Functional brain-wide network mapping during acute stress exposure in rats: Interaction between the lateral habenula and cortical, amygdalar, hypothalamic and monoaminergic regions.

Upon stress exposure, a broad network of structures comes into play in order to provide adequate responses and restore homeostasis. It has been known for decades that the main structures engaged during the stress response are the medial prefrontal cortex, the amygdala, the hippocampus, the hypothalamus, the monoaminergic systems (noradrenaline, dopamine and serotonin) and the periaqueductal gray. The lateral habenula (LHb) is an epithalamic structure directly connected to prefrontal cortical areas and to the amygdala, whereas it functionally interacts with the hippocampus. Also, it is a main modulator of monoaminergic systems. The LHb is activated upon exposure to basically all types of stressors, suggesting it is also involved in the stress response. However, it remains unknown if and how the LHb functionally interacts with the broad stress response network. In the current study we performed in rats a restraint stress procedure followed by immunohistochemical staining of the c-Fos protein throughout the brain. Using graph theory-based functional connectivity analyses, we confirm the principal hubs of the stress network (e.g., prefrontal cortex, amygdala and periventricular hypothalamus) and show that the LHb is engaged during stress exposure in close interaction with the medial prefrontal cortex, the lateral septum and the medial habenula. In addition, we performed DREADD-induced LHb inactivation during the same restraint paradigm in order to explore its consequences on the stress response network. This last experiment gave contrasting results as the DREADD ligand alone, clozapine-N-oxide, was able to modify the network.

Somatostatin analogue pasireotide (SOM230) inhibits catecholamine secretion in human pheochromocytoma cells.

Increasingly common, neuroendocrine tumors (NETs) are regarded nowadays as neoplasms potentially causing debilitating symptoms and life-threatening medical conditions. Pheochromocytoma is a NET that develops from chromaffin cells of the adrenal medulla, and is responsible for an excessive secretion of catecholamines. Consequently, patients have an increased risk for clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Somatostatin analogues are among the main anti-secretory medical drugs used in current clinical practice in patients with NETs. However, their impact on pheochromocytoma-associated catecholamine hypersecretion remains incompletely explored. This study investigated the potential efficacy of octreotide and pasireotide (SOM230) on human tumor cells directly cultured from freshly resected pheochromocytomas using an implemented catecholamine secretion measurement by carbon fiber amperometry. SOM230 treatment efficiently inhibited nicotine-induced catecholamine secretion both in bovine chromaffin cells and in human tumor cells whereas octreotide had no effect. Moreover, SOM230 specifically decreased the number of exocytic events by impairing the stimulation-evoked calcium influx as well as the nicotinic receptor-activated inward current in human pheochromocytoma cells. Altogether, our findings indicate that SOM230 acts as an inhibitor of catecholamine secretion through a mechanism involving the nicotinic receptor and might be considered as a potential anti-secretory treatment for patients with pheochromocytoma.

The endocannabinoid system is modulated in reward and homeostatic brain regions following diet-induced obesity in rats: a cluster analysis approach.

OBJECTIVES: Increased availability of high-calorie palatable food in most countries has resulted in overconsumption of these foods, suggesting that excessive eating is driven by pleasure, rather than metabolic need. The behavior contributes to the rise in eating disorders, obesity, and associated pathologies like diabetes, cardiac disease, and cancers. The mesocorticolimbic dopamine and homeostatic circuits are interconnected and play a central role in palatable food intake. The endocannabinoid system is expressed in these circuits and represents a potent regulator of feeding, but the impact of an obesogenic diet on its expression is not fully known. METHODS: Food intake and body weight were recorded in male Wistar rats over a 6-week free-choice regimen of high fat and sugar; transcriptional regulations of the endocannabinoid system were examined post-mortem in brain reward regions (prefrontal cortex, nucleus accumbens, ventral tegmental area, and arcuate nucleus). K-means cluster analysis was used to classify animals based on individual sensitivity to obesity and palatable food intake. Endocannabinoid levels were quantified in the prefrontal cortex and nucleus accumbens. Gene expression in dopamine and homeostatic systems, including ghrelin and leptin receptors, and classical homeostatic peptides, were also investigated. RESULTS: The free-choice high-fat -and sugar diet induced hyperphagia and obesity in rats. Cluster analysis revealed that the propensity to develop obesity and excessive palatable food intake was differently associated with dopamine and endocannabinoid system gene expression in reward and homeostatic brain regions. CB2 receptor mRNA was increased in the nucleus accumbens of high sugar consumers, whereas CB1 receptor mRNA was decreased in obesity prone rats. CONCLUSIONS: Transcriptional data are consistent with observations of altered dopamine function in rodents that have access to an obesogenic diet and point to cannabinoid receptors as GPCR targets involved in neuroplasticity mechanisms associated with maladaptive intake of palatable food.

