Pharmacological and phytochemical insights on the pancreatic ß-cell modulation by Angelica L. roots.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica roots are a significant source of traditional medicines for various cultures around the northern hemisphere, from indigenous communities in North America to Japan. Among its many applications, the roots are used to treat type 2 diabetes mellitus; however, this application is not mentioned often. Ethnopharmacological studies have reported the use of A. japonica var. hirsutiflora, A. furcijuga, A. shikokiana, and A. keiskei to treat diabetes symptoms, and further reports have demonstrated the three angelica roots, i.e., A. japonica var. hirsutiflora, A. reflexa, and A. dahurica, exhibit insulin secretagogue activity. AIM OF THE STUDY: This study aimed to phytochemically characterize and compare angelica roots monographed in the European Pharmacopeia 11th, isolate major plant metabolites, and assess extracts and isolates' capability to modulate pancreatic ß-cell function. MATERIALS AND METHODS: Root extracts of Angelica archangelica, Angelica dahurica, Angelica biserrata, and Angelica sinensis were phytochemically profiled using liquid chromatography method coupled with mass spectrometry. Based on this analysis, simple and furanocoumarins were isolated using chromatography techniques. Extracts (1.6-50 µg/mL) and isolated compounds (5-40 µmol/L) were studied for their ability to modulate insulin secretion in the rat insulinoma INS-1 pancreatic ß-cell model. Insulin was quantified by the homogeneous time-resolved fluorescence method. RESULTS: Forty-one secondary metabolites, mostly coumarins, were identified in angelica root extracts. A. archangelica, A. dahurica, and A. biserrata root extracts at concentration of 12.5-50 µg/mL potentiated glucose-induced insulin secretion, which correlated with their high coumarin content. Subsequently, 23 coumarins were isolated from these roots and screened using the same protocol. Coumarins substituted with the isoprenyl group were found to be responsible for the extracts' insulinotropic effect. CONCLUSIONS: Insulinotropic effects of three pharmacopeial angelica roots were found, the metabolite profiles and pharmacological activities of the roots were correlated, and key structures responsible for the modulation of pancreatic ß-cell function were identified. These findings may have implications for the traditional use of angelica roots in treating diabetes. Active plant metabolites may also become lead structures in the search for new antidiabetic treatments.

Design and characterization of a triazole-based growth hormone secretagogue receptor modulator inhibiting the glucoregulatory and feeding actions of ghrelin.

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that regulates essential physiological functions. In particular, activation of GHSR in response to its endogenous agonist ghrelin promotes food intake and blood glucose increase. Therefore, compounds aimed at blocking GHSR signaling constitute potential options against obesity-related metabolic disorders. We have previously developed potent ligands of GHSR based on a triazole scaffold. Here, we report a new 3,4,5-trisubstituted 1,2,4-triazole compound, named JMV 6616, that potently blocks GHSR activity in vitro and in vivo. Specifically, in HEK293T cells JMV 6616 behaves as an inverse agonist since it binds to GHSR and inhibits its ghrelin-independent signaling. Accordingly, using purified labeled GHSR assembled into lipid nanodiscs we found that JMV 6616 decreases GHSR-catalyzed G protein activation and stabilizes an inactive receptor conformation. Importantly, JMV 6616 also acts on native GHSR since it blocks the insulinostatic effect of ghrelin in pancreatic islets. In mice, JMV 6616 inhibits blood glucose-raising effects of ghrelin treatment and the orexigenic actions of acute ghrelin administration. Together, our data suggest that this triazole-derived modulator of GHSR holds promise to mitigate several pathological features associated with eating and metabolic disorders.

Liver-Expressed Antimicrobial Peptide 2 antagonizes the insulinostatic effect of ghrelin in rat isolated pancreatic islets.

The hormone ghrelin is the endogenous agonist of the G protein-coupled receptor (GPCR) termed growth hormone secretagogue receptor (GHSR). Ghrelin inhibits glucose-stimulated insulin secretion by activating pancreatic GHSR. Recently, Liver-Expressed Antimicrobial Peptide 2 (LEAP2) was recognized as an endogenous GHSR ligand that blocks ghrelin-induced actions. Nonetheless, the effect of LEAP2 on glucose-stimulated insulin secretion from pancreatic islets is unknown. We aimed at exploring the activity of LEAP2 on glucose-stimulated insulin secretion. Islets of Langerhans isolated from rat pancreas were exposed to glucose in the presence or in the absence of LEAP2 and ghrelin and then insulin secretion was assayed. LEAP2 did not modulate glucose-stimulated insulin secretion. However, LEAP2 blocked the insulinostatic action of ghrelin. Our data show that LEAP2 behaves as an antagonist of pancreatic GHSR.

