OS064. Contribute of the L-cysteine/ H2S pathway in placenta homeostasisin hypertensive disorders.

INTRODUCTION: Hydrogen sulfide (H2S) is considered the third endogenous gas transmitter besides nitric oxide and carbon monoxide [1]. It is produced from L-cysteine or L-methionine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). H2S is involved in the control of vascular homeostasis, having either relaxant or contractant effect on smooth muscle cells. The H2S involvement in rat and human intrauterine tissues has also been shown [2]. OBJECTIVES: The aim of our study was to investigate the L-cysteine/ H2S pathway in rat and human placenta in hypertensive state. METHODS: Placental samples were collected from spontaneous hypertensive rats (SHR) and normotensive rat (Wistar Kyoto; WKY). In parallel, placental samples were collected from 10 pre-eclamptic women and 5 controls after caesarean sections. Pre-eclamptic women were divided into two subgroups: Group1 (women who developed Early Preeclampsia, n=4); Group2 (women who developed Late Preeclampsia, n=6). The expression of CBS and CSE was evaluated in sample tissues by Western blotting analysis. The enzymatic activity was assessed in basal and stimulated (L- cysteine) condiction by a colorimetric assay. Statistical analysis was performed by using Student's t test. P<0,05 was considered as statistically significant. RESULTS: The expression of CBS and CSE in placenta of SHR rats were significantly reduced (p<0.05) compared to WKY. The H2S production resulted significantly (p<0,05) lower in SHR than WKY rats. In human placenta, the basal H2S production was similar in the three groups; interestingly the H2S production by adding L-cysteine, was higher in Late Preeclampsia compared to control group. CONCLUSION: H2S was produced in rat and human placenta. CBS and CSE, the enzymes involved in the production of H2S, were down-regulated in SHR rats and, as a consequence the H2S production was significantly reduced. Starting from these data, we tried to analyze the role of hydrogen sulfide in preeclampsia to assess the contribute of this gas transmitter in the development of this condition. Unexpectedly, preliminary data demonstrated that in women developing Late Preeclampsia there was an higher production of H2S after stimulation with L-cysteine, not revealed in Early Preeclampsia or in healthy control group. Our results indicated that the L-cysteine/H2S pathway could contribute to the development of preeclampsia condition.

Palmitoylethanolamide stimulation induces allopregnanolone synthesis in C6 Cells and primary astrocytes: involvement of peroxisome-proliferator activated receptor-α.

Palmitoylethanolamide (PEA) regulates many pathophysiological processes in the central nervous system, including pain perception, convulsions and neurotoxicity, and increasing evidence points to its neuroprotective action. In the present study, we report that PEA, acting as a ligand of peroxisome-proliferator activated receptor (PPAR)-α, might regulate neurosteroidogenesis in astrocytes, which, similar to other glial cells and neurones, have the enzymatic machinery for neurosteroid de novo synthesis. Accordingly, we used the C6 glioma cell line and primary murine astrocytes. In the mitochondrial fraction from cells stimulated with PEA, we demonstrated an increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 enzyme (P450scc) expression, both comprising proteins considered to be involved in crucial steps of neurosteroid formation. The effects of PEA were completely blunted by GW6471, a selective PPAR-α antagonist, or by PPAR-α silencing by RNA interference. Accordingly, allopregnanolone (ALLO) levels were increased in supernatant of PEA-treated astrocytes, as revealed by gas chromatography-mass spectrometry, and this effect was inhibited by GW6471. Moreover, PEA showed a protective effect, reducing malondialdehyde formation in cells treated with l-buthionine-(S,R)-sulfoximine, a glutathione depletor and, interestingly, the effect of PEA was partially inhibited by finasteride, a 5α-reductase inhibitor. A similar profile of activity was demonstrated by ALLO and the lack of an additive effect with PEA suggests that the reduction of oxidative stress by PEA is mediated through ALLO synthesis. The present study provides evidence indicating the involvement of the saturated acylethanolamide PEA in ALLO synthesis through PPAR-α in astrocytes and explores the antioxidative activity of this molecule, confirming its homeostatic and protective role both under physiological and pathological conditions.

Differential modification of inflammatory enzymes in J774A.1 macrophages by ochratoxin A alone or in combination with lipopolysaccharide.

