M. D. Barrachina

The role of nitric oxide and platelet-activating factor in the inhibition by endotoxin of pentagastrin-stimulated gastric acid secretion.

Administration of E. coli endotoxin (1 mg/kg i.v.) abolished the acid secretory response induced by a bolus injection of pentagastrin (100 micrograms/kg i.v.) in the continuously perfused stomach of the anaesthetized rat. Endotoxin administration did not modify mean systemic arterial blood pressure. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME; 5-20 mg/kg i.v.), but not dexamethasone (5 mg/kg s.c. twice) or indomethacin (5 mg/kg i.m.), substantially restored the secretory responses to pentagastrin. The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.v.), but not by its enantiomer D-arginine (100 mg/kg i.v.). L-NAME (10 mg/kg i.v.) increased blood pressure but this does not seem to be the mechanism by which endotoxin-induced acid inhibition was prevented, since similar systemic pressor responses induced by noradrenaline (15 micrograms/kg per min i.v.) had no such effect. The platelet-activating factor (PAF) receptor antagonist, WEB 2086 (2 mg/kg), induced a partial reversal of the inhibition by endotoxin of acid responses to pentagastrin. In endotoxin-treated rats, the combined administration of L-NAME (10 mg/kg) and WEB 2086 (2 mg/kg) completely restored the degree of H+ output induced by pentagastrin to levels similar to those of control, vehicle-treated animals. These findings suggest that endotoxin-induced acute inhibition of acid responses to pentagastrin involves NO synthesis and the release of PAF.

M2 macrophages activate WNT signaling pathway in epithelial cells: relevance in ulcerative colitis.

Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2 macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in enterocyte differentiation.

ER stress in human hepatic cells treated with Efavirenz: Mitochondria again

BACKGROUND & AIMS ER stress is associated with a growing number of liver diseases, including drug-induced hepatotoxicity. The non-nucleoside analogue reverse transcriptase inhibitor Efavirenz, a cornerstone of the multidrug strategy employed to treat HIV1 infection, has been related to the development of various adverse events, including metabolic disturbances and hepatic toxicity, the mechanisms of which remain elusive. Recent evidence has pinpointed a specific mitochondrial effect of Efavirenz in human hepatic cells. This study assesses the induction of ER stress by Efavirenz in the same model and the implication of mitochondria in this process. METHODS Primary human hepatocytes and Hep3B were treated with clinically relevant concentrations of Efavirenz and parameters of ER stress were studied using standard cell biology techniques. RESULTS ER stress markers, including CHOP and GRP78 expression (both protein and mRNA), phosphorylation of eIF2α, and presence of the spliced form of XBP1 were upregulated. Efavirenz also enhanced cytosolic Ca(2+) content and induced morphological changes in the ER suggestive of ER stress. This response was greatly attenuated in cells with altered mitochondrial function (Rho°). The effects of Efavirenz on the ER, and particularly in regard to the mitochondrial involvement, differed from those elicited by a standard pharmacological ER stressor. CONCLUSIONS This newly discovered mechanism of cellular insult involving ER stress and UPR response may help comprehend the hepatic toxicity that has been associated with the widespread and life-long use of Efavirenz. In addition, the specificity of the actions of Efavirenz observed expands our knowledge of the mechanisms that trigger ER stress and shed some light on the mitochondria/ER interplay in drug-induced hepatic challenge.

Induction of CD36 and Thrombospondin-1 in Macrophages by Hypoxia-Inducible Factor 1 and Its Relevance in the Inflammatory Process