Development of Nonpeptidic Inverse Agonists of the Ghrelin Receptor (GHSR) Based on the 1,2,4-Triazole Scaffold.

GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding, and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4, and 5, this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.

Urolithin C increases glucose-induced ERK activation which contributes to insulin secretion.

Polyphenols exert pharmacological actions through protein-mediated mechanisms and by modulating intracellular signalling pathways. We recently showed that a gut-microbial metabolite of ellagic acid named urolithin C is a glucose-dependent activator of insulin secretion acting by facilitating L-type Ca2+ channel opening and Ca2+ influx into pancreatic ß-cells. However, it is still unknown whether urolithin C regulates key intracellular signalling proteins in ß-cells. Here, we report that urolithin C enhanced glucose-induced extracellular signal-regulated kinases 1/2 (ERK1/2) activation as shown by higher phosphorylation levels in INS-1 ß-cells. Interestingly, inhibition of ERK1/2 with two structurally distinct inhibitors led to a reduction in urolithin C effect on insulin secretion. Finally, we provide data to suggest that urolithin C-mediated ERK1/2 phosphorylation involved insulin signalling in INS-1 cells. Together, these data indicate that the pharmacological action of urolithin C on insulin secretion relies, in part, on its capacity to enhance glucose-induced ERK1/2 activation. Therefore, our study extends our understanding of the pharmacological action of urolithin C in ß-cells. More generally, our findings revealed that urolithin C modulated the activation of key multifunctional intracellular signalling kinases which participate in the regulation of numerous biological processes.

GPCR and Alcohol-Related Behaviors in Genetically Modified Mice.

G protein-coupled receptors (GPCRs) constitute the largest class of cell surface signaling receptors and regulate major neurobiological processes. Accordingly, GPCRs represent primary targets for the treatment of brain disorders. Several human genetic polymorphisms affecting GPCRs have been associated to different components of alcohol use disorder (AUD). Moreover, GPCRs have been reported to contribute to several features of alcohol-related behaviors in animal models. Besides traditional pharmacological tools, genetic-based approaches mostly aimed at deleting GPCR genes provided substantial information on how key GPCRs drive alcohol-related behaviors. In this review, we summarize the alcohol phenotypes that ensue from genetic manipulation, in particular gene deletion, of key GPCRs in rodents. We focused on GPCRs that belong to fundamental neuronal systems that have been shown as potential targets for the development of AUD treatment. Data are reviewed with particular emphasis on alcohol reward, seeking, and consumption which are behaviors that capture essential aspects of AUD. Literature survey indicates that in most cases, there is still a gap in defining the intracellular transducers and the functional crosstalk of GPCRs as well as the neuronal populations in which their signaling regulates alcohol actions. Further, the implication of only a few orphan GPCRs has been so far investigated in animal models. Combining advanced pharmacological technologies with more specific genetically modified animals and behavioral preclinical models is likely necessary to deepen our understanding in how GPCR signaling contributes to AUD and for drug discovery.

The ellagitannin metabolite urolithin C is a glucose-dependent regulator of insulin secretion through activation of L-type calcium channels.

BACKGROUND AND PURPOSE: The pharmacology of polyphenol metabolites on beta-cell function is largely undetermined. We sought to identify polyphenol metabolites that enhance the insulin-secreting function of beta-cells and to explore the underlying mechanisms. EXPERIMENTAL APPROACH: INS-1 beta-cells and rat isolated islets of Langerhans or perfused pancreas preparations were used for insulin secretion experiments. Molecular modelling, intracellular Ca2+ monitoring, and whole-cell patch-clamp recordings were used for mechanistic studies. KEY RESULTS: Among a set of polyphenol metabolites, we found that exposure of INS-1 beta-cells to urolithins A and C enhanced glucose-stimulated insulin secretion. We further characterized the activity of urolithin C and its pharmacological mechanism. Urolithin C glucose-dependently enhanced insulin secretion in isolated islets of Langerhans and perfused pancreas preparations. In the latter, enhancement was reversible when glucose was lowered from a stimulating to a non-stimulating concentration. Molecular modelling suggested that urolithin C could dock into the Cav 1.2 L-type Ca2+ channel. Calcium monitoring indicated that urolithin C had no effect on basal intracellular Ca2+ but enhanced depolarization-induced increase in intracellular Ca2+ in INS-1 cells and dispersed cells isolated from islets. Electrophysiology studies indicated that urolithin C dose-dependently enhanced the L-type Ca2+ current for levels of depolarization above threshold and shifted its voltage-dependent activation towards more negative potentials in INS-1 cells. CONCLUSION AND IMPLICATIONS: Urolithin C is a glucose-dependent activator of insulin secretion acting by facilitating L-type Ca2+ channel opening and Ca2+ influx into pancreatic beta-cells. Our work paves the way for the design of polyphenol metabolite-inspired compounds aimed at ameliorating beta-cell function.

