Graphical Models of Psychosocial Factors in Chronic Somatic Diseases

AbstractIn this paper we describe a graph, tree and forest model of psychosocial factors dependencies of chronically ill patients, called graphical models. Foundation of the study was the theory of meaningfulness of suffering by V. E. Frankl. 181 patients with either arterial hypertension or neoplasms with bad prognosis were examined thrice: 0-10 days from the time of diagnosis (stage I), about 5 weeks from the diagnosis (stage II) and at a follow-up about 5 months since stage II (stage III). 75 factors were available for consideration: 17 in stage I, 28 in stage II, 27 in stage III and 4 sets of data that describe populations: age, gender, education, number of stages executed. For both diseases graphs and trees are built under assumption that factors are vertices and significant correlations are edges, leading to model of dependencies between factors. Usefulness of this approach to analysis of difference between diseases is discussed

Epithelial sodium channel and n-3 polyunsatured fatty acids.

I. DESCRIPTION DE PROJET DE RECHERCHELe canal sodium épithélial bloquable par l’amiloride (ENaC) est une protéine intégrale de la membrane apicale des épithéliums impliqués dans l’absorption du sodium. Deux fonctions majeures sont directement liées au fonctionnement d’ENaC. D’une part, la régulation de la balance sodée par le rein et donc de la pression artérielle et d’autre part, la clairance du fluide alvéolaire pulmonaire.Le transport vectoriel de sel et d’eau à travers ces épithéliums à jonctions serrées repose sur un transport actif de sodium entraînant un flux osmotique d’eau. Ce transport de sodium s’effectue en deux étapes: l’entrée apicale, par diffusion, facilitée via ENaC, et la sortie basolatérale, active, par les pompes Na+/K+ ATPases.Ces dernières années, un intérêt grandissant est porté sur les acides gras polyinsaturés à longues chaînes de type oméga 3 (PUFAs) et leurs implications dans divers processus physiologiques. Entre autres effets, les PUFAs modulent différents types de canaux ioniques (canaux Na+ dépendant du voltage, Ca++ L-type, K+).Les études in vivo impliquant un effet à long terme des PUFAs décrivent des mécanismes inhibiteurs. Cependant, lors d’une étude précédente, axée sur la composition lipidique des membranes de cellules rénales en culture et l’influence de l’ajout d’acides gras saturés et insaturés sur le transport du sodium, nous avons constaté que les acides gras polyinsaturés à longues chaînes de type oméga 3 augmentaient la réabsorption du sodium. Ces résultats pourraient être intéressants, car les canaux sodiques de l’épithélium alvéolaire sont en contact direct avec le surfactant, dont la composition lipidique varie en fonction de l’apport alimentaire en PUFAs. Chez les prématurés humains, le syndrome de détresse respiratoire est une des causes les plus fréquentes de mortalité. Dans un certain nombre de cas, on peut restaurer une fonction pulmonaire satisfaisante par l’administration de surfactant.Dans ce travail, nous avons opté pour une approche fondamentale des mécanismes de régulation du canal sodium épithélial par l’acide eicosapentanoïque (EPA, C 20:5, n-3). Des études électrophysiologiques, biochimiques et d’imagerie cellulaire ont été réalisées sur la lignée cellulaire A6 de rein d’amphibien, qui sert d’épithélium modèle pour l’étude d’ENaC depuis plus de 25 ans. Cette lignée exprime des canaux sodiques très sélectifs et possède des propriétés électrophysiologiques facilitant l’étude de leur régulation.Ce travail nous a permis de mettre en évidence de nouveaux mécanismes fondamentaux dont la pertinence physiologique et /ou clinique ne pourra être établie qu’en transposant cette étude sur un modèle in vivo, comme nous le proposons dans les perspectives.Dans le présent travail, nous avons étudié :1.\Doctorat en Sciences biomédicales et pharmaceutiquesinfo:eu-repo/semantics/nonPublishe

