for understanding what circuit dysregulations may occur in depression, as well as possible targets for the development of new classes of antidepressant drugs.

Mitragynina speciosa, more commonly known as kratom, is a plant native to Southeast Asia, the leaves of which have been used traditionally as a stimulant, analgesic, and treatment for opioid addiction. Recently, growing use of the plant in the United States and concerns that kratom represents an uncontrolled drug with potential abuse liability, have highlighted the need for more careful study of its pharmacological activity. The major active alkaloid found in kratom, mitragynine, has been reported to have opioid agonist and analgesic activity in vitro and in animal models, consistent with the purported effects of kratom leaf in humans. However, preliminary research has provided some evidence that mitragynine and related compounds may act as atypical opioid agonists, inducing therapeutic effects such as analgesia, while limiting the negative side effects typical of classical opioids. Here we report evidence that an active metabolite plays an important role in mediating the analgesic effects of mitragynine. We find that mitragynine is converted in vitro in both mouse and human liver preparations to the much more potent mu-opioid receptor agonist 7-hydroxymitragynine, and that this conversion is mediated by cytochrome P450 3A isoforms. Further, we show that 7-hydroxymitragynine is formed from mitragynine in mice and that brain concentrations of this metabolite are sufficient to explain most or all of the opioid-receptor-mediated analgesic activity of mitragynine. At the same time, mitragynine is found in the brains of mice at very high concentrations relative to its opioid receptor binding affinity, suggesting that it does not directly activate opioid receptors. The results presented here provide a metabolism-dependent mechanism for the analgesic effects of mitragynine and clarify the importance of route of administration for determining the activity of this compound. Further, they raise important questions about the interpretation of existing data on mitragynine and highlight critical areas for further research in animals and humans.

Substance use and related mental health epidemics are causing increasing suffering and death in diverse communities.1,2Despite extensive efforts focused on developing pharmacotherapies for treating substance use disorders, there is an urgent need for radically different therapeutic approaches.3,4Ibogaine provides an important drug prototype in this direction, as a psychoactive iboga alkaloid suggested to have the ability to interrupt opioid use in drug-dependent humans.5However, ibogaine and its major metabolite noribogaine present considerable safety risk associated with cardiac arrhythmias.6We introduce a new class of iboga alkaloids - “oxa-iboga” - defined as benzofuran-containing iboga analogs and created via structural editing of the iboga skeleton. The oxa-iboga compounds act as potent kappa opioid receptor agonistsin vitroandin vivo, but exhibit atypical behavioral features compared to standard kappa psychedelics. We show that oxa-noribogaine has greater therapeutic efficacy in rat models of opioid use, and no cardiac pro-arrhythmic potential, in contrast to noribogaine. Oxa-noribogaine induces long-lasting suppression of morphine and fentanyl intake after a single dose, persistent reduction of morphine intake and reinforcing efficacy after a short treatment regimen, and suppression of morphine and fentanyl drug seeking in relapse models. Oxa-noribogaine also induces a lasting elevation of neurotrophin proteins in the ventral tegmental area and medial prefrontal cortex, consistent with targeted neuroplasticity induction and alteration of addiction-like states. As such, oxa-iboga compounds represent candidates for a novel type of pharmacotherapy for treatment of opioid use disorder.

Mitragynine (MG) is the most abundant alkaloid component of the psychoactive plant material “kratom”, which according to numerous anecdotal reports shows efficacy in self-medication for pain syndromes, depression, anxiety, and substance use disorders. We have developed a synthetic method for selective functionalization of the unexplored C11 position of the MG scaffold (C6 position in indole numbering) via the use of an indole-ethylene glycol adduct and subsequent iridium-catalyzed borylation. Through this work we discover that C11 represents a key locant for fine-tuning opioid receptor signaling efficacy. 7-Hydroxymitragynine (7OH), the parent compound with low efficacy on par with buprenorphine, is transformed to an even lower efficacy agonist by introducing a fluorine substituent in this position (11-F-7OH), as demonstrated in vitro at both mouse and human mu opioid receptors (mMOR/hMOR) and in vivo in mouse analgesia tests. Low efficacy opioid agonists are of high interest as candidates for generating safer opioid medications with mitigated adverse effects., Nature Communications, 12 (1), ISSN:2041-1723

We have previously shown that topical opioids including morphine and its congeners promote healing of full thickness ischemic wounds in rats. We examined the contribution of mu opioid receptor (MOPr)-mediated healing of full thickness ischemic wounds using MOPr and delta or kappa opioid receptor (DOPr or KOPr) knockout (KO) mice. Wound closure in the early (day 5) as well as later phases was delayed in topical morphine or PBS treated MOPr-KO mice compared to reciprocal treatments of wounds in wild-type (WT) mice. MOPr expression was significantly upregulated at 30 min in the wound margins and colocalized with wound margins and vasculature in the epidermal and dermal layers of the skin. We next examined whether neuropeptide expression was involved in the mechanism of MOPr-mediated wound closure. Substance P (SP) and calcitonin gene related peptide (CGRP) immunoreactivity (ir) was significantly increased in the skin of MOPr-KO mice as compared to WT mice. Neuropeptide-ir was increased significantly in PBS-treated wounds of MOPr and WT mice, but morphine treatment reduced neuropeptide immunoreactivity in both as compared to PBS. Wounding of keratinocytes led to the release of opioid peptide beta-endorphin (β-END) in conditioned medium, which stimulated the proliferation of endothelial cells. MOPr-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, CTOP) and non-selective OPr antagonist naloxone inhibited endothelial proliferation induced by wounded keratinocyte conditioned medium. Additionally, accelerated wound area closure in vitro by morphine was suppressed by methylnaltrexone, a non-selective OPr antagonist with high affinity for MOPr. Morphine and its congeners stimulated the proliferation of endothelial cells from WT mice but not those from MOPr-KO mice. Furthermore, morphine-induced mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) phosphorylation in endothelial cells was significantly decreased in MOPr-KO mice as compared to WT mice. Collectively, these data suggest that MOPr plays a critical role in the proliferation phase with the formation of granulation tissue during wound healing.

