Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vi, 52 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 42-49).

The KATP channels play an important role in the membrane excitability and vascular tone regulation. Previous studies indicate that the function of KATP channels is disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrate S-glutathionylation to be a modulation mechanism underlying the oxidant-mediated vascular KATP channel inhibition, the molecular basis for the channel inhibition and the alleviation of the channel inhibition by vasoactive intestinal peptide (VIP). We found that an exposure of isolated mesenteric rings to H2O2 impaired the KATP channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, micromolar H2O2 or diamide caused a strong inhibition of the vascular KATP channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH), indicating S-glutathionylation. By co-expressions of Kir6.1 or Kir6.2 with SUR2B subunits, we found that the oxidant sensitivity of the KATP channel relied on the Kir6.1 subunit. Systematic mutational analysis revealed three cysteine residues (Cys43, Cys120 and Cys176) to be important. Among them, Cys176 was prominent, contributing to >80% oxidant sensitivity. Biochemical pull-down assay with biotinylated glutathione ethyl ester (BioGEE) showed that mutations of Cys176 impaired the oxidant-induced incorporation of GSH to the Kir6.1 subunit. Simulation modeling of Kir6.1 S-glutathionylation revealed that after incorporation to residue 176, the GSH moiety occupied a space between slide helix and two transmembrane helices. This prevented the necessary conformational change of the inner helix for channel gating, and retained the channel in its closed state. VIP is a potent vasodilator, and is shown to have protective role against oxidative stress. We found that the channel was strongly augmented by VIP and the channel activation relied on PKA phosphorylation. These results therefore indicate that 1) the vascular KATP channel is strongly inhibited in oxidative stress, 2) S-glutathionylation underlies the oxidant-mediated KATP channel inhibition, 3) Cys176 in the Kir6.1 subunit is the major site for S-glutathionylation, and 4) the Kir6.1/SUR2B channel is activated in a PKA-dependent manner by VIP that has been previously shown to alleviate oxidative stress.

Le tissu adipeux sous-cutané (TAsc) est le site préférentiel du stockage postprandial des triglycérides (TG). Quand les capacités d’accrétion sont dépassées, le stockage des TG se fait dans des sites ectopiques du TA et dans des tissus non adipocytaires, par exemple foie et muscles, ce qui entraine de multiples dysfonctionnements dans ces organes et tissus, et permet le développement du syndrome d’insulinorésistance. Chez les sujets obèses, la période postprandiale est caractérisée par des anomalies métaboliques, immunitaires, hormonales, et également par une diminution importante du flot sanguin dans le tissu adipeux (FSTA) sous-cutané. Ce blocage de la perfusion postprandiale du TA a aussi été montrée chez des individus minces qui avaient de très lourds antécédents familiaux de maladies cardiométabolique (obésité, diabète de type 2, maladies cardiovasculaires). Dans cette thèse, on classifiera ces individus comme « non-répondeurs ». À ce jour, peu d’attention a été accordée à ce phénomène. L’hypothèse qui sous-tend cette thèse est que les anomalies du FSTA sont innées ou primaires et sont impliquées très tôt dans le développement de la résistance à l’insuline (RI), du diabète de type 2 et du syndrome métabolique. Le but de notre recherche était donc de vérifier si les altérations du FSTA sont présentes chez les personnes saines et minces, mais à très haut risque de développer une RI ou une maladie cardiométabolique. Nous avons aussi cherché à déterminer les facteurs liés à la non-réponse. Pour cela il nous a fallu explorer certains facteurs hormonaux impliqués dans la régulation du FSTA. Nos résultats montrent que le FSTA est très diminué, à jeun et en postprandial, chez les sujets à haut risque de maladies cardiométaboliques mais encore minces et métaboliquement sains, sans RI. Nous avons aussi montré, pour la première fois, l’effet vasodilatateur du peptide intestinal vasoactif (VIP) dans le TAsc, tout comme le rôle stimulant du système cholinergique dans la régulation postprandiale du FSTA. Cependant, aucun de ces facteurs ne participe au dysfonctionnement du FSTA postprandial chez les non-répondeurs. Des taux répétés de TG plus élevés chez les non-répondeurs et l’association du FSTA avec certains indices de la RI décrits dans la littérature suggèrent que l’altération du métabolisme lipidique suite à la diminution du FSTA puisse servir de médiateur à la détérioration de la sensibilité à l’insuline. / Abstract : Subcutaneous adipose tissue (SCAT) is the preferential site of triacylglycerols (TAG) postprandial disposal. When the buffering capacity of SCAT for lipids is exceeded, TAG are disposed in ectopic adipose tissue depots and in non-adipose tissues, such as liver and muscles. Consequently, multiple dysfunctions of these organs and tissues develop including insulin resistance (IR). In obese people, the postprandial period is characterized by metabolic, immune and hormonal alterations, but also by severely altered adipose tissue blood flow (ATBF). Nevertheless, significant alteration of postprandial ATBF was also found in lean individuals with highly positive familiar history of cardiometabolic diseases (obesity, type 2 diabetes, cardiovascular diseases). In the thesis, we term them as "non-responders". Up to date, little attention has been payed to this phenomenon. The underlying hypothesis of this thesis is that alterations in ATBF are inborne or very early and that they participate on the development of IR, type 2 diabetes and metabolic syndrome. Consequently, the aim of our research was to verify if the alterations in ATBF are present in healthy, normal-weight subjects, but at very high risk for development of IR or cardiometabolic diseases. Simultaneously, we searched for factors linked with nonresponsiveness phenomenon. To do this, we examined some hormonal factors in ATBF regulation. Our results confirm the presence of altered fasting and postprandial ATBF in at highrisk subjects for cardiometabolic diseases, but still lean and metabolically healthy, without IR. For the first time, we have also demonstrated the role of cholinergic system in postprandial ATBF regulation, and vasodilatory effect of vasoactive intestinal peptide (VIP) in SCAT. However, none of these factors takes part in postprandial ATBF dysfunction in non-responders. Higher TAG levels repeatedly found in non-responders and the association of ATBF with some indices of insulin sensitivity described in the literature suggest that alteration of lipid metabolism as a result of low ATBF may mediate deterioration of insulin sensitivity.

