Opioid-induced mitogen-activated protein kinase signaling in rat enteric neurons following chronic morphine treatment.

Opioids, acting at μ opioid receptors, are commonly used for pain management. Chronic opioid treatment induces cellular adaptations, which trigger long-term side effects, including constipation mediated by enteric neurons. We tested the hypothesis that chronic opioid treatment induces alterations of μ opioid receptor signaling in enteric neurons, which are likely to serve as mechanisms underlying opioid-induced constipation. In cultured rat enteric neurons, either untreated (naïve) or exposed to morphine for 4 days (chronic), we compared the effect of morphine and DAMGO (D-Ala2,MePhe4,Gly-ol5 enkephalin) on μ opioid receptor internalization and downstream signaling by examining the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinases 1 and 2 (MAPK/ERK) pathway, cAMP accumulation and transcription factor cAMP Response Element-Binding protein (CREB) expression. μ opioid receptor internalization and MAPK/ERK phosphorylation were induced by DAMGO, but not morphine in naïve neurons, and by both opioids in chronic neurons. MAPK/ERK activation was prevented by the receptor antagonist naloxone, by blocking receptor trafficking with hypertonic sucrose, dynamin inhibitor, or neuronal transfection with mutated dynamin, and by MAPK inhibitor. Morphine and DAMGO inhibited cAMP in naïve and chronic enteric neurons, and induced desensitization of cAMP signaling. Chronic morphine treatment suppressed desensitization of cAMP and MAPK signaling, increased CREB phosphorylation through a MAPK/ERK pathway and induced delays of gastrointestinal transit, which was prevented by MAPK/ERK blockade. This study showed that opioids induce endocytosis- and dynamin-dependent MAPK/ERK activation in enteric neurons and that chronic morphine treatment triggers changes at the receptor level and downstream signaling resulting in MAPK/ERK-dependent CREB activation. Blockade of this signaling pathway prevents the development of gastrointestinal motility impairment induced by chronic morphine treatment. These findings suggest that alterations in μ opioid receptor downstream signaling including MAPK/ERK pathway in enteric neurons chronically treated with morphine contribute to the development of opioid-induced constipation

PKG1α oxidation negatively regulates food seeking behaviour and reward

Genes that are highly conserved in food seeking behaviour, such as protein kinase G (PKG), are of interest because of their potential role in the global obesity epidemic. PKG1α can be activated by binding of cyclic guanosine monophosphate (cGMP) or oxidant-induced interprotein disulfide bond formation between the two subunits of this homodimeric kinase. PKG1α activation by cGMP plays a role in reward and addiction through its actions in the ventral tegmental area (VTA) of the brain. ‘Redox dead’ C42S PKG1α knock-in (KI) mice, which are fully deficient in oxidant- induced disulfide-PKG1α formation, display increased food seeking and reward behaviour compared to wild-type (WT) littermates. Rewarding monoamines such as dopamine, which are released during feeding, are metabolised by monoamine oxidase to generate hydrogen peroxide that was shown to mediate PKG1α oxidation. Indeed, inhibition of monoamine oxidase, which prevents it producing hydrogen peroxide, attenuated PKG1α oxidation and increased sucrose preference in WT, but not KI mice. The deficient reward phenotype of the KI mice was rescued by expressing WT kinase that can form the disulfide state in the VTA using an adeno-associated virus, consistent with PKG1α oxidation providing a break on feeding behaviour. In conclusion, disulfide-PKG1α in VTA neurons acts as a negative regulator of feeding and therefore may provide a novel therapeutic target for obesity

Desensitization of μOR signaling in enteric neurons.

<p>A: Single exposure to DAMGO (1 µM, 5 min) induced significant MAPK activation in naïve enteric neurons, whereas a second exposure to the same DAMGO dose following 2 hours DAMGO pretreatment abolished DAMGO-mediated MAPK response, indicating desensitization. B: Single exposure to DAMGO (1 µM) or morphine (10 µM) activated MAPK in chronic neurons. A second exposure to the same dose of DAMGO or morphine following 2 hours DAMGO or morphine pretreatment induced the same effect in chronic neurons as single exposures, indicating suppression of desensitization. (** p<0.01 vs. control in A and B). C and D: DAMGO and morphine inhibit forskolin-stimulated cAMP in naïve (C) and chronic (D) enteric neurons. This effect was not observed in naïve enteric neurons (C) with a second opioid stimulation following a prior 2 hour exposure, indicative of desensitization. D: Note the over 2 fold increase in cAMP in unstimulated chronic neurons (cAMP superactivation or “overshooting”) vs. naïve control; DAMGO and morphine inhibition of cAMP was not prevented by 2 hours DAMGO or morphine pretreatment in chronic neurons, indicating suppression of desensitization. **p<0.01 vs. controls. N = 5–7 experiments performed in duplicate per group.</p

Opioid-induced MAPK activation in naïve (A) and chronically treated (B) enteric neurons.