Action of mefloquine/amitriptyline THN101 combination on neuropathic mechanical hypersensitivity in mice.

ABSTRACT: Tricyclic antidepressants that inhibit serotonin and noradrenaline reuptake, such as amitriptyline, are among the first-line treatments for neuropathic pain, which is caused by a lesion or disease affecting the somatosensory nervous system. These treatments are, however, partially efficient to alleviate neuropathic pain symptoms, and better treatments are still highly required. Interactions between neurons and glial cells participate in neuropathic pain processes, and importantly, connexins-transmembrane proteins involved in cell-cell communication-contribute to these interactions. In a neuropathic pain model in rats, mefloquine, a connexin inhibitor, has been shown to potentiate the antihyperalgesic effect of amitriptyline, a widely used antidepressant. In this study, we further investigated this improvement of amitriptyline action by mefloquine, using the cuff model of neuropathic pain in mice. We first observed that oral mefloquine co-treatment prolonged the effect of amitriptyline on mechanical hypersensitivity by 12 hours after administration. In addition, we showed that this potentiation was not due to pharmacokinetic interactions between the 2 drugs. Besides, lesional and pharmacological approaches showed that the prolonged effect was induced through noradrenergic descending pathways and the recruitment of α2 adrenoceptors. Another connexin blocker, carbenoxolone, also improved amitriptyline action. Additional in vitro studies suggested that mefloquine may also directly act on serotonin transporters and on adenosine A1 and A2A receptors, but drugs acting on these other targets failed to amplify amitriptyline action. Together, our data indicate that pharmacological blockade of connexins potentiates the therapeutic effect of amitriptyline in neuropathic pain.

Unveiling the Impact of Morphine on Tamoxifen Metabolism in Mice in vivo.

Background: Tamoxifen is used to treat breast cancer and cancer recurrences. After administration, tamoxifen is converted into two more potent antitumor compounds, 4OH-tamoxifen and endoxifen by the CYP3A4/5 and 2D6 enzymes in human. These active compounds are inactivated by the same UDP-glucuronosyltransferase isoforms as those involved in the metabolism of morphine. Importantly, cancer-associated pain can be treated with morphine, and the common metabolic pathway of morphine and tamoxifen suggests potential clinically relevant interactions. Methods: Mouse liver microsomes were used to determine the impact of morphine on 4OH-tamoxifen metabolism in vitro. For in vivo experiments, female mice were first injected with tamoxifen alone and then with tamoxifen and morphine. Blood was collected, and LC-MS/MS was used to quantify tamoxifen, 4OH-tamoxifen, N-desmethyltamoxifen, endoxifen, 4OH-tamoxifen-glucuronide, and endoxifen-glucuronide. Results: In vitro, we found increased K m values for the production of 4OH-tamoxifen-glucuronide in the presence of morphine, suggesting an inhibitory effect on 4OH-tamoxifen glucuronidation. Conversely, in vivo morphine treatment decreased 4OH-tamoxifen levels in the blood while dramatically increasing the formation of inactive metabolites 4OH-tamoxifen-glucuronide and endoxifen-glucuronide. Conclusions: Our findings emphasize the need for caution when extrapolating results from in vitro metabolic assays to in vivo drug metabolism interactions. Importantly, morphine strongly impacts tamoxifen metabolism in mice. It suggests that tamoxifen efficiency could be reduced when both drugs are co-administered in a clinical setting, e.g., to relieve pain in breast cancer patients. Further studies are needed to assess the potential for tamoxifen-morphine metabolic interactions in humans.

A Nonpeptide Oxytocin Receptor Agonist for a Durable Relief of Inflammatory Pain.

Oxytocin possesses several physiological and social functions, among which an important analgesic effect. For this purpose, oxytocin binds mainly to its unique receptor, both in the central nervous system and in the peripheral nociceptive terminal axon in the skin. However, despite its interesting analgesic properties and its current use in clinics to facilitate labor, oxytocin is not used in pain treatment. Indeed, it is rapidly metabolized, with a half-life in the blood circulation estimated at five minutes and in cerebrospinal fluid around twenty minutes in humans and rats. Moreover, oxytocin itself suffers from several additional drawbacks: a lack of specificity, an extremely poor oral absorption and distribution, and finally, a lack of patentability. Recently, a first non-peptide full agonist of oxytocin receptor (LIT-001) of low molecular weight has been synthesized with reported beneficial effect for social interactions after peripheral administration. In the present study, we report that a single intraperitoneal administration of LIT-001 in a rat model induces a long-lasting reduction in inflammatory pain-induced hyperalgesia symptoms, paving the way to an original drug development strategy for pain treatment.