N-Terminal Liver-Expressed Antimicrobial Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the Ghrelin Receptor.

The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

Activation of the cAMP Pathway Induces RACK1-Dependent Binding of ß-Actin to BDNF Promoter.

RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified ß-actin as a direct RACK1 binding partner and found that the association between ß-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that ß-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of ß-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that ß-actin is a new RACK1 binding partner and that the RACK1 and ß-actin association participate in the cAMP-dependent regulation of BDNF transcription.

The novel nonapeptide acein targets angiotensin converting enzyme in the brain and induces dopamine release.

BACKGROUND AND PURPOSE: Using an in-house bioinformatics programme, we identified and synthesized a novel nonapeptide, H-Pro-Pro-Thr-Thr-Thr-Lys-Phe-Ala-Ala-OH. Here, we have studied its biological activity, in vitro and in vivo, and have identified its target in the brain. EXPERIMENTAL APPROACH: The affinity of the peptide was characterized using purified whole brain and striatal membranes from guinea pigs and rats . Its effect on behaviour in rats following intra-striatal injection of the peptide was investigated. A photoaffinity UV cross-linking approach combined with subsequent affinity purification of the ligand covalently bound to its receptor allowed identification of its target. KEY RESULTS: The peptide bound with high affinity to a single class of binding sites, specifically localized in the striatum and substantia nigra of brains from guinea pigs and rats. When injected within the striatum of rats, the peptide stimulated in vitro and in vivo dopamine release and induced dopamine-like motor effects. We purified the target of the peptide, a ~151 kDa protein that was identified by MS/MS as angiotensin converting enzyme (ACE I). Therefore, we decided to name the peptide acein. CONCLUSION AND IMPLICATIONS: The synthetic nonapeptide acein interacted with high affinity with brain membrane-bound ACE. This interaction occurs at a different site from the active site involved in the well-known peptidase activity, without modifying the peptidase activity. Acein, in vitro and in vivo, significantly increased stimulated release of dopamine from the brain. These results suggest a more important role for brain ACE than initially suspected.

mTOR complex 1: a key player in neuroadaptations induced by drugs of abuse.

The mammalian (or mechanistic) target of rapamycin (mTOR) complex 1 (mTORC1) is a serine and threonine kinase that regulates cell growth, survival, and proliferation. mTORC1 is a master controller of the translation of a subset of mRNAs. In the central nervous system mTORC1 plays a crucial role in mechanisms underlying learning and memory by controlling synaptic protein synthesis. Here, we review recent evidence suggesting that the mTORC1 signaling pathway promotes neuroadaptations following exposure to a diverse group of drugs of abuse including stimulants, cannabinoids, opiates, and alcohol. We further describe potential molecular mechanisms by which drug-induced mTORC1 activation may alter brain functions. Finally, we propose that mTORC1 is a focal point shared by drugs of abuse to mediate drug-related behaviors such as reward seeking and excessive drug intake, and offer future directions to decipher the contribution of the kinase to mechanisms underlying addiction. Recent studies suggesting that exposure to diverse classes of drugs of abuse as well as exposure to drug-associated memories lead to mTORC1 kinase activation in the limbic system. In turn, mTORC1 controls the onset and the maintenance of pathological neuroadaptions that underlie several features of drug addiction such as drug seeking and relapse. Therefore, we propose that targeting mTORC1 and its effectors is a promising strategy to treat drug disorders.

Disruption of alcohol-related memories by mTORC1 inhibition prevents relapse.