Ochratoxin A (OTA) is a fungal metabolite with controversial immunomodulatory effects. A prolonged in vivo exposure to the mycotoxin may result in impaired immunity and decreased resistance to infections. In the present study, OTA modulation of lipopolysaccharide (LPS)-induced inflammatory process is described in the macrophagic cell line, J774A.1 in order to better understand the mechanisms underlying OTA immunotoxicity. OTA (30 nM-100 microM) induces a time and concentration dependent cytotoxic effect, increased when cells were co-stimulated with LPS (100 ng/ml), a concentration that alone did not modify the cellular viability. Moreover, OTA (3 microM) alone induces a significant increase in cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, while at the highest concentration (10 microM) a reduced expression of both enzymes was shown, consistently with the mycotoxin cytotoxic profile. The role of nuclear factor-kB (NF-kB) in the mycotoxin effect was also demonstrated. Conversely, when cells were co-stimulated with LPS, OTA showed a concentration-dependent reduction of COX-2 and iNOS expression and their respective metabolites (PGE(2) and NO). These results confirm the pro-inflammatory role of OTA by itself, and demonstrate the impaired capability of OTA-treated macrophages to respond properly to noxious stimuli, such as LPS, mimicking the environmental co-exposure to both compounds.

AM404, an anandamide transport inhibitor, reduces plasma extravasation in a model of neuropathic pain in rat: role for cannabinoid receptors.

Neuropathic pain consequent to peripheral nerve injury has been associated with local inflammation. Following noxious stimulation afferent fibres release substance P (SP) and calcitonin-gene related peptide (CGRP), which are closely related to oedema formation and plasma leakage. The effect of the anandamide transport blocker AM404 has been studied on plasma extravasation after chronic constriction injury (CCI) which consists in a unilateral loose ligation of the rat sciatic nerve (Bennett and Xie, 1988). AM404 (1-3-10 mg kg(-1)) reduced plasma extravasation in the legated paw, measured as mug of Evans Blue per gram of fresh tissue. A strong effect on vascular permeability was also produced by the synthetic cannabinoid agonist WIN 55,212-2 (0.1-0.3-1 mg kg(-1)). Using specific antagonists or enzyme inhibitors, we demonstrate that cannabinoids act at several levels: data on the 3rd day suggest a strong involvement of substance P (SP) and calcitonin gene-related peptide (CGRP) in the control of vascular tone, whereas at the 7th and 14th days the major role seems to be played by prostaglandins (PGs) and nitric oxide (NO). Capsaicin injection in ligated paws of AM404- or WIN 55,212-2-treated rats resulted in an increase of Evans Blue extravasation, suggesting the involvement of the cannabinergic system in the protective effect of C fibres of ligated paws. Taken together, these data demonstrate the efficacy of cannabinoids in controlling pain behaviour through the modulation of several pain mediators and markers of vascular reactivity, such as SP, CGRP, PGs and NO.

Modulation of neuropathic and inflammatory pain by the endocannabinoid transport inhibitor AM404 [N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide].

The endocannabinoid system may serve important functions in the central and peripheral regulation of pain. In the present study, we investigated the effects of the endocannabinoid transport inhibitor AM404 [N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide] on rodent models of acute and persistent nociception (intraplantar formalin injection in the mouse), neuropathic pain (sciatic nerve ligation in the rat), and inflammatory pain (complete Freund's adjuvant injection in the rat). In the formalin model, administration of AM404 (1-10 mg/kg i.p.) elicited dose-dependent antinociceptive effects, which were prevented by the CB(1) cannabinoid receptor antagonist rimonabant (SR141716A; 1 mg/kg i.p.) but not by the CB2 antagonist SR144528 (1 mg/kg i.p.) or the vanilloid antagonist capsazepine (30 mg/kg i.p.). Comparable effects were observed with UCM707 [N-(3-furylmethyl)-eicosa-5,8,11,14-tetraenamide], another anandamide transport inhibitor. In both the chronic constriction injury and complete Freund's adjuvant model, daily treatment with AM404 (1-10 mg/kg s.c.) for 14 days produced a dose-dependent reduction in nocifensive responses to thermal and mechanical stimuli, which was prevented by a single administration of rimonabant (1 mg/kg i.p.) and was accompanied by decreased expression of cyclooxygenase-2 and inducible nitric-oxide synthase in the sciatic nerve. The results provide new evidence for a role of the endocannabinoid system in pain modulation and point to anandamide transport as a potential target for analgesic drug development.