<p>DAMGO (1 µM, black bars) induced a transient MAPK/ERK1/2 activation in naïve (A) and chronic (B) neurons at 5 and 10 minutes, whereas morphine (grey bars) induced MAPK/ERK1/2 activation only in chronic (B) neurons. **p<0.01 compared to controls (white bars). N = 4–7 experiments in triplicate. Representative gels of pERK1/2 and tERK are shown at the bottom of each graph. tERK was used to verify that the treatment did not affect the total level of this protein and to confirm equal gel loading.</p

Effect of opioids on CREB phosphorylation in enteric neurons.

<p>Naïve (A) and chronic (B) neurons were stimulated with 1 µM DAMGO, 10 µM morphine or medium (control) for 0–20 minutes. DAMGO and morphine induced a significant, transient CREB activation in chronic, but not naïve enteric neurons. CREB phosphorylation in chronic neurons was blocked by the MEK1/2 inhibitor (U0126) treatment. *p<0.05 and **p<0.01 vs. control; n = 4–7 experiments in triplicate per group. Representative gels of pCREB and CREB are shown at the bottom of the figure. Total CREB was used to verify that the treatment did not affect the total level of this protein and to confirm equal gel loading.</p

μOR immunoreactivity in naïve enteric neurons (A–C) and in neurons chronically treated with morphine (D–F).

<p>μOR immunoreactivity is at the cell surface in unstimulated and morphine-stimulated neurons (A, C arrows), and it is in the cytosol following stimulation with DAMGO (B) in naïve enteric neurons. μOR immunoreactivity is at the cell surface in unstimulated neurons (D, arrows), but in the cytosol following DAMGO or morphine stimulation (E, F) in chronic enteric neurons.</p

Increased expression of the HDAC 9 gene is associated with antiestrogen‐resistance of breast cancers

International audienceEstrogens play a pivotal role in breast cancer etiology, and endocrine therapy remains the main first line treatment for estrogen receptor‐alpha (ER α)‐positive breast cancer. ER are transcription factors whose activity is finely regulated by various regulatory complexes, including histone deacetylases (HDACs). Here, we investigated the role of HDAC 9 in ER α signaling and response to antiestrogens in breast cancer cells. Various Michigan Cancer Foundation‐7 (MCF 7) breast cancer cell lines that overexpress class II a HDAC 9 or that are resistant to the partial antiestrogen 4‐hydroxy‐tamoxifen (OHTam) were used to study phenotypic changes in response to ER ligands by using transcriptomic and gene set enrichment analyses. Kaplan–Meier survival analyses were performed using public transcriptomic datasets from human breast cancer biopsies. In MCF 7 breast cancer cells, HDAC 9 decreased ER α mRNA and protein expression and inhibited its transcriptional activity. Conversely, HDAC 9 mRNA was strongly overexpressed in OHT am‐resistant MCF 7 cells and in ER α‐negative breast tumor cell lines. Moreover, HDAC 9‐overexpressing cells were less sensitive to OHT am antiproliferative effects compared with parental MCF 7 cells. Several genes (including MUC 1, SMC 3 and S100P) were similarly deregulated in OHT am‐resistant and in HDAC 9‐overexpressing MCF 7 cells. Finally, HDAC 9 expression was positively associated with genes upregulated in endocrine therapy‐resistant breast cancers and high HDAC 9 levels were associated with worse prognosis in patients treated with OHT am. These results demonstrate the complex interactions of class II a HDAC 9 with ER α signaling in breast cancer cells and its effect on the response to hormone therap

Mu-opioid receptors and dietary protein stimulate a gut-brain neural circuitry limiting food intake.

International audienceIntestinal gluconeogenesis is involved in the control of food intake. We show that mu-opioid receptors (MORs) present in nerves in the portal vein walls respond to peptides to regulate a gut-brain neural circuit that controls intestinal gluconeogenesis and satiety. In vitro, peptides and protein digests behave as MOR antagonists in competition experiments. In vivo, they stimulate MOR-dependent induction of intestinal gluconeogenesis via activation of brain areas receiving inputs from gastrointestinal ascending nerves. MOR-knockout mice do not carry out intestinal gluconeogenesis in response to peptides and are insensitive to the satiety effect induced by protein-enriched diets. Portal infusions of MOR modulators have no effect on food intake in mice deficient for intestinal gluconeogenesis. Thus, the regulation of portal MORs by peptides triggering signals to and from the brain to induce intestinal gluconeogenesis are links in the satiety phenomenon associated with alimentary protein assimilation