Possible role of lysophosphatidic acid in rat model of hypoxic pulmonary vascular remodeling

Pulmonary hypertension is characterized by cellular and structural changes in the vascular wall of pulmonary arteries. We hypothesized that lysophosphatidic acid (LPA), a bioactive lipid, is implicated in this vascular remodeling in a rat model of hypoxic pulmonary hypertension. Exposure of Wistar rats to 10% O2 for 3 weeks induced an increase in the mean serum levels of LPA, to 40.9 (log-detransformed standard deviations: 23.4 -71.7) μM versus 21.6 (11.0-42.3) μM in a matched control animal group (P = 0.037). We also observed perivascular LPA immunohistochemical staining in lungs of hypoxic rats colocalized with the secreted lysophospholipase D autotaxin (ATX). Moreover, ATX colocalized with mast cell tryptase, suggesting implication of these cells in perivascular LPA production. Hypoxic rat lungs expressed more ATX transcripts (2.4-fold) and more transcripts of proteins implicated in cell migration: β2 integrin (1.74-fold), intracellular adhesion molecule 1 (ICAM-1; 1.84-fold), and αM integrin (2.70-fold). Serum from the hypoxic group of animals had significantly higher chemoattractant properties toward rat primary lung fibroblasts, and this increase in cell migration could be prevented by the LPA receptor 1 and 3 antagonists. LPA also increased adhesive properties of human pulmonary artery endothelial cells as well as those of human peripheral blood mononuclear cells, via the activation of LPA receptor 1 or 3 followed by the stimulation of gene expression of ICAM-1, β-1, E-selectin, and vascular cell adhesion molecule integrins. In conclusion, chronic hypoxia increases circulating and tissue levels of LPA, which might induce fibroblast migration and recruitment of mononuclear cells in pulmonary vasculature, both of which contribute to pulmonary vascular remodeling.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone

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Modulation of epithelial Na+ channel activity by long-chain n-3 fatty acids.

The epithelial sodium channel is found in apical membranes of a variety of native epithelial tissues, where it regulates sodium and fluid balance. In vivo, a number of hormones and other endogenous factors, including polyunsaturated fatty acids (PUFAs), regulate these channels. We tested the effects of essential n-3 and n-6 PUFAs on amiloride-sensitive sodium transport in A6 epithelial cells. Eicosapentaenoic acid [EPA; C20:5(n-3)] transiently stimulated amiloride-sensitive open-circuit current (I(Na)) from 4.0 +/- 0.3 to 7.7 +/- 0.3 microA/cm2 within 30 min (P < 0.001). No activation was seen in the presence of 10 microM amiloride. In cell-attached but not in cell-excised patches, EPA acutely increased the open probability of sodium channels from 0.45 +/- 0.08 to 0.63 +/- 0.10 (P = 0.02, paired t-test). n-6 PUFAs, including linoleic acid (C18:2), eicosatetraynoic acid (C20:4), and docosapentanoic acid (C22:5) had no effect, whereas n-3 docosahexanoic acid (C22:6) activated amiloride-sensitive I(Na) in a manner similar to EPA. Activation of I(Na) by EPA was prevented by H-89, a PKA inhibitor. Similarly, PKA activity was stimulated by EPA. Nonspecific stimulation of phosphodiesterase activity by CoCl2 completely prevented the effect of EPA on sodium transport. We conclude that n-3 PUFAs activate epithelial sodium channels downstream of cAMP in a cAMP-dependent pathway also involving PKA.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Epithelial Na+ channel stimulation by n-3 fatty acids requires proximity to a membrane-bound A-kinase-anchoring protein complexed with protein kinase A and phosphodiesterase