Endomorphins have been proposed as the endogenous ligand agonists of the μ-opioid receptor; however, no propeptide precursor protein for endomorphins has been identified. Here, to identify the presumed precursor of endomorphins, we designed an immunoscreening assay using specific affinity-purified rabbit antisera raised against synthetic endomorphins in a whole mouse brain cDNA library. Following this approach, we identifiy a DNA sequence encoding a protein precursor, which we name proMexneurin, that contains three different peptide sequences: Mexneurin-1 (an endomorphin-like peptide), Mexneurin-2, and Mexneurin-3, a peptide which appears to be unrelated to endomorphins. RT-PCR analysis and in situ hybridization reveal a widespread distribution of proMexneurin mRNA throughout the mouse brain. Both Mexneurin-1 and Mexneurin-3 peptides display biological activities in the mouse CNS.

BACKGROUND: The nociceptin/orphanin FQ opioid peptide (NOP) receptor and its endogenous ligand N/OFQ have been implicated in the regulation of drug and alcohol use disorders. In particular, evidence demonstrated that NOP receptor activation blocks reinforcing and motivating effects of alcohol across a range of behavioral measures, including alcohol intake, conditioned place preference and vulnerability to relapse. METHODS: Here, we show the effects of pharmacological activation and inhibition of NOP receptors on binge-like alcohol consumption, as measured by the “drinking in the dark” (DID) model in C57BL/6J mice. RESULTS: We found that two potent and selective NOP agonists AT-202 (0, 0.3, 1, 3 mg/kg) and AT-312 (0, 0.3, 1 mg/kg) did not affect binge alcohol drinking at doses that do not affect locomotor activity. AT-202 also failed to alter DID behavior when administered to mice previously exposed to chronic alcohol treatment with an alcohol-containing liquid diet. Conversely, treatment with either the high affinity NOP receptor antagonist SB-612111 (0, 3, 10, 30 mg/kg) or the selective antagonist LY2817412 (0, 3, 10, 30 mg/kg) decreased binge drinking. SB-612111 was effective at all doses examined, LY2817412 was effective at 30 mg/kg. Consistently, NOP receptor knockout mice consumed less alcohol compared to wild type. SB-612111 reduced DID and increased sucrose consumption at doses that do not appear to affect locomotor activity. However, the high dose of SB-612111 (30 mg/kg) reduced alcohol intake but failed to inhibit preference in a two-bottle choice DID model that can assess moderate alcohol intake. CONCLUSIONS: The present results suggest that NOP receptor inhibition rather than activation may represent a valuable approach for treatment of alcohol use disorders characterized by excessive alcohol consumption such as binge drinking.

AIMS/HYPOTHESIS: Peptide hormones are first synthesised as larger, inactive precursors that are converted to their active forms by endopeptidase cleavage and post-translational modifications, such as amidation. Recent, large-scale genome-wide studies have suggested that two coding variants of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), are associated with impaired insulin secretion and increased type 2 diabetes risk. We aimed to elucidate the role of PAM in modulating beta cell peptide amidation, beta cell function and the development of diabetes. METHODS: PAM transcript and protein levels were analysed in mouse islets following induction of endoplasmic reticulum (ER) or cytokine stress, and PAM expression patterns were examined in human islets. To study whether haploinsufficiency of PAM accelerates the development of diabetes, Pam(+/−) and Pam(+/+) mice were fed a low-fat diet (LFD) or high-fat diet (HFD) and glucose homeostasis was assessed. Since aggregates of the PAM substrate human islet amyloid polypeptide (hIAPP) lead to islet inflammation and beta cell failure, we also investigated whether PAM haploinsufficiency accelerated hIAPP-induced diabetes and islet amyloid formation in Pam(+/−) and Pam(+/+) mice with beta cell expression of hIAPP. RESULTS: Immunostaining revealed high expression of PAM in alpha, beta and delta cells in human pancreatic islets. Pam mRNA and PAM protein expression were reduced in mouse islets following administration of an HFD, and in isolated islets following induction of ER stress with thapsigargin, or cytokine stress with IL-1β, IFN-γ and TFN-α. Despite Pam(+/−) only having 50% PAM expression and enzyme activity as compared with Pam(+/+) mice, glucose tolerance and body mass composition were comparable in the two models. After 24 weeks of HFD, both Pam(+/−) and Pam(+/+) mice had insulin resistance and impaired glucose tolerance, but no differences in glucose tolerance, insulin sensitivity or plasma insulin levels were observed in PAM haploinsufficient mice. Islet amyloid formation and beta cell function were also similar in Pam(+/−) and Pam(+/+) mice with beta cell expression of hIAPP. CONCLUSIONS/INTERPRETATION: Haploinsufficiency of PAM in mice does not accelerate the development of diet-induced obesity or hIAPP transgene-induced diabetes.

The mu opioid receptor gene undergoes extensive alternative splicing. Mu opioids can be divided into three classes based on the role of different groups of splice variants. Morphine and methadone require only full length 7 transmembrane (7TM) variants for analgesia, whereas IBNtxA (3’-iodobenzyol-6β-naltrexamide) needs only truncated 6TM variants. A set of endomorphin analogs fall into a third group that requires both 6TM and 7TM splice variants. Unlike morphine, endomorphin 1 and 2, DAPP (Dmt,D-Ala-Phe-Phe-NH2) and IDAPP (3’-iodo-Dmt-D-Ala-Phe-Phe-NH2) analgesia was lost in an exon 11 knockout mouse lacking 6TM variants. Restoring 6TM variant expression in a knockout mouse lacking both 6TM and 7TM variants failed to rescue DAPP or IDAPP analgesia. However, re-establishing 6TM expression in an exon 11 knockout mouse that still expressed 7TM variants did rescue the response, consistent with the need for both 6TM and 7TM variants. In receptor binding assays, 125I-IDAPP labeled more sites (Bmax) than 3H-DAMGO in wildtype mice. In exon 11 knockout mice 125I-IDAPP binding was lowered to levels similar to 3H-DAMGO, which remained relatively unchanged compared to wildtype mice. 125I-IDAPP binding was totally lost in an exon 1/exon 11 knockout model lacking all Oprm1 variant expression, confirming the drug was not cross labeling non-mu opioid receptors. These findings suggested that 125I-IDAPP labeled two populations of mu binding sites in wildtype mice, one corresponding to 7TM variants and the second dependent upon 6TM variants. Together, these data indicate that endomorphin analogs represent a unique, genetically defined and distinct class of mu opioid analgesic.