Relapse to alcohol abuse is an important clinical issue that is frequently caused by cue-induced drug craving. Therefore, disruption of the memory for the cue-alcohol association is expected to prevent relapse. It is increasingly accepted that memories become labile and erasable soon after their reactivation through retrieval during a memory reconsolidation process that depends on protein synthesis. Here we show that reconsolidation of alcohol-related memories triggered by the sensory properties of alcohol itself (odor and taste) activates mammalian target of rapamycin complex 1 (mTORC1) in select amygdalar and cortical regions in rats, resulting in increased levels of several synaptic proteins. Furthermore, systemic or central amygdalar inhibition of mTORC1 during reconsolidation disrupts alcohol-associated memories, leading to a long-lasting suppression of relapse. Our findings provide evidence that the mTORC1 pathway and its downstream substrates are crucial in alcohol-related memory reconsolidation and highlight this pathway as a therapeutic target to prevent relapse.

The small G protein H-Ras in the mesolimbic system is a molecular gateway to alcohol-seeking and excessive drinking behaviors.

Uncontrolled consumption of alcohol is a hallmark of alcohol abuse disorders; however, the central molecular mechanisms underlying excessive alcohol consumption are still unclear. Here, we report that the GTP binding protein, H-Ras in the nucleus accumbens (NAc) plays a key role in neuroadaptations that underlie excessive alcohol-drinking behaviors. Specifically, acute (15 min) systemic administration of alcohol (2.5 g/kg) leads to the activation of H-Ras in the NAc of mice, which is observed even 24 h later. Similarly, rat operant self-administration of alcohol (20%) also results in the activation of H-Ras in the NAc. Using the same procedures, we provide evidence suggesting that the exchange factor GRF1 is upstream of H-Ras activation by alcohol. Importantly, we show that infection of mice NAc with lentivirus expressing a short hairpin RNA that targets the H-Ras gene produces a significant reduction of voluntary consumption of 20% alcohol. In contrast, knockdown of H-Ras in the NAc of mice did not alter water, quinine, and saccharin intake. Furthermore, using two-bottle choice and operant self-administration procedures, we show that inhibiting H-Ras activity by intra-NAc infusion of the farnesyltransferase inhibitor, FTI-276, produced a robust decrease of rats' alcohol drinking; however, sucrose consumption was unaltered. Finally, intra-NAc infusion of FTI-276 also resulted in an attenuation of seeking for alcohol. Together, these results position H-Ras as a central molecular mediator of alcohol's actions within the mesolimbic system and put forward the potential value of the enzyme as a novel target to treat alcohol use disorders.

Direct interaction between scaffolding proteins RACK1 and 14-3-3ζ regulates brain-derived neurotrophic factor (BDNF) transcription.

RACK1 is a scaffolding protein that spatially and temporally regulates numerous signaling cascades. We previously found that activation of the cAMP signaling pathway induces the translocation of RACK1 to the nucleus. We further showed that nuclear RACK1 is required to promote the transcription of the brain-derived neurotrophic factor (BDNF). Here, we set out to elucidate the mechanism underlying cAMP-dependent RACK1 nuclear translocation and BDNF transcription. We identified the scaffolding protein 14-3-3ζ as a direct binding partner of RACK1. Moreover, we found that 14-3-3ζ was necessary for the cAMP-dependent translocation of RACK1 to the nucleus. We further observed that the disruption of RACK1/14-3-3ζ interaction with a peptide derived from the RACK1/14-3-3ζ binding site or shRNA-mediated 14-3-3ζ knockdown inhibited cAMP induction of BDNF transcription. Together, these data reveal that the function of nuclear RACK1 is mediated through its interaction with 14-3-3ζ. As RACK1 and 14-3-3ζ are two multifunctional scaffolding proteins that coordinate a wide variety of signaling events, their interaction is likely to regulate other essential cellular functions.

AKT signaling pathway in the nucleus accumbens mediates excessive alcohol drinking behaviors.