Essential polyunsatured fatty acids have been shown to modulate enzymes, channels and transporters, to interact with lipid bilayers and to affect metabolic pathways. We have previously shown that eicosapentanoic acid (EPA, C20:5, n-3) activates epithelial sodium channels (ENaCs) in a cAMP-dependent manner involving stimulation of cAMP-dependent protein kinase (PKA). In the present study, we explored further the mechanism of EPA stimulation of ENaC in A6 cells. Fluorescence resonance energy transfer experiments confirmed activation of PKA by EPA. Consistent with our previous studies, EPA had no further stimulatory effect on amiloride-sensitive transepithelial current (INa) in the presence of CPT-cAMP. Thus, we investigated the effect of EPA on cellular pathways which produce cAMP. EPA did not stimulate adenylate cyclase activity or total cellular cAMP accumulation. However, membrane-bound phosphodiesterase activity was inhibited by EPA from 2.46 pmol/mg of protein/min to 1.3 pmol/mg of protein/min. To investigate the potential role of an A-kinase-anchoring protein (AKAP), we used HT31, an inhibitor of the binding between PKA and AKAPs as well as cerulenin, an inhibitor of myristoylation and palmitoylation. Both agents prevented the stimulatory effect of EPA and CPT-cAMP on INa and drastically decreased the amount of PKA in the apical membrane. Colocalization experiments in A6 cells cotransfected with fluorescently labeled ENaC β subunit and PKA regulatory subunit confirmed the close proximity of the two proteins and the membrane anchorage of PKA. Last, in A6 cells transfected with a dead mutant of Sgk, an enzyme which up-regulates ENaCs, EPA did not stimulate Na+ current. Our results suggest that stimulation of ENaCs by EPA occurs via SGK in membrane-bound compartments containing an AKAP, activated PKA, and a phosphodiesterase. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Electrophysiological characterization of rat type II pneumocytes in situ

Optimal aeration of the lungs is dependent on an alveolar fluid clearance, a process that is governed by Na+ and Cl- transport. However, the specific contribution of various ion channels in different alveolar cell types under basal or stimulated conditions is not exactly known. We established a novel functional model of rat lung slices suitable for nystatin-perforated whole-cell patch-clamp experiments. Lung slices retained a majority of live cells for up to 72 hours. Type II pneumocytes in situ had a mean capacitance of 8.8 +/- 2.5 pF and a resting membrane potential of -4.4 +/- 1.9 mV. Bath replacement of Na+ with NMDG+ decreased inward whole-cell currents by 70%, 21% and 52% of which were sensitive to 10 microM and 1 mM of amiloride, respectively. Exposure of slices to 0.5 microM dexamethasone for 1 hour did not affect ion currents, while chronic exposure (0.5 microM, 24-72 h) induced an increase in both total Na+-entry currents and amiloride-sensitive currents. Under acute exposure to 100 microM cpt-cAMP, Type II cells in situ rapidly hyperpolarized by 25-30 mV, due to activation of whole-cell Cl- currents sensitive to 0.1 mM of 5-Nitro-2-(3-phenylpropylamino)benzoic acid. In addition, in the presence of cpt-cAMP, total sodium currents and currents sensitive to 10 microM amiloride increased by 32% and 70%, respectively. Thus, in Type II pneumocytes in situ: (1) amiloride-sensitive sodium channels contribute to only half of total Na+-entry and are stimulated by chronic exposure to glucocorticoids; (2) acute increase in cellular cAMP content simultaneously stimulates the entry of Cl- and Na+ ions.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Melatonin down-regulates volume-sensitive chloride channels in fibroblasts.