// Tiffany Scully 1 , Sue M. Firth 1 , Carolyn D. Scott 1 , Hasanthi C. de Silva 1 , John E. Pintar 2 , Tailoi Chan-Ling 3 , Stephen M. Twigg 4 , Robert C. Baxter 1 1 Hormones and Cancer Laboratories, Kolling Institute, University of Sydney, Royal North Shore Hospital, Sydney, New South Wales 2065, Australia 2 Department of Neuroscience and Cell Biology, Rutgers University, Robert Wood Johnson Medical School, New Brunswick, NJ 08854, USA 3 Department of Anatomy, Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia 4 Charles Perkins Centre, Sydney Medical School, University of Sydney, Sydney, New South Wales 2006, Australia Correspondence to: Robert C. Baxter, email: robert.baxter@sydney.edu.au Keywords: IGFBP-3, obesity, breast cancer, BP3KO mouse, T-cell Received: January 21, 2016 Accepted: June 16, 2016 Published: July 18, 2016 ABSTRACT Obesity is associated epidemiologically with poor breast cancer prognosis, but the mechanisms remain unclear. Since IGF binding protein-3 (IGFBP-3) influences both breast cancer growth and adipocyte maturation, it may impact on how obesity promotes breast oncogenesis. This study investigated the role of endogenous IGFBP-3 on the development of obesity and subsequently on breast tumor growth. Wild-type (WT) C57BL/6 or IGFBP-3-null (BP3KO) mice were fed a high-fat diet (HFD) or control chow-diet for 15 weeks before orthotopic injection with syngeneic EO771 murine breast cancer cells. When the largest tumor reached 1000 mm 3 , tissues and tumors were excised for analysis. Compared to WT, BP3KO mice showed significantly reduced weight gain and mammary fat pad mass (contralateral to tumor) in response to HFD, despite similar food intake. EO771 tumor weight and volume were increased by HFD and decreased by BP3KO. Despite differences in tumor size, tumors in BP3KO mice showed no differences from WT in the number of mitotically active (Ki67 + ) and apoptotic (cleaved caspase-3 + ) cells, but had greater infiltration of CD3 + T-cells. These data suggest that endogenous (circulating and/or stromal) IGFBP-3 is stimulatory to adipose tissue expansion and enhances mammary tumor growth in immune-competent mice, potentially by suppressing T-cell infiltration into tumors.

Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit β-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors.

Unfortunately, the human islet checklist was omitted from the electronic supplementary material (ESM) linked to this paper.

IGFBP-3 has both stimulatory and inhibitory effects on cancer progression. The growth of EO771 mammary carcinoma cells as syngeneic tumors in C57BL/6 mice is reduced in Igfbp3-null (BP3KO) mice, suggesting that systemic IGFBP-3 enhances tumor progression. In this study we assessed the growth of EO771 cells expressing human IGFBP-3 in BP3KO mice. Cells expressing hIGFBP-3 showed decreased proliferation in vitro and increased levels of IGF-1 receptor (IGF1R) protein but not mRNA, consistent with sequestration of endogenous IGF by IGFBP-3. The growth rate of these cells was restored by exposure to IGF-1 or analogues with reduced affinity for IGFBP-3 (long Arg3-IGF-1) or IGF1R (Leu24-IGF-1). In EO771 cells implanted orthotopically into mice, hIGFBP-3 expression by the cells inhibited tumor establishment in BP3KO but not wild-type mice. For tumors that successfully established, final weight was not affected significantly by hIGFBP-3 expression. However, final tumor weight was inversely related to intratumoral T cell counts, and sera from BP3KO mice with tumors showed low-titer immunoreactivity against IGFBP-3. The contrasting effects on tumor establishment and progression of IGFBP-3 expressed by mammary carcinoma cells, compared to systemic stromal and circulating IGFBP-3, highlights the complexity of growth regulation by IGFBP-3 in mammary tumors.

Nociceptin/orphanin FQ (N/OFQ), a 17 amino acid peptide, is the endogenous ligand of the ORL1/nociceptin-opioid-peptide (NOP) receptor. N/OFQ appears to regulate a variety of physiologic functions including stimulating feeding behavior. Recently, a new class of thienospiro-piperidine-based NOP antagonists was described. One of these molecules, LY2940094 has been identified as a potent and selective NOP antagonist that exhibited activity in the central nervous system. Herein, we examined the effects of LY2940094 on feeding in a variety of behavioral models. Fasting-induced feeding was inhibited by LY2940094 in mice, an effect that was absent in NOP receptor knockout mice. Moreover, NOP receptor knockout mice exhibited a baseline phenotype of reduced fasting-induced feeding, relative to wild-type littermate controls. In lean rats, LY2940094 inhibited the overconsumption of a palatable high-energy diet, reducing caloric intake to control chow levels. In dietary-induced obese rats, LY2940094 inhibited feeding and body weight regain induced by a 30% daily caloric restriction. Last, in dietary-induced obese mice, LY2940094 decreased 24-hour intake of a high-energy diet made freely available. These are the first data demonstrating that a systemically administered NOP receptor antagonist can reduce feeding behavior and body weight in rodents. Moreover, the hypophagic effect of LY2940094 is NOP receptor dependent and not due to off-target or aversive effects. Thus, LY2940094 may be useful in treating disorders of appetitive behavior such as binge eating disorder, food choice, and overeating, which lead to obesity and its associated medical complications and morbidity.