A Dual Noradrenergic Mechanism for the Relief of Neuropathic Allodynia by the Antidepressant Drugs Duloxetine and Amitriptyline.

In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. Despite the widespread use of these drugs, the mechanism underlying their therapeutic action in this pain context remains partly elusive. The present study combined data collected in male and female mice from a model of neuropathic pain and data from the clinical setting to understand how antidepressant drugs act. We show two distinct mechanisms by which the selective inhibitor of serotonin and noradrenaline reuptake duloxetine and the tricyclic antidepressant amitriptyline relieve neuropathic allodynia. One of these mechanisms is acute, central, and requires descending noradrenergic inhibitory controls and α2A adrenoceptors, as well as the mu and delta opioid receptors. The second mechanism is delayed, peripheral, and requires noradrenaline from peripheral sympathetic endings and ß2 adrenoceptors, as well as the delta opioid receptors. We then conducted a transcriptomic analysis in dorsal root ganglia, which suggested that the peripheral component of duloxetine action involves the inhibition of neuroimmune mechanisms accompanying nerve injury, including the downregulation of the TNF-α-NF-κB signaling pathway. Accordingly, immunotherapies against either TNF-α or Toll-like receptor 2 (TLR2) provided allodynia relief. We also compared duloxetine plasma levels in the animal model and in patients and we observed that patients' drug concentrations were compatible with those measured in animals under chronic treatment involving the peripheral mechanism. Our study highlights a peripheral neuroimmune component of antidepressant drugs that is relevant to their delayed therapeutic action against neuropathic pain.SIGNIFICANCE STATEMENT In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. However, the mechanism by which antidepressant drugs can relieve neuropathic pain remained in part elusive. Indeed, preclinical studies led to contradictions concerning the anatomical and molecular substrates of this action. In the present work, we overcame these apparent contradictions by highlighting the existence of two independent mechanisms. One is rapid and centrally mediated by descending controls from the brain to the spinal cord and the other is delayed, peripheral, and relies on the anti-neuroimmune action of chronic antidepressant treatment.

Light rescues circadian behavior and brain dopamine abnormalities in diurnal rodents exposed to a winter-like photoperiod.

Seasonal affective disorder (SAD), beyond mood changes, is characterized by alterations in daily rhythms of behavior and physiology. The pathophysiological conditions of SAD involve changes in day length and its first-line treatment is bright light therapy. Animal models using nocturnal rodents have been studied to elucidate the neurobiological mechanisms of depression, but might be ill suited to study the therapeutic effects of light in SAD since they exhibit light-aversive responses. Here Arvicanthis ansorgei, a diurnal rodent, was used to determine behavioral, molecular and brain dopamine changes in response to exposure to a winter-like photoperiod consisting of a light-dark cycle with 8 h of light, under diminished light intensity, and 16 h of darkness. Furthermore, we evaluated whether timed-daily bright light exposure has an effect on behavior and brain physiology of winter-like exposed animals. Arvicanthis under a winter-like condition showed alterations in the synchronization of the locomotor activity rhythm to the light-dark cycle. Moreover, alterations in day-night activity of dopaminergic neurotransmission were revealed in the nucleus accumbens and the dorsal striatum, and in the day-night clock gene expression in the suprachiasmatic nucleus. Interestingly, whereas dopamine disturbances were reversed in animals exposed to daily light at early or late day, altered phase of the daily rhythm of locomotion was reverted only in animals exposed to light at the late day. Moreover, Per2 gene expression in the SCN was also affected by light exposure at late day in winter-like exposed animals. These findings suggest that light induces effects on behavior by mechanisms that rely on both circadian and rhythm-independent pathways influencing the dopaminergic circuitry. This last point might be crucial for understanding the mechanisms of non-pharmacological treatment in SAD.

Sleep Deprivation and Caffeine Treatment Potentiate Photic Resetting of the Master Circadian Clock in a Diurnal Rodent.