Melatonin has been reported to present with vasorelaxant and anti-fibrotic properties. We hypothesized that melatonin may down-regulate volume-regulated anion channels (VRAC) in fibroblasts to limit their migration and proliferation. While acute exposure of L929 fibroblasts to melatonin did not result in a significant decrease in VRAC current, pretreatment with 100 μM melatonin for 1 h decreased swelling-dependent activation of anion currents by 83% as measured by whole-cell perforated patch-clamp technique. This down-regulation of VRAC currents was dose-dependent with a half-maximal inhibition of 3.02 ± 0.48 μM. Overnight treatment of cells with 100 nM melatonin had the same inhibitory potency as a 1-h treatment with 100 μM. A similar down-regulatory effect of melatonin on VRAC was observed in primary rat lung fibroblasts. The effect of melatonin was prevented by luzindole and K185 that suggests implication of MT2 receptor. GF109203X, a protein kinase C inhibitor, blocked melatonin's action on VRAC, indicating that MT2 receptor activation results in stimulation of PKC. Consequently, melatonin inhibited regulatory volume decrease following hypotonic swelling of cells. Melatonin also decreased the migration of L929 fibroblasts through the same pathways that blocked VRAC. There was no significant inhibition of cell proliferation. Our study suggests that the attenuation of fibrosis and vascular remodeling by melatonin seen in animal models of hypertension and pulmonary fibrosis might be, in part, related to blunted fibroblast migration possibly through protein kinase C-mediated decrease in chloride channel activity.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Opposing effects of bone morphogenetic protein-2 and endothelin-1 on lung fibroblast chloride currents.

Alteration in the control of bone morphogenetic protein (BMP)-regulated genes and increased expression of endothelin (ET)-1 are both believed to play important roles in the still incompletely understood pathobiology of pulmonary vascular remodeling and fibrosis. Recent studies have drawn attention to the contribution of adventitial fibroblast activation in these phenomena. Because chloride channels are involved in the control of physiological function of fibroblasts, we hypothesized that these channels are differentially regulated by BMPs and ET. We measured chloride ion currents by whole-cell path-clamping in cultured primary human pulmonary fibroblasts. The application of BMP2 prevented activation of these currents by hypotonic challenge in a time- and dose-dependent manner, partially via protein kinase C signaling. Maximal inhibition was observed after 45-minute incubation of cells in the presence of 10 ng/ml of BMP2. ET-1 did not activate chloride channels acutely; however, prolonged treatment of cells with ET-1 (100 nM, 2 h) induced the appearance of lysophosphatidic acid-activated chloride currents (a marker of differentiated myofibroblasts), and this induction could be effectively blocked by BMP2 pretreatment (10 ng/ml). BMP2 also prevented stimulation of α-smooth muscle actin gene expression and cell migration of fibroblasts induced by ET-1. We conclude that ET-1 and BMP2 have opposing effects on chloride channel activity in human fibroblasts. This is a potentially relevant mechanism involved in pulmonary vascular remodeling and fibrosis.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Aldosterone-induced and GTP-stimulated methylation of a 90-kDa polypeptide in the apical membrane of A6 epithelia

Aldosterone treatment of A6 cultured renal epithelial cells methylates the apical membrane, and we examined the aldosterone-induced carboxymethylation of the apical membrane of these cells to determine the targeted polypeptides. Methionine-deprived A6 cells were incubated with aldosterone and [3H]methionine. Homogenates and apical membranes were solubilized and analyzed by SDS-polyacrylamide gel electrophoresis. Label incorporation in a 90-kDa polypeptide was more intense (4-fold) in membranes after aldosterone compared to control. For in vitro methylation, membranes were isolated, incubated with S-adenosyl-L-[methyl-3H]methionine, and analyzed for 3H-methyl uptake. Label incorporation was low in control membranes but markedly stimulated (4-fold) in membrane preparations from aldosterone-treated cells. Guanosine 5′-O-(3-thiotriphosphate) increased in vitro methylation of a 90-kDa polypeptide 5-fold in control membranes but after aldosterone, where methylation was already stimulated, little change was observed. We conclude that aldosterone induces methylation of an apical membrane 90-kDa polypeptide, possibly a subunit of the epithelial Na+ channel, in a GTP-dependent manner, and this may be one of the final steps in a cascade of reactions leading to the natriferic action of this hormone.SCOPUS: ar.jinfo:eu-repo/semantics/publishe