Epidemiological studies show an association between obesity and poor breast cancer prognosis. We previously demonstrated that global IGFBP-3 deficiency, in IGFBP-3-null mice, resulted in a 50% reduction in mammary tumour growth over 3 weeks relative to tumours in wild-type (WT) C57BL/6 mice. This growth reduction was ameliorated by high fat feeding-induced obesity. This study aimed to examine how IGFBP-3 promotes tumour growth by influencing the immune tumour microenvironment in healthy and obese mice. Syngeneic EO771 cells, which lack detectable IGFBP-3 expression, were grown as orthotopic tumours in WT and IGFBP-3-null C57BL/6 mice placed on either a control chow or a high-fat diet (HFD), and examined by quantitative PCR and immunohistochemistry. In WT mice, increased stromal expression of IGFBP-3 was positively associated with tumour growth, supporting the hypothesis that IGFBP-3 in the microenvironment promotes tumour progression. Examining markers of immune cell subsets, gene expression of Ifng, Cd8a, Cd8b1 and Tnf and CD8 measured by immunohistochemistry were elevated in tumours of IGFBP-3-null mice compared to WT, indicating an accumulation of CD8+ T cells, but this increase was absent if the IGFBP-3-null mice had been exposed to HFD. Expression of these genes was negatively associated with tumour growth. Although similar among groups overall, Nkg2d and Tnfsf10 tumoural expression was associated with decreased tumour growth. Overall, the results of this study provide an immune-based mechanism by which host IGFBP-3 may promote breast tumour growth in the EO771 murine breast cancer model, and suggest that targeting IGFBP-3 might make a novel contribution to immune therapy for breast cancer.

IBNtxA (3′-iodobenzoyl-6β-naltrexamide) is a potent analgesic in mice lacking many traditional opioid side effects. In mice, it displays no respiratory depression, does not produce physical dependence with chronic administration, and shows no cross-tolerance to morphine. It has limited effects on gastrointestinal transit and shows no reward behavior. Biochemical studies indicate its actions are mediated through a set of μ-opioid receptor clone MOR-1 splice variants associated with exon 11 that lack exon 1 and contain only six transmembrane domains. Like the mouse and human, rats express exon 11–associated splice variants that also contain only six transmembrane domains, raising the question of whether IBNtxA would have a similar pharmacologic profile in rats. When given systemically, IBNtxA is a potent analgesic in rats, with an ED50 value of 0.89 mg/kg s.c., approximately 4-fold more potent than morphine. It shows no analgesic cross-tolerance in morphine-pelleted rats. IBNtxA displays no respiratory depression as measured by blood oxygen saturation. In contrast, oximetry shows that an equianalgesic dose of morphine lowers blood oxygen saturation values by 30%. IBNtxA binding is present in a number of brain regions, with the thalamus standing out with very high levels and the cerebellum with low levels. As in mice, IBNtxA is a potent analgesic in rats with a favorable pharmacologic profile and reduced side effects.

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide whose receptor is designated ORL1 or nociceptin receptor (NOP). We utilized a potent, selective, and orally bioavailable antagonist with documented engagement with NOP receptors in vivo to assess antidepressant‐ and anxiolytic‐related pharmacological effects of NOP receptor blockade along with measures of cognitive and motor impingement. LY2940094 ([2‐[4‐[(2‐chloro‐4,4‐difluoro‐spiro[5H‐thieno[2,3‐c]pyran‐7,4′‐piperidine]‐1′‐yl)methyl]‐3‐methyl‐pyrazol‐1‐yl]‐3‐pyridyl]methanol) displayed antidepressant‐like behavioral effects in the forced‐swim test in mice, an effect absent in NOP −/− mice. LY2940094 also augmented the behavioral effect of fluoxetine without changing target occupancies (NOP and serotonin reuptake transporter [SERT]). LY2940094 did not have effects under a differential‐reinforcement of low rate schedule. Although anxiolytic‐like effects were not observed in some animal models (conditioned suppression, 4‐plate test, novelty‐suppressed feeding), LY2940094 had effects like that of anxiolytic drugs in three assays: fear‐conditioned freezing in mice, stress‐induced increases in cerebellar cGMP in mice, and stress‐induced hyperthermia in rats. These are the first reports of anxiolytic‐like activity with a systemically viable NOP receptor antagonist. LY2940094 did not disrupt performance in either a 5‐choice serial reaction time or delayed matching‐to‐position assay. LY2940094 was also not an activator or suppressor of locomotion in rodents nor did it induce failures of rotarod performance. These data suggest that LY2940094 has unique antidepressant‐ and anxiolytic‐related pharmacological effects in rodents. Clinical proof of concept data on this molecule in depressed patients have been reported elsewhere.

Depression is a debilitating chronic illness that affects around 350 million people worldwide. Current treatments, such as selective serotonin reuptake inhibitors, are not ideal because only a fraction of patients achieve remission. Tianeptine is an effective antidepressant with a previously unknown mechanism of action. We recently reported that tianeptine is a full agonist at the mu opioid receptor (MOR). Here we demonstrate that the acute and chronic antidepressant-like behavioral effects of tianeptine in mice require MOR. Interestingly, while tianeptine also produces many opiate-like behavioral effects such as analgesia and reward, it does not lead to tolerance or withdrawal. Furthermore, the primary metabolite of tianeptine (MC5), which has a longer half-life, mimics the behavioral effects of tianeptine in a MOR-dependent fashion. These results point to the possibility that MOR and its downstream signaling cascades may be novel targets for antidepressant drug development.