Circadian rhythms in nocturnal and diurnal mammals are primarily synchronized to local time by the light/dark cycle. However, nonphotic factors, such as behavioral arousal and metabolic cues, can also phase shift the master clock in the suprachiasmatic nuclei (SCNs) and/or reduce the synchronizing effects of light in nocturnal rodents. In diurnal rodents, the role of arousal or insufficient sleep in these functions is still poorly understood. In the present study, diurnal Sudanian grass rats, Arvicanthis ansorgei, were aroused at night by sleep deprivation (gentle handling) or caffeine treatment that both prevented sleep. Phase shifts of locomotor activity were analyzed in grass rats transferred from a light/dark cycle to constant darkness and aroused in early night or late night. Early night, but not late night, sleep deprivation induced a significant phase shift. Caffeine on its own induced no phase shifts. Both sleep deprivation and caffeine treatment potentiated light-induced phase delays and phase advances in response to a 30 min light pulse, respectively. Sleep deprivation in early night, but not late night, potentiated light-induced c-Fos expression in the ventral SCN. Caffeine treatment in midnight triggered c-Fos expression in dorsal SCN. Both sleep deprivation and caffeine treatment potentiated light-induced c-Fos expression in calbindin-containing cells of the ventral SCN in early and late night. These findings indicate that, in contrast to nocturnal rodents, behavioral arousal induced either by sleep deprivation or caffeine during the sleeping period potentiates light resetting of the master circadian clock in diurnal rodents, and activation of calbindin-containing suprachiasmatic cells may be involved in this effect.SIGNIFICANCE STATEMENT Arousing stimuli have the ability to regulate circadian rhythms in mammals. Behavioral arousal in the sleeping period phase shifts the master clock in the suprachiasmatic nuclei and/or slows down the photic entrainment in nocturnal animals. How these stimuli act in diurnal species remains to be established. Our study in a diurnal rodent, the Grass rat, indicates that sleep deprivation in the early rest period induces phase delays of circadian locomotor activity rhythm. Contrary to nocturnal rodents, both sleep deprivation and caffeine-induced arousal potentiate the photic entrainment in a diurnal rodent. Such enhanced light-induced circadian responses could be relevant for developing chronotherapeutic strategies.

The TLR4-Active Morphine Metabolite Morphine-3-Glucuronide Does Not Elicit Macrophage Classical Activation In Vitro.

Macrophages are abundant in the tumor microenvironment where they adopt a pro-tumor phenotype following alternative polarization induced by paracrine factors from cancer and stromal cells. In contrast, classically activated macrophages have tumoricidal activities, such that the polarization of tumor-associated macrophages has become a novel therapeutic target. Toll-like receptor 4 engagement promotes classical activation of macrophages, and recent literature suggests TLR4 agonism to prevent metastasis and promote survival in experimental metastasis models. A growing number of studies indicate that TLR4 can respond to opioids, including the opioid receptor-inactive morphine metabolite morphine-3-glucuronide (M3G). We measured the activation of TLR4 in a reporter cell line exogenously expressing TLR4 and TLR4 co-receptors, and confirmed that M3G weakly but significantly activates TLR4. We hypothesized that M3G would promote the expression of classical activation signature genes in macrophages in vitro. We exposed mouse and human macrophage cell lines to M3G or the TLR4 activator lipopolysaccharide (LPS), alone or in combination with interferon gamma (IFN-γ). The classical macrophage activation markers tested were iNOS, CD86, IL-6, or TNF-α in RAW 264.7 cells and IL-6, IL-12, IL-23, TNF-α, CXCL10, and CXCL11 in THP1 cells. Our results show that despite exhibiting TLR4-activation ability, M3G does not elicit the expression of classical activation markers in LPS-responsive macrophages.

Morphine decreases the pro-angiogenic interaction between breast cancer cells and macrophages in vitro.

Interactions between the various cell types that constitute a solid tumour are essential to the biology of the tumour. We evaluated the effect of morphine on the proangiogenic interaction taking place between macrophages and breast cancer cells in vitro. The conditioned medium (CM) from breast cancer cells co-cultured with macrophages elicited endothelial cell proliferation and tube formation. This effect was inhibited if the co-culture occurred in the presence of morphine. The CM from breast cancer cells or macrophages grown individually, whether or not prepared in the presence of morphine, was ineffective in stimulating EC proliferation or tube formation. Using a mouse antibody array, we identified several angiogenesis-regulating factors differentially expressed in the CM of co-cultured cells prepared in the presence or absence of morphine, amongst which interleukin (IL)-6, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF)-A. VEGF was induced in both cell types by the co-culture and this was prevented by morphine in a non-naloxone reversible fashion. The effect of CM from co-cultured cells on endothelial tube formation, but not proliferation, was prevented by anti-VEGF neutralizing antibody. Our results indicate that morphine prevents, in part via modulating VEGF-A expression, the pro-angiogenic interaction between macrophages and breast cancer cells.

