Diss. (sammanfattning) Stockholm : Karol. inst., 1999. / Härtill 7 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.

In this thesis the in vitro release of immunoreactive CCK₈ (iCCK₈) from rat central nervous system preparations and the regulation of this release have been studied. Rat brain was dissected into the following regions; hypothalamus, cerebral cortex, striatum and thalamus, according to the method of Brownstein, Arimura, Sato et. al. (1975). CCK₈ was found to be distributed throughout these regions (range of 9 - 300 pmoles), with the highest concentration in cortex (300 pmol). In addition, low levels (range 9 - 30 pmoles) of CCK₈ were found in spinal cord, brain stem and cerebellum, in agreement with other workers. Immunohistochemical techniques have demonstrated CCK-like immunoreactivity in nerve cell bodies and fibres throughout brain, particularly in the cortex. Subcellular fractionation of rat brain was used to study the subcellular localisation of CCK. Tissue was homogenised to shear off nerve terminals (synaptosomes) which were purified and used to study the release of the peptide from hypothalamic and extrahypothalamic nerve endings.

The effects of cholecystokinin octapeptide (CCK-8), and its antagonist proglumide, upon conditioned behavior in the rat was studied. First, the effects of CCK-8 and proglumide upon passive avoidance behavior was investigated. Rats were trained to avoid a darkened chamber by presenting electrical footshock (two seconds of intensity levels) inside the chamber. Directly following the footshock, injections of CCK-8 or proglumide were given, with avoidance behavior measured 24 hours following the injection. CCK-8 was found to produce reductions in the passive avoidance latency at doses ranging from 30 ug/Kg to 500 ug/Kg. This effect was found to be dependent upon the current intensity used during conditioning. The CCK-8 effect was found when the current was at 0.25 mA, but at no other current setting tested. Proglumide (5 mg/Kg) was found to block the CCK-8 effect upon passive avoidance behavior. A lower dose of proglumide (2 mg/Kg) was found to produce reductions in the passive avoidance latency. These results suggest that CCK-8 may play a role in passive avoidance conditioning in rats. The effects of CCK-8 upon an appetitively conditioned behavior were then investigated. Rats were trained to locate and drink from a drinking tube that contained a 10 percent sucrose solution. Following 30 seconds exposure to the solution, injections of CCK-8 were given, with the latency to begin drinking from the tube measured 24 hours later. CCK-8 was found to produce increases in the latency to begin drinking, at doses of 20 ug/Kg and 100 ug/Kg. CCK-8 also produced a reduction in the amount of sucrose solution consumed during the test period. When CCK-8 was given following exposure to regular tap water, no increase in drinking latency or reduction in consumption was found. These results suggest that CCK-8 can act as an aversive stimulus and is capable of producing conditioned taste aversions. The results of this dissertation project demonstrate that CCK-8 can influence the acquisition of conditioned behavior in the rat when the octapeptide is paired with the presentation of an unconditioned stimulus (shock or sucrose).

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Cholecystokinin (CCK) is - a multifunctional peptide hormone that is widely distributedthroughout the body. Initially discovered as a gut hormone, CCK is important in integrating many digestive functions. In the nervous system cholecystokinin functions as a neurotransmitter or neuromodulator. It is also considered by many to be a naturally occurring satiety factor, important for the termination of a meal. Recently, our lab has identified the presence of CCK-like immunoreactivity in taste receptor cells of Sprague-Dawley rats. Preliminary in situ hybridization experiments appeared to demonstrate that the mRNA for cholecystokinin may also be expressed in the lingual epithelium and the taste cells of the circumvallate and foliate papillae of the rat tongue. To provide confirrnatory evidence for the presence of CCK in taste epithelium and to investigate its role in taste receptor cells, we further examined the expression of cholecystokinin mRNA in rat lingual epithelium using Northern blot analysis and RT-PCR. Northern analysis proved to be difficult using standard non-radiographic techniques and small oligonucleotide (35 bp) probes. Generating a 535 by radio-labeled probe with random primed labeling, Northern analysis demonstrated positive bands in control tissue (cerebral cortex and duodenum) but failed to demonstrate binding to lingual tissue. Since expression of CCK mRNA in taste cells could be below the level of detection of Northern analysis, the more sensitive technique of RT-PCR was employed. Similar results were obtained with RT-PCR PCR products were observed in cortical and duodenal tissues, but not in gustatory tissue. Therefore immunocytochemical and in situ hybridization results appear to be in conflict with those obtained with Northern and RT-PCR techniques. There remain many caveats in the collective interpretation of these results and further experimentation, particularly with probes designed to hybridize with differing regions of the CCK gene, will be required to more fully understand the putative presence and processing the CCK mRNA in taste receptor cells. / Department of Biology