Pain remains a pervasive problem throughout medicine, transcending all specialty boundaries. Despite the extraordinary insights into pain and its mechanisms over the past few decades, few advances have been made with analgesics. Most pain remains treated by opiates, which have significant side effects that limit their utility. We now describe a potent opiate analgesic lacking the traditional side effects associated with classical opiates, including respiratory depression, significant constipation, physical dependence, and, perhaps most important, reinforcing behavior, demonstrating that it is possible to dissociate side effects from analgesia. Evidence indicates that this agent acts through a truncated, six-transmembrane variant of the G protein-coupled mu opioid receptor MOR-1. Although truncated splice variants have been reported for a number of G protein-coupled receptors, their functional relevance has been unclear. Our evidence now suggests that truncated variants can be physiologically important through heterodimerization, even when inactive alone, and can comprise new therapeutic targets, as illustrated by our unique opioid analgesics with a vastly improved pharmacological profile.

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [(35)S]GTPγS functional assays were performed in the presence of putative δ(1)-(DPDPE: agonist, BNTX: antagonist), δ(2)-(agonist: deltorphin II, Ile(5,6)-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ(2)- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K(D)=0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [(3)H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [(3)H]Ile(5,6)-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49±0.06 and 0.30±0.01 nM potency against DPDPE and deltorphin II in the [(35)S]GTPγS functional assay, respectively. The rank order of potency of putative δ(1)- and δ(2)-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ(1)- and δ(2)-opioid receptors could not be unequivocally distinguished in vitro.

We investigated D1, D2 receptors and dopamine transporter (DAT) binding levels in mice lacking all three opioid receptors and wild-type (WT) mice on three different genetic backgrounds. Quantitative autoradiography was used to determine the level of radioligand binding to the D1 and D2 receptors and DAT labeled with [3H]SCH23390, [3H]raclopride, and [3H]mazindol, respectively in triple-opioid receptor knockout (KO) and WT maintained on C57BL/6 (B6) and 129/SvEvTac (129) as well as C57BL/6 × 129/SvPas (B6 × 129) strains. No significant genotype effect was observed in D1, D2 receptors and DAT binding in any regions analyzed in any of the strains studied, suggesting that a lack of all three opioid receptors does not influence D1, D2 receptors and DAT expression, irrespective of their genetic strain background. However, strain differences were observed in D1 binding between the three strains of mice studied. Lower levels of D1 binding were observed in the substantia nigra of B6 × 129 WT mice compared with the 129 WT mice and in the olfactory tubercle of B6 × 129 WT compared with B6 WT and 129 WT mice. Lower levels of D1 binding were observed in the caudate putamen of B6 × 129 KO mice compared with 129 KO mice. In contrast, no significant strain differences were observed in D2 and DAT binding between the three strains of mice in any regions analyzed. Overall, these results indicate a lack of modulation of the dopaminergic system by the deletion of all three opioid receptors regardless of different background strains. Synapse 64:520–527, 2010. © 2010 Wiley-Liss, Inc.

Prohormone convertase 2 (PC2) functions in the generation of neuropeptides from their precursors. A quantitative peptidomics approach was used to evaluate the role of PC2 in the processing of peptides in a variety of brain regions. Altogether, 115 neuropeptides or other peptides derived from secretory pathway proteins were identified. These peptides arise from 28 distinct secretory pathway proteins, including proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, and many others. Forty one of the peptides found in wild type mice were not detectable in any of the brain regions of PC2 knockout mice, and another twenty four peptides were present at levels ranging from 20–79% of wild type levels. Most of the other peptides were not substantially affected by the mutation, with levels ranging from 80–120% of wild type levels, and only three peptides were found to increase in one or more brain regions of PC2 knock-out mice. Taken together, these results are consistent with a broad role for PC2 in neuropeptide processing, but with functional redundancy for many of the cleavages. Comparison of the cleavage sites affected by the absence of PC2 confirms previous suggestions that sequences with a Trp, Tyr and/or Pro in the P1′ or P2′ position are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

Opioid receptor like-1 receptor (ORL 1 ) is selective for orphaninFQ/nociceptin (OFQ/N), a peptide linked to stress. Since immunologic stimuli exert stressor-like effects, the neuroendocrine and behavioral effects of the T-cell superantigen staphylococcal enterotoxin A (SEA) were tested in ORL 1 −/− and ORL 1 +/+ wildtype 129S6 mice. Within 2 h of SEA challenge both genotypes showed elevated corticosterone, but only wildtypes were elevated after 4 h, and had altered hypothalamic CRH mRNA. Although amygdaloid CRH and TNFα mRNA was increased by SEA, this did not vary with genotype. Interestingly, gustatory neophobia due to SEA challenge was augmented in ORL 1 −/− mice, although object neophobia tested 4 days later was abrogated. These results suggest differential requirements for ORL 1 in the mediation of neuroimmune effects exerted at different times after an immune challenge.

Amidated peptides are critically involved in many physiological functions. Genetic deletion of peptidylglycine alpha-amidating monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T(4) and pituitary TSH-beta mRNA levels, mice heterozygous for PAM (PAM(+/-)) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM(+/-) mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM(+/-) mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM(+/-) mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM(+/-) mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM(+/-) mice. This deficit was eliminated in PAM(+/-) mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans.

On the basis of numerous studies that have described interactions between the dopaminergic and opioidergic systems, we have investigated whether genetic deletion of dopamine D2 receptors (D2R) might influence the expression of central opioid receptors. The levels of mu, delta, kappa and nociceptin opioid peptide receptors were determined in the brains and spinal cords of D2R knockout mice using quantitative autoradiography. The significant changes in opioid receptor binding found in the brains of heterozygous and homozygous mice were mainly restricted to the basal ganglia. In homozygous mice, a down-regulation of mu and delta receptors was observed in the striatal and pallidal areas. This alteration may be an adaptive response to the increase in enkephalin levels previously described in the striatum of these mutant mice. On the contrary, an up-regulation of kappa receptors was found in the striatal and nigral regions and might be related to a change in dynorphin levels. Significant increases in nociceptin receptor binding were also observed in homozygous mice in brain areas involved in motor behavior. At the spinal level, only kappa and nociceptin receptor binding showed significant overall differences between genotypes. The functional consequences of these adaptive changes are discussed in relation to the findings of behavioral and neurochemical studies reported to date in D2R knockout mice.