Inflammation is part of a complex biological response of vascular tissue to pathogens or damaged cells. First inflammatory cells attempt to remove the injurious stimuli and this is followed by a healing process mediated principally by phagocytosis of senescent cells. Hypoxia and p38-MAPK are associated with inflammation, and hypoxia inducible factor 1 (HIF-1) has been detected in inflamed tissues. We aimed to analyse the role of p38-MAPK and HIF-1 in the transcriptional regulation of CD36, a class B scavenger receptor, and its ligand thrombospondin (TSP-1) in macrophages and to evaluate the involvement of this pathway in phagocytosis of apoptotic neutrophils. We have also assessed HIF-1α, p38-MAPK and CD36 immunostaining in the mucosa of patients with inflammatory bowel disease. Results show that hypoxia increases neutrophil phagocytosis by macrophages and induces the expression of CD36 and TSP-1. Addition of a p38-MAPK inhibitor significantly reduced the increase in CD36 and TSP-1 expression provoked by hypoxia and decreased HIF-1α stabilization in macrophages. Transient transfection of macrophages with a miHIF-1α-targeting vector blocked the increase in mRNA expression of CD36 and TSP-1 during hypoxia and reduced phagocytosis, thus highlighting a role for the transcriptional activity of HIF-1. CD36 and TSP-1 were necessary for the phagocytosis of neutrophils induced by hypoxic macrophages, since functional blockade of these proteins undermined this process. Immunohistochemical studies revealed CD36, HIF-1α and p38-MAPK expression in the mucosa of patients with inflammatory bowel disease. A positive and significant correlation between HIF-1α and CD36 expression and CD36 and p38-MAPK expression was observed in cells of the lamina propria of the damaged mucosa. Our results demonstrate a HIF-1-dependent up-regulation of CD36 and TSP-1 that mediates the increased phagocytosis of neutrophils by macrophages during hypoxia. Moreover, they suggest that CD36 expression in the damaged mucosa of patients with inflammatory bowel disease depends on p38-MAPK and HIF-1 activity.

Analgesic and central depressor effects of the dichloromethanol extract from Schinus molle L.

The analgesic and central depressor effects of the dichloromethanol extract of Schinus molle L. were analysed in in vivo models. This extract showed low acute toxicity, CNS depressor activity and analgesic effect. Following further fractionation, the hexane/dichloromethane (75/25) fraction showed the most interesting results. Thus, this fraction caused a total inhibition of motor activity and significantly reduced the threshold of pain to chemical stimulus.

Endotoxin inhibition of distension-stimulated gastric acid secretion in rat: mediation by NO in the central nervous system.

1 The involvement of nitric oxide in the acute inhibitory effects of low doses of endotoxin, following intracerebroventricular (i.c.v.) or intravenous (i.v.) administration, on gastric acid secretion stimulated by distension or i.v. infusion of pentagastrin has been investigated in the continuously perfused stomach of the anaesthetized rat. 2 The i.c.v. administration of E. coli endotoxin (800 ng kg−1) abolished the acid secretory response induced by gastric distension (20 cm water intragastric pressure) within 30 min of administration. 3 By contrast, submaximal rates of acid secretion induced by i.v. infusion of pentagastrin (8 μg kg−1 h−1) were not inhibited by i.c.v. administration of endotoxin (800 ng kg−1). 4 Prior i.c.v. administration of the NO synthase inhibitor, NG‐nitro‐l‐arginine methyl ester (l‐NAME, 800 μg kg−1) restored the acid secretory responses to distension in rats treated with endotoxin (i.c.v.). 5 Likewise, i.v. administration of endotoxin (5 μg kg−1) abolished the acid secretory response induced by gastric distension within 30 min of administration. Prior i.c.v injection of l‐NAME (800 μg kg−1) or its i.v. administration (10 mg kg−1) restored acid secretory responses in rats receiving i.v. endotoxin. 6 The reversal by l‐NAME (i.v.) of the acid inhibitory effects of endotoxin (i.v.) was prevented by l‐arginine (12 mg kg−1, i.c.v. or 100 mg kg−1, i.v), but not by its enantiomer d‐arginine. 7 The present results imply the existence of an acute response to endotoxin involving NO synthesis in the brain. NO may act as a neuromodulator or neurotransmitter in a nervous reflex leading to the inhibition of acid secretion stimulated by gastric distension.

Synthesis of nitric oxide in postganglionic myenteric neurons during endotoxemia: implications for gastric motor function in rats

We have investigated the mechanisms underlying acute changes in gastric motor function triggered by endotoxemia. In fundal strips from rats pre‐treated with endotoxin (40 µg/kg, i.p. 30 min), mechanical activity was analyzed and the source of nitric oxide (NO) was visualized by confocal microscopy of tissue loaded with the fluorescent dye DAF‐FM. NOS expression was determined by quantitative RT‐PCR and Western blot, and enzyme activity by the citrulline assay. Strips from endotoxin‐treated rats were hypo‐contractile. This was prevented by pre‐incubation with the neurotoxin tetrodotoxin, the gangliar blocker hexamethonium, or non‐selective and neuronal‐specific NOS inhibitors (L‐NOARG and TRIM, respectively). The soluble guanylyl cyclase (sGC) inhibitor ODQ and the inhibitor of small conductance Ca2+‐activated K+ channels apamin prevented relaxation induced by endotoxin, nicotine, exogenous NO (DETA‐NONOate), and the NO‐independent sGC activator BAY 41‐2272. NO synthesis was observed in neuronal soma, axons, and nerve endings of the myenteric plexus in the fundus of endotoxin‐treated rats and was prevented by L‐NAME, tetrodotoxin, and hexamethonium. nNOS and iNOS mRNA and protein contents were unchanged. Our findings demonstrate synthesis of NO in post‐ganglionic myenteric neurons during early endotoxemia that mediates gastric hypo‐contractility. The effect of NO is mediated via sGC and small conductance Ca2+‐activated K+channels.