Cortical malformations as a result of altered development are a common cause of human epilepsy. The cellular mechanisms that render neurons of malformed cortex epileptogenic remain unclear. Using a rat model of the malformation of microgyria, a previous study showed an alteration in the number of immunocytochemically-identified parvalbumin cells, a GABAergic inhibitory interneurons subtype (Rosen et al., 1998). A second study showed no change in the total number of GABAergic neurons (Schwarz et al., 2000). Consequently, we hypothesize that interneuron subtypes are differentially affected by maldevelopment. The present study investigated (1) whether interneuron subtype identity is retained in malformed cortex, based on chemical content, and (2) whether the proportion of three chemical subtypes is altered in malformed cortex. Here we demonstrate that three non-overlapping subtype markers remain non-overlapping in malformed cortex, but show altered distributions. These findings suggest that an increase in one subpopulation of interneurons may compensate for a corresponding decrease in a second subset.

Crohns sjukdom är en kronisk inflammatorisk tarmsjukdom av okänd orsak. Det tidigaste tecknet på Crohns sjukdom är mikroskopiska sår i det s.k. follikelassocierade epitelet (FAE) som täcker ansamlingar av immunceller i tarmen. FAE är specialiserat för att fånga innehåll från tarmen och transportera det till underliggande immunvävnad. Denna funktion är viktig för att inducera skyddande immunsvar, men den utgör också en ingångsväg för sjukdomsalstrande bakterier. Crohns sjukdom är associerat med ett kraftigt ökat immunsvar mot bakterier, och sjukdomsförloppet kan ändras av stress. Det övergripande syftet med avhandlingen var att studera effekterna av stress på FAE samt att undersöka rollen av FAE vid utvecklingen av tarminflammation, särskilt vid Crohns sjukdom. Inledningsvis studerades effekterna av psykologisk stress på FAE. Stressade råttor uppvisade ökad genomsläpplighet av bakterier efter stress, och passagen var högre i FAE än i vanligt epitel. Efterföljande experiment visade att stressförändringarna i slemhinnan regleras via kortikotropinfrisättande hormon och mastceller. Vidare visade det sig att vasoaktiv intestinal peptid kunde efterlikna stressens effekter på genomsläppligheten, och att detta kunde förhindras genom att blockera mastcellerna. Studier av tunntarmsslemhinna från patienter med icke-inflammatorisk tarmsjukdom och friska kontroller visade en högre passage av bakterier i FAE än i vanligt epitel. Hos patienter med Crohns sjukdom var bakteriepassagen genom FAE betydligt ökad jämfört med kontroller. Resultaten från detta avhandlingsarbete visar att stress kan förändra upptaget av bakterier från tarmen via FAE, med mekanismer som innefattar kortikotropinfrisättande hormon och mastceller. Detta har gett nya kunskaper kring regleringen av slemhinnebarriären. Vidare presenterar denna avhandling nya insikter i sjukdomsuppkomsten vid Crohns sjukdom genom att påvisa en tidigare okänd defekt i barriärfunktionen i FAE. / The earliest observable signs of Crohn’s disease are microscopic erosions in the follicle-associated epithelium (FAE) covering the Peyer’s patches. The FAE, which contains M cells, is specialised in sampling of luminal content and delivery to underlying immune cells. This sampling is crucial for induction of protective immune responses, but it also provides a route of entry for microorganisms into the mucosa. Crohn’s disease is associated with an increased immune response to bacteria, and the disease course can be altered by stress. The overall aim of this thesis was to study the effects of stress on the FAE and elucidate the role of FAE in the development of intestinal inflammation, specifically Crohn’s disease. Initially, rats were submitted to acute and chronic water avoidance stress to study the effects of psychological stress on the FAE. Stressed rats showed enhanced antigen and bacterial passage, and the passage was higher in FAE than in regular villus epithelium (VE). Further, stress gave rise to ultrastructural changes. Subsequent experiments revealed the stress-induced increase in permeability to be regulated by corticotropin-releasing hormone and mast cells. Furthermore, vasoactive intestinal peptide (VIP) mimicked the stress effects on permeability, and the VIP effects were inhibited by a mast cell stabiliser. Human studies of ileal mucosa from patients with non-inflammatory disease and healthy controls showed a higher antigen and bacterial passage in FAE than in VE. In patients with Crohn’s disease, the bacterial passage across the FAE was significantly increased compared to non-inflammatory and inflammatory controls (ulcerative colitis). Furthermore, there was an enhanced uptake of bacteria into dendritic cells, and augmented TNF-α release in Crohn’s disease mucosa. Taken together this thesis shows that stress can modulate the uptake of luminal antigens and bacteria via the FAE, through mechanisms involving CRH and mast cells. It further shows that human ileal FAE is functionally distinct from VE, and that Crohn’s disease patients exhibit enhanced FAE permeability compared to inflammatory and non-inflammatory controls. This thesis presents novel insights into regulation of the FAE barrier, as well as into the pathophysiology of Crohn’s disease by demonstrating a previously unrecognised defect of the FAE barrier function in ileal Crohn’s disease.

Mes études doctorales ont permis de démontrer que les souris déficientes en VIP présentent une microcéphalie ayant principalement une origine maternelle qui affecte secondairement le développement de la substance blanche. Cette production placentaire par les lymphocytes T pourrait être affectée dans des pathologies du système immunitaire. De plus, nos données indiquent qu’une déficience en VIP prédispose à l'apparition de troubles sensoriels, en particulier de la nociception. Il est donc possible que les déficits précoces de développement du cerveau murin et l'apparition de l'hypersensibilité cutanée mécanique et thermique froide soient deux facettes d'une même pathologie. Des mesures d'activité de décharge spontanée des neurones dans le thalamus sensoriel chez des mâles adultes anesthésiés ont montré que les neurones des animaux KO sont hyper-excités, ce qui suggère un traitement aberrant des informations, notamment nociceptives, ou que l'activité inhibitrice des interneurones des réseaux locaux est réduite. / The studies carried out during my PhD demonstrate that VIP-deficient mice suffer from microcephaly and as well as white matter deficits mainly due to the absence of maternal VIP during embryogenesis, Placental secretion of VIP is dependent on T lymphocytes and could be altered in pathologies of the immune system. Moreover, our data links VIP deficiency to sensory alterations, specifically, the nociceptive system. Thus, it is possible that early developmental defects and hypersensitivity to mechanical and cold stimuli are two manifestations of the same pathology. This hypothesis was reinforced following analysis of spontaneous firing patterns of neurons in the sensory thalamus of anesthetized adult males. Neurons from VIP-KO mice are hyperactive, which suggests aberrant local processing of nociceptive input or that the inhibitory inputs from local interneuron networks is reduced.