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. It is currently unknown whether epidermal stem cells influence or are affected by skin aging. We therefore compared young and aged skin stem cell abundance, organization, and proliferation. We discovered that despite age-associated differences in epidermal proliferation, dermal thickness, follicle patterning, and immune cell abundance, epidermal stem cells were maintained at normal levels throughout life. These findings, coupled with observed dermal gene expression changes, suggest that epidermal stem cells themselves are intrinsically aging resistant and that local environmental or systemic factors modulate skin aging.

The mu opioid receptor (MOR) in the rat and mouse caudate putamen (CPu) and thalamus was demonstrated as diffuse and broad bands by Western blot with a polyclonal antibody against a C-terminal peptide of MOR, which were absent in the cerebellum and brains of MOR-knockout mice. The electrophoretic mobility of MOR differed in the two brain regions with median relative molecular masses (Mr's) of 75 kDa (CPu) vs. 66 kDa (thalamus) for the rat, and 74 kDa (CPu) vs. 63 kDa (thalamus) for the mouse, which was due to its differential N-glycosylation. Rat MOR in CPu was found mainly associated with low-density cholesterol- and ganglioside M1 (GM1)-enriched membrane subdomains (lipid rafts), while the MOR in the thalamus was present in rafts and non-rafts without preference. Cholesterol reduction by methyl-beta-cyclodextrin decreased DAGMO-induced [35S]GTPgammaS binding in rat CPu membranes to a greater extent than in the thalamus membranes.

Analgesic effects of delta opioid receptor (DOR) -selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) -knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266-273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II-induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR's effects was also independent of beta-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.

Vessel loss precipitates many diseases. In particular, vessel loss resulting in hypoxia induces retinal neovascularization in diabetic retinopathy and in retinopathy of prematurity (ROP), major causes of blindness. Here we define insulin-like growth factor binding protein-3 (IGFBP3) as a new modulator of vascular survival and regrowth in oxygen-induced retinopathy. In IGFBP3-deficient mice, there was a dose-dependent increase in oxygen-induced retinal vessel loss. Subsequent to oxygen-induced retinal vessel loss, Igfbp3 −/− mice had a 31% decrease in retinal vessel regrowth versus controls after returning to room air. No difference in serum insulin-like growth factor 1 (IGF1) levels was observed among groups. Wild-type mice treated with exogenous IGFBP3 had a significant increase in vessel regrowth. This correlated with a 30% increase in endothelial progenitor cells in the retina at postnatal day 15, indicating that IGFBP3 could be serving as a progenitor cell chemoattractant. In a prospective clinical study, we measured IGFBP3 (and IGF1) plasma levels weekly and examined retinas in all premature infants born at gestational ages , n = 13) was 802 μg/liter, and for infants with no ROP (ROP stage 0, n = 38) the mean level was 974 μg/liter ( P < 0.03). These results suggest that IGFBP3, acting independently of IGF1, helps to prevent oxygen-induced vessel loss and to promote vascular regrowth after vascular destruction in vivo in a dose-dependent manner, resulting in less retinal neovascularization.

IGF binding protein-5 (IGFBP-5) modulates the availability of IGF-I to its receptor and potentiates the intestinotrophic action of IGF-I. Our aim was to test the hypothesis that stimulation of intestinal growth due to coinfusion of IGF-I with total parenteral nutrition (TPN) solution is dependent on increased expression of IGFBP-5 through conducting our studies in IGFBP-5 knockout (KO) mice. IGFBP-5 KO, heterozygote (HT) and wild type (WT) male and female mice were maintained with TPN or TPN plus coinfusion of IGF-I [recombinant human (rh)IGF-I; 2.5 mg·kg−1·day−1] for 5 days. The concentration of IGF-I in serum was 73% greater ( P < 0.0001) in mice given TPN + IGF-I infusion compared with TPN alone. IGF-I attenuated the 2–3 g loss of body weight associated with TPN in WT mice, whereas KO and HT mice did not show improvement in body weight with IGF-I treatment. KO and HT mice had significantly greater levels of circulating IGF-I binding proteins (IGFBPs) compared with WT mice. Intestinal growth due to IGF-I was observed in all groups treated with IGF-I based on greater concentrations of protein and DNA in small intestine and colon and significantly greater crypt depth and muscularis thickness in jejunum. Jejunal expression of IGFBP-5 mRNA was greater in WT mice, whereas IGFBP-3 mRNA was greater in KO mice treated with IGF-I. In summary, the absence of the IGFBP-5 gene did not block the ability of IGF-I to stimulate intestinal growth, possibly because greater jejunal expression of IGFBP-3 compensates for the absence of IGFBP-5.

Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo , affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the β-endorphin peptides derived from the proopiomelanocortin ( Pomc ) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid β-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.

Opioids are extensively used analgesics yet can paradoxically increase pain sensitivity in humans and rodents. This hyperalgesia is extensively conceptualized to be a consequence of opioid receptor activity, perhaps providing an adaptive response to analgesia, and to utilize N -methyl- d -aspartate (NMDA) receptors. These assumptions were tested here in opioid receptor triple knock-out (KO) mice lacking all three genes encoding opioid receptors (μ, δ, and κ) by comparing their thermal nociceptive responses to the opioids morphine and oxymorphone with those of B6129F 1 controls. Injecting acute opioid bolus doses in controls caused maximal analgesia that was completely abolished in KO mice, confirming the functional consequence of the KO mouse opioid receptor deficiency. Continuous opioid infusion by osmotic pump in control mice also initially caused several consecutive days of analgesia that was shortly thereafter followed by several consecutive days of hyperalgesia. In contrast, continuously infusing KO mice with opioids caused no detectable analgesic response, but only immediate and steady declines in nociceptive thresholds culminating in several days of unremitting hyperalgesia. Finally, injecting the non-competitive NMDA receptor antagonist MK-801 during opioid infusion markedly reversed hyperalgesia in control but not KO mice. These data demonstrate that sustained morphine and oxymorphone delivery causes hyperalgesia independently of prior or concurrent opioid or NMDA receptor activity or opioid analgesia, indicating the contribution of mechanisms outside of current conceptions, and are inconsistent with proposals of hyperalgesia as a causative factor of opioid analgesic tolerance.