Morphine Modulates Interleukin-4- or Breast Cancer Cell-induced Pro-metastatic Activation of Macrophages.

Interactions between cancer cells and stromal cells in the tumour microenvironment play a key role in the control of invasiveness, metastasis and angiogenesis. Macrophages display a range of activation states in specific pathological contexts and alternatively activated (M2) macrophages can promote tumour aggressiveness. Opioids are able to modulate tumour growth and metastasis. We tested whether morphine modulates the activation of macrophages induced by (i) interleukin-4 (IL-4), the prototypical M2 polarization-inducing cytokine, or (ii) coculture with breast cancer cells. We showed that IL-4 causes increased MMP-9 production and expression of the alternative activation markers arginase-1 and MRC-1. Morphine prevented IL-4-induced increase in MMP-9 in a naloxone- and methylnaltrexone-reversible fashion. Morphine also prevented IL-4-elicited alternative activation of RAW264.7 macrophages. Expression of MMP-9 and arginase-1 were increased when RAW264.7 were subjected to paracrine activation by 4T1 cells, and this effect was prevented by morphine via an opioid receptor-mediated mechanism. Morphine further decreased 4T1 breast cancer cell invasion elicited by co-culture with RAW264.7. Reduction of MMP-9 expression and alternative activation of macrophages by morphine was confirmed using mouse bone marrow-derived macrophages. Taken together, our results indicate that morphine may modulate tumour aggressiveness by regulating macrophage protease production and M2 polarization within the tumour microenvironment.

CTIP2 is a negative regulator of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the ß-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.

Endogenous morphine-like compound immunoreactivity increases in parkinsonism.

Morphine is endogenously synthesized in the central nervous system and endogenous dopamine is thought to be necessary for endogenous morphine formation. As Parkinson's disease results from the loss of dopamine and is associated with central pain, we considered how endogenous morphine is regulated in the untreated and l-DOPA-treated parkinsonian brain. However, as the cellular origin and overall distribution of endogenous morphine remains obscure in the pathological adult brain, we first characterized the distribution of endogenous morphine-like compound immunoreactive cells in the rat striatum. We then studied changes in the endogenous morphine-like compound immunoreactivity of medium spiny neurons in normal, Parkinson's disease-like and l-DOPA-treated Parkinson's disease-like conditions in experimental (rat and monkey) and human Parkinson's disease. Our results reveal an unexpected dramatic upregulation of neuronal endogenous morphine-like compound immunoreactivity and levels in experimental and human Parkinson's disease, only partially normalized by l-DOPA treatment. Our data suggest that endogenous morphine formation is more complex than originally proposed and that the parkinsonian brain experiences a dramatic upregulation of endogenous morphine immunoreactivity. The functional consequences of such endogenous morphine upregulation are as yet unknown, but based upon the current knowledge of morphine signalling, we hypothesize that it is involved in fatigue, depression and pain symptoms experienced by patients with Parkinson's disease.

Endogenous morphine in SH-SY5Y cells and the mouse cerebellum.

BACKGROUND: Morphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca(2+)-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10(-10) M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies. CONCLUSIONS/SIGNIFICANCE: The presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.

Identification of morphine-6-glucuronide in chromaffin cell secretory granules.

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.

Characterization of a morphine-like molecule in secretory granules of chromaffin cells.

BACKGROUND: Morphine and morphine-like molecules were identified in numerous mammalian tissues. In particularly, morphine was present in adrenal gland and in PC-12 cells, corresponding to the cell line derived from rat adrenal medullary chromaffin cells. MATERIAL/METHODS: Morphine-like compounds were isolated after a deproteinization followed by a reverse phase high pressure chromatography purification coupled to a UV detector. This alkaloid was identified using nano-eletrospray ionization quadripole time of flight tandem mass spectrometry. RESULTS: We demonstrate the presence of a morphine-like molecule inside the bovine chromaffin cell secretory granules. This alkaloid was also detected into the secretion medium of primary chromaffin cells in culture following nicotinic stimulation. CONCLUSIONS: Our findings demonstrate that a morphine-like component is synthesized in chromaffin cells and secreted into the incubation medium upon stimulation. This finding strongly suggests that this material may be released from adrenal tissues in response to stressors.

The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine-binding protein, possesses a new biological activity on cardiac physiology.

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.