Hypoxic macrophages impair autophagy in epithelial cells through Wnt1: relevance in IBD

A defective induction of epithelial autophagy may have a role in the pathogenesis of inflammatory bowel diseases. This process is regulated mainly by extracellular factors such as nutrients and growth factors and is highly induced by diverse situations of stress. We hypothesized that epithelial autophagy is regulated by the immune response that in turn is modulated by local hypoxia and inflammatory signals present in the inflamed mucosa. Our results reveal that HIF-1α and Wnt1 were co-localized with CD68 in cells of the mucosa of IBD patients. We have observed increased protein levels of β-catenin, phosphorylated mTOR, and p62 and decreased expression of LC3II in colonic epithelial crypts from damaged mucosa in which β-catenin positively correlated with phosphorylated mTOR and negatively correlated with autophagic protein markers. In cultured macrophages, HIF-1 mediated the increase in Wnt1 expression induced by hypoxia, which enhanced protein levels of β-catenin, activated mTOR, and decreased autophagy in epithelial cells in co-culture. Our results demonstrate a HIF-1-dependent induction of Wnt1 in hypoxic macrophages that undermines autophagy in epithelial cells and suggest a role for Wnt signaling and mTOR pathways in the impaired epithelial autophagy observed in the mucosa of IBD patients.

Profile of leukocyte-endothelial cell interactions induced in venules and arterioles by nucleoside reverse transcriptase inhibitors in vivo

BACKGROUND There is controversy regarding cardiovascular (CV) toxicity of the nucleoside reverse-transcriptase inhibitors used to treat human immunodeficiency virus infection. METHODS We evaluated the effects of nucleoside reverse-transcriptase inhibitors on leukocyte-endothelium interactions, a hallmark of CV diseases, in rat mesenteric vessels using intravital microscopy and in human arterial cells using a flow chamber system. RESULTS Abacavir and didanosine increased rolling, adhesion and emigration in rat vessels. These effects were reversed with antibodies against Macrophage-1 antigen (Mac-1) or intercellular adhesion molecule 1 and were reproduced in human cells. Lamivudine, zidovudine, emtricitabine, and tenofovir had no effects. CONCLUSIONS Our results support the association of abacavir and didanosine with CV diseases.

A cerebral nitrergic pathway modulates endotoxin-induced changes in gastric motility

This study analyses the neural pathway involved in the modulation of gastric motor function by stress. Systemic administration of low doses of endotoxin (40 μg kg−1, i.v.) prevents the increase in gastric tone induced by 2‐deoxy‐D‐glucose (200 mg kg−1, i.v., 2‐DG) in urethane‐anaesthetized rats. Functional inhibition of afferent neurones by systemic administration of capsaicin (20+30+50 mg kg−1, i.m.) in adult rats prevented the inhibitory effects of endotoxin. Pre‐treatment with the nitric oxide synthase (NOS) inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME), both i.v. (10 mg kg−1) and i.c. (200 μg rat−1), prevented the inhibitory effects of endotoxin on gastric tone induced by 2‐DG. Immunohistochemical studies show Fos expression in the dorsal vagal complex (DVC) of the brainstem of 2‐DG‐treated animals. Peripheral administration of endotoxin (40 μg kg−1, i.p.) increased the number of Fos‐immunoreactive cells induced by 2‐DG, both in the nucleus tractus solitarii (NTS) and in the dorsal motor nucleus (DMN) of the DVC. Pre‐treatment with L‐NAME prevented the increase in Fos expression induced by endotoxin in both nuclei. Endotoxin (40 μg kg−1, i.p.) increased Ca2+‐dependent nitric oxide synthase (cNOS) activity in the brainstem. Addition of 7‐nitroindazole (600 μM, 7‐NI) to the assay significantly inhibited the increase in cNOS activity caused by endotoxin. No change in NOS activity of any isoform was observed in the stomach of animals treated with endotoxin. The present study suggests that inhibition of gastric motor function by low doses of endotoxin involves activation of capsaicin‐sensitive afferent neurones and neuronal NOS in the brainstem.