BACKGROUND: Neuroadaptations within the nucleus accumbens (NAc) have been implicated in molecular mechanisms underlying the development and/or maintenance of alcohol abuse disorders. We recently reported that the activation of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in the NAc of rodents, after exposure to alcohol, contributes to alcohol drinking behaviors. The kinase AKT is a main upstream activator of the mTORC1 pathway. We therefore hypothesized that the activation of AKT in the NAc in response to alcohol exposure plays an important role in mechanisms that underlie excessive alcohol consumption. METHODS: Western blot analysis was used to assess the phosphorylation levels of enzymes. Acute exposure of mice to alcohol was achieved by the administration of 2 g/kg alcohol intraperitoneally (i.p.). Two-bottle choice and operant self-administration procedures were used to assess drinking behaviors in rats. RESULTS: We found that acute systemic administration of alcohol and recurring cycles of excessive voluntary consumption of alcohol and withdrawal result in the activation of AKT signaling in the NAc of rodents. We show that inhibition of AKT or its upstream activator, phosphatidylinositol-3-kinase (PI3K), within the NAc of rats attenuates binge drinking as well as alcohol but not sucrose operant self-administration. CONCLUSIONS: Our results suggest that the activation of the AKT pathway in the NAc in response to alcohol exposure is an important contributor to the molecular mechanisms underlying alcohol-drinking behaviors. AKT signaling pathway inhibitors are therefore potential candidates for drug development for the treatment of alcohol use and abuse disorders.

Role for mammalian target of rapamycin complex 1 signaling in neuroadaptations underlying alcohol-related disorders.

Alcohol addiction is a chronically relapsing disorder that includes certain maladaptive learning and memory. The serine and threonine kinase complex, mammalian target of rapamycin complex 1 (mTORC1), has been implicated in synaptic plasticity, learning, and memory by controlling protein translation. Here we show that administration of alcohol and excessive voluntary consumption of alcohol induce the activation of the mTORC1-mediated signaling pathway in the nucleus accumbens (NAc) of rodents. We further show that the protein expression levels of GluR1 and Homer, two synaptic proteins whose translation has been shown to be modulated by mTORC1, are up-regulated in the NAc of rodents with a history of excessive alcohol consumption. In addition, our results document that the Food and Drug Administration-approved inhibitor of mTORC1, rapamycin, decreases expression of alcohol-induced locomotor sensitization and place preference, as well as excessive alcohol intake and seeking in preclinical rodent models of alcohol abuse. Together, our results suggest that mTORC1 within the NAc is a contributor to molecular mechanisms underlying alcohol-drinking behaviors. Furthermore, despite its massive health and socioeconomic impact worldwide, pharmacotherapies for alcohol abuse and addiction remain limited. Our data therefore put forward the possibility that targeting the mTORC1 signaling cascade is an innovative and valuable strategy for the treatment of alcohol use and abuse disorders.

Epigenetic regulation of BDNF expression via the scaffolding protein RACK1.

Scaffolding proteins are major contributors to the spatial and temporal orchestration of signaling cascades and hence cellular functions. RACK1 is a scaffolding protein that plays an important role in the regulation of, and cross-talk between, various signaling pathways. Here we report that RACK1 is a mediator of chromatin remodeling, resulting in an exon-specific expression of the brain-derived neurotrophic factor (BDNF) gene. Specifically, we found that following the activation of the cAMP pathway, nuclear RACK1 localizes at the promoter IV region of the BDNF gene by its association with histones H3 and H4, leading to the dissociation of the transcription repressor methyl-CpG-binding protein 2 (MeCP2) from the promoter, resulting in the acetylation of histone H4. These chromatin modifications lead to the activation of the promoter and to the subsequent promoter-controlled transcription of BDNF exon IV. Our findings expand our knowledge regarding the function of scaffolding proteins such as RACK1. Furthermore, this novel mechanism for the regulation of exon-specific expression of the BDNF gene by RACK1 could have implications on the neuronal functions of the growth factor including synaptic plasticity, learning, and memory.

Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells.

Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF(2)) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF(2) receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.

Effect of long-term exposure of SH-SY5Y cells to morphine: a whole cell proteomic analysis.

BACKGROUND: Opiate addiction reflects plastic changes that endurably alter synaptic transmission within relevant neuronal circuits. The biochemical mechanisms of these adaptations remain largely unknown and proteomics-based approaches could lead to a broad characterization of the molecular events underlying adaptations to chronic drug exposure. RESULTS: Thus, we have started proteomic analyses of the effects of chronic morphine exposure in a recombinant human neuroblastoma SH-SY5Y clone that stably overexpresses the mu-opioid receptor. Cells were treated with morphine for 6, 24 and 72 hours, the proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue, and the protein map was compared with that obtained from untreated cells. Spots showing a statistically significant variation were selected for identification using mass spectrometric analyses. CONCLUSION: A total of 45 proteins were identified, including proteins involved in cellular metabolism, cytoskeleton organization, vesicular trafficking, transcriptional and translational regulation, and cell signaling.

Long-term morphine treatment enhances proteasome-dependent degradation of G beta in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization.

The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role.