IGFs (IGF-I and IGF-II) are essential for development, and their bioactivities are tightly regulated by six related IGF-binding proteins (IGFBPs). IGFBP-5 is the most highly conserved binding protein and is expressed in several key developmental lineages as well as in multiple adult tissues including the mammary gland. To explore IGFBP-5 actions in vivo, we produced IGFBP-5 knockout (KO) mice. Whole-body growth, selected organ weights, and body composition were essentially normal in IGFBP-5 KO mice, presumably because of substantial compensation by remaining IGFBP family members. The IGFBP-5 KO mice also exhibited normal mammary gland development and were capable of nursing their pups. We then directly evaluated the proposed role of IGFBP-5 in apoptosis and remodeling of mammary gland during involution. We found that the process of involution after forced weaning was delayed in IGFBP-5 KO mice, with both the appearance of apoptotic cells and the reappearance of adipocytes retarded in mutant mice, compared with controls. We also determined the effects of IGFBP-5 deletion on mammary gland development in pubertal females after ovariectomy and stimulation with estradiol/progesterone. In this paradigm, IGFBP-5 KO mammary glands exhibited enhanced alveolar bud formation consistent with enhanced IGF-I action. These results demonstrate that IGFBP-5, although not essential for normal growth, is required for normal mammary gland involution and can regulate mammary gland morphogenesis in response to hormone stimulation.

Prohormone convertase (PC) 1/3 and 2 are involved in the generation of neuropeptides from their precursors. A quantitative peptidomic approach was used to explore the role PC2 plays in the processing of hypothalamic peptides. In this approach, extracts from mice lacking PC2 activity and from wild-type littermates were labeled with isotopic tags, combined, fractionated on a reverse phase HPLC column, and analyzed by electrospray ionization mass spectrometry. Altogether, 53 neuropeptides or other peptides derived from secretory pathway proteins were identified and sequenced using tandem mass spectrometry. These peptides arise from 21 distinct proteins: proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, promelanin-concentrating hormone, proneurotensin, proneuropeptide Y, provasopressin, pronociceptin/orphanin, prothyrotropin-releasing hormone, cocaine- and amphetamine-regulated transcript, chromogranin A and B, secretogranin II, prohormone convertase 1 and 2, propeptidyl-amidating monooxygenase, and proteins designated proSAAS and VGF. Approximately one third of the peptides found in wild-type mice were not detectable in PC2 knock-out mice, and another third were present at levels ranging from 25 to 75% of wild-type levels. Comparison of the cleavage sites suggests that sequences with a Trp, Tyr and/or Pro in the P1' or P2' position, or a basic residue in the P3 position, are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

Salvia divinorum is a natural occurring hallucinogen that is traditionally used by the Mazatec Indians of central Mexico. The diterpene salvinorin A was identified as an active component of S. divinorum over 20 years ago, but only recently has biochemical screening indicated that a molecular target of salvinorin A in vitro is the kappa-opioid receptor. We have examined whether salvinorin A, the C2-substituted derivative salvinorinyl-2-propionate, and salvinorin B can act as kappa-opioid receptor agonists in vivo. We found that following intracerebroventricular injection over a dose range of 1 to 30 microg of both salvinorin A and salvinorinyl-2-propionate produces antinociception in wild-type mice but not in a novel strain of kappa-opioid receptor knockout mice. Moreover, both salvinorin A and salvinorinyl-2-propionate reduce rectal body temperature, similar to conventional kappa-opioid receptor agonists, in a genotype-dependent manner. In addition, we determined that salvinorin A has high affinity for kappa 1- but not kappa 2-opioid receptors, demonstrating selectivity for this receptor subclass. Finally, treatment over the same dose range with salvinorin B, which is inactive in vitro, produced neither antinociceptive nor hypothermic effects in wild-type mice. These data demonstrate that salvinorin A is the active component of S. divinorum, selective for kappa(1)-opioid receptors, and that salvinorin A and specific structurally related analogs produce behavioral effects that require the kappa-opioid receptor.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of peptide hormones. We previously had found high expression of PAM in several regions of the developing rodent. To determine the function of PAM during mouse embryogenesis, we produced a null mutant of the PAM gene. Homozygous mutants die in utero between e14.5 and e15.5 with severe edema that is likely due to cardiovascular deficits. These defects include thinning of the aorta and carotid arteries and are very similar to those of the recently characterized adrenomedullin (AM) gene KO despite the presence of elevated immunoreactive AM in PAM KO embryos. No peptide amidation activity was detected in PAM mutant embryos, and there was no moderation of the AM-like phenotype that could be expected if any alternative peptide amidation mechanism exists in the mouse. Despite the proposed contribution of amidated peptides to neuronal cell proliferation, no alteration in neuroblast proliferation was observed in homozygous mutant embryos prior to lethality. Mice heterozygous for the mutant PAM allele develop normally and express wildtype levels of several amidated peptides despite having one half the wildtype levels of PAM activity and PAM protein. Nonetheless, both an increase in adiposity and a mild glucose intolerance developed in aged (>10 months) heterozygous mice compared to littermate controls. Ablation of PAM thus demonstrates an essential function for this gene during mouse development, while alterations in PAM activity in the adult may underlie more subtle physiologic effects.

Repeated forced-swim stress (FSS) produced analgesia, immobility and potentiation of cocaine-conditioned place preference (CPP) in wild-type C57Bl/6 mice, but not in littermates lacking the kappa opioid receptor (KOR) gene. These results were surprising because kappa agonists are known to produce conditioned place aversion and to suppress cocaine-CPP when coadministered with cocaine. The possibility that disruption of the kappa system blocked the stress response by adversely affecting the hypothalamic-pituitary axis was examined by measuring plasma corticosterone levels. However, disruption of the dynorphin/kappa system by gene deletion or receptor antagonism did not reduce the FSS-induced elevation of plasma corticosterone levels. A second explanation for the difference is that kappa receptor activation caused by FSS occurred prior to cocaine conditioning rather than contemporaneously. To test this hypothesis, we measured the effects of the kappa agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) administered to mice at various intervals preceding cocaine conditioning. The results showed that the interaction between the kappa system and cocaine reinforcement depended on the timing of the drug pairing. Mice given U50,488 60 min prior to cocaine showed a robust, nor-BNI-sensitive potentiation of cocaine-CPP, whereas administration 15 min before cocaine significantly suppressed cocaine-CPP. In the absence of cocaine, U50,488 given 60 min prior to saline conditioning produced no place preference, whereas administration 15 min before saline conditioning produced significant place aversion. The results of this study suggest that kappa receptor activation induced by FSS prior to the cocaine-conditioning session may be both necessary and sufficient for potentiation of the reinforcing actions of cocaine.

Genetic and pharmacological approaches were used to examine κ-opioid receptor (KOR-1) regulation of dopamine (DA) dynamics in the nucleus accumbens and vulnerability to cocaine. Microdialysis revealed that basal DA release and DA extraction fraction (Ed), an indirect measure of DA uptake, are enhanced in KOR-1 knock-out mice. Analysis of DA uptake revealed a decreasedKmbut unchangedVmaxin knock-outs. Knock-out mice exhibited an augmented locomotor response to cocaine, which did not differ from that of wild-types administered a behavioral sensitizing cocaine treatment. The ability of cocaine to increase DA was enhanced in knock-outs, whereas c-fos induction was decreased. Although repeated cocaine administration to wild types produced behavioral sensitization, knock-outs exhibited no additional enhancement of behavior. Administration of the long-acting KOR antagonist nor-binaltorphimine to wild-type mice increased DA dynamics. However, the effects varied with the duration of KOR-1 blockade. Basal DA release was increased whereasEdwas unaltered after 1 h blockade. After 24 h, release andEdwere increased. The behavioral and neurochemical effects of cocaine were enhanced at both time points.These data demonstrate the existence of an endogenous KOR-1 system that tonically inhibits mesoaccumbal DA neurotransmission. Its loss induces neuroadaptations characteristic of “cocaine-sensitized” animals, indicating a critical role of KOR-1 in attenuating responsiveness to cocaine. The increased DA uptake after pharmacological inactivation or gene deletion highlights the plasticity of mesoaccumbal DA neurons and suggests that loss of KOR-1 and the resultant disinhibition of DA neurons trigger short- and long-term DA transporter adaptations that maintain normal DA levels, despite enhanced release.

Dopamine neurons originating in the midbrain and projecting to cortico-limbic and motor structures are one of the major neuronal substrates implicated in the reinforcing properties of drugs of abuse. The output of this system is largely determined by its impulse activity (amount and pattern of firing activity). Several intrinsic and synaptic factors can influence dopamine neuronal activity and, consequently, addiction liability. Pharmacological studies indicate that mu-opioid receptors and their activation by endogenous opioids may play an important role. In the present study, we use a genetic approach to better understand the role of mu-opioid receptors in modulating dopamine neuronal activity in vivo. Using in vivo extracellular single-unit recordings, we show that mice lacking mu-opioid receptors exhibit lower firing rates of dopamine neurons compared with their wild-type littermates. Although we observed no overall changes in bursting activity compared with wild-type mice, animals lacking mu-opioid receptors exhibited a higher proportion of regular-spiking cells that lacked bursting activity. These findings are the first to emphasize the critical role of mu-opioid receptors in modulating action potential output of dopamine neurons in vivo using a genetic approach. They also provide a possible underlying mechanism for the decreased reinforcing properties of drugs of abuse that was previously observed in mice lacking mu-opioid receptors.

In vitro assays have demonstrated that peptides derived from the recently-identified proSAAS precursor inhibit prohormone convertase 1 (PC1) suggesting that this novel peptide may function as an endogenous inhibitor of PC1. To further understand the role of proSAAS in vivo, we have investigated the expression of proSAAS mRNA and processing of proSAAS during pre- and early postnatal rodent development. In situ hybridization showed that, by embryonic day 12.5 (e12.5) in the rat, proSAAS mRNA was present in essentially all differentiating neurons in the mantle layer of the myelencephalon, metencephalon, diencephalon, spinal cord and several sympathetic ganglia. During later stages of prenatal development, widespread proSAAS expression continues in post-mitotic neurons of both the CNS and PNS and begins in endocrine cells of the anterior and intermediate pituitary. Although proSAAS expression overlaps with PC1 in several regions, its overall expression pattern is significantly more extensive, suggesting that proSAAS may be multifunctional during development. Processed forms of proSAAS are present by at least mid-gestation with marked accumulation of two C-terminal forms, comprising the PC1 inhibitory fragment of proSAAS.

Peptide growth factors regulate cell fate by activating distinct signal transduction pathways that ultimately influence gene expression. Insulin-like growth factors (IGFs) play central roles in controlling somatic growth and participate in skeletal muscle development and regeneration. In cultured muscle cells, IGF action is critical both for maintaining viability during the transition from proliferating to differentiating myoblasts and for facilitating differentiation. By contrast, platelet-derived growth factor (PDGF) can sustain cell survival but inhibits differentiation. Here we examine the genetic programs that accompany IGF and PDGF action in myoblasts. Through analysis of high-density oligonucleotide arrays containing approximately 36,000 mouse probe sets, we identify 90 transcripts differentially induced by IGF-I, including 28 muscle-specific genes and 33 previously unannotated mRNAs, and 55 transcripts specifically stimulated by PDGF, including 14 unknowns. Detailed study of one IGF-induced mRNA shows that it encodes a protein related to a recently characterized repulsive guidance molecule postulated to regulate neuronal targeting during development. Our results demonstrate the power of transcriptional profiling for gene discovery and provide opportunities for investigating new proteins potentially involved in different aspects of growth factor action in muscle.