La maladie cœliaque est la principale pathologie liée à un déficit en cholécystokinine (CCK) occasionnant une diminution de la fonction pancréatique et une réduction de la contraction vésiculaire. La maladie cœliaque ou intolérance au gluten est une affection auto-immunitaire qui se caractérise par une malabsorption intestinale causée par une atteinte inflammatoire de l’intestin grêle, suite à l’ingestion de gluten présent dans la farine de blé, d’orge ou de seigle. Le but de ce travail est d’étudier la raison de ce déficit en CCK chez des patients adultes souffrant de maladie cœliaque. La diminution de sécrétion de CCK a d’abord été attribuée à une réduction du nombre de cellules à CCK intestinales et de leur contenu hormonal dans le cadre d’une muqueuse intestinale atrophique, de type destructif ou hypoplasique. Elle pourrait cependant être liée à d’autres mécanismes: une diminution de la stimulation des cellules endocrines suite à une moindre hydrolyse des nutriments dans la lumière intestinale, un trouble fonctionnel des cellules, une altération de l’interaction avec d’autres cellules intestinales (endocrines, épithéliales ou nerveuses), l’absence d’un facteur stimulant la sécrétion de CCK ou la présence de facteurs inhibiteurs associés au processus inflammatoire dans la muqueuse intestinale. Nous avons montré que la sécrétion de CCK n’est pas seulement diminuée chez les patients cœliaques présentant une muqueuse duodénale de type hypoplasique ou destructif mais également chez les patients dont la muqueuse ne présente qu’un infiltrat excessif de lymphocytes intraépithéliaux (IEL). Seuls les patients cœliaques traités, sans caractère desctructif ou hypoplasique de leur muqueuse, et sans infiltrat inflammatoire, présentent une sécrétion de CCK normale et semblable aux volontaires sains. En utilisant l’immunocytochimie et la PCR semi-quantitative, nous avons démontré que le déficit en sécrétion de CCK n’est pas lié à une diminution du nombre de cellules à CCK dans la muqueuse duodénale mais plutôt à une diminution du taux d’ARN messager, et de la synthèse de CCK, ceci tant chez les patients avec des lésions muqueuses de type destructif ou hypoplasique que chez les patients ne présentant qu’un infiltrat de lymphocytes intraépithéliaux. Ces données suggèrent que l’infiltrat inflammatoire affecte l’expression du gène de la CCK dans les cellules entéroendocrines intestinales. La partie suivante de notre travail a consisté à investiguer le rôle d’un déficit d’hydrolyse intraduodénale des nutriments sur la sécrétion de CCK chez des patients cœliaques adultes. Nous avons montré que la sécrétion postprandiale de CCK n’est pas améliorée par l’ingestion d’un repas prédigéré, chez des patients présentant des lésions destructives ou hypoplasiques comme ceux présentant des lésions muqueuses infiltratives. De plus l’hydrolyse alimentaire intraduodénale devrait être normale en cas de lésion muqueuse limitée à un infiltrat de IEL. Les taux plasmatiques réduits de CCK dans ce groupe de patients suggèrent clairement que le déficit de sécrétion de CCK pourrait être lié au processus inflammatoire et à des facteurs provenant de l’infiltrat de lymphocytes intraépithéliaux. Pour cette raison, nous avons étudié les effets de surnageants de cultures lymphocytaires et de cytokines produites par les lymphocytes de la muqueuse intestinale sur des biopsies duodénales maintenues en culture de courte durée et sur des cellules sécrétant de la CCK et provenant d’une lignée cellulaire murine neuroendocrine STC-1. Nous avons montré un effet inhibiteur significatif sur la sécrétion de CCK du surnageant de cultures de lymphocytes de la lamina propria stimulés par de la phytohémagglutinine A, ainsi que de l’interleukine-1 et du TNF-. En conclusion, nous avons montré que le déficit de sécrétion de CCK est présent dès le stade initial infiltratif de la lésion muqueuse cœliaque et que ce déficit pourrait être lié au processus inflammatoire présent dans la muqueuse intestinale et à certains facteurs suppresseurs associés à ces cellules inflammatoires. La modulation de la sécrétion de CCK doit être considérée comme un processus complexe impliquant une co-régulation par les nutriments, des peptides stimulants, des neurotransmetteurs et des facteurs solubles comme les cytokines. Les données décrites dans ce travail nous font entrevoir de nouveaux mécanismes de régulation et de nouvelles pistes pour améliorer la prise en charge de patients cœliaques continuant à présenter des troubles digestifs malgré un régime sans gluten bien suivi. / Celiac sprue is the principal disease associated with impaired cholecystokinin (CCK) release that accounts for the diminished gallbladder contraction and pancreatic function. Celiac sprue, also known as celiac disease or gluten-sensitive enteropathy, is an autoimmune disorder characterized by malabsorption resulting from inflammatory injury to the small intestine mucosa after the ingestion of wheat gluten or related rye and barley proteins. The aim of our work was to study the defective CCK release that is found in adult patients with celiac disease. The CCK decreased secretion was first thought to be caused by a reduction in the number of intestinal CCK cells in the presence of mucosal atrophy, but could also be related to other mechanisms: a lack of stimulation of the mucosal CCK cells due to impaired intra-duodenal hydrolysis of nutrients, a functional abnormality of the CCK-cell, an impaired interaction of the CCK-cell with other intestinal cells (endocrine, epithelial or nervous cells), the absence of a stimulating factor (luminal cholecystokinin releasing factor) or the presence of inhibitory factors associated with the inflammatory process taking place in the mucosa. We have shown that CCK release is not only impaired in untreated celiac patients with a destructive/hypoplastic type duodenal mucosa but also in patients whose mucosa only shows a significant intraepithelial lymphocytic (IEL) infiltrate. Treated celiac patients without hypoplastic or destructive mucosa and without any increase in IEL infiltrate exhibit a basal and postprandial CCK release similar to the control group. Using immunocytochemistry and semi-quantitative PCR we have demonstrated that the defective release of CCK observed in patients with coeliac disease is not related to a decrease in the number of CCK cells present in the proximal part of the small intestine but rather to a decrease in CCK synthesis related to a decrease in mRNA content, even in patients with an infiltrative type of celiac disease. These data suggest that the inflammatory infiltrate in the mucosa of celiac patients affects the expression of the CCK gene. The next part of our work included the investigation of the role of the intraluminal nutrient hydrolysis in adult celiac patients. Our data indicates that ingestion of a pre-digested meal does not correct the defective CCK release in patients with a destructive or an infiltrative type of celiac mucosal lesion. Moreover, intraduodenal food digestion should be considered normal in celiac patients with mucosal lesions limited to an intraepithelial lymphocytic infiltrate. The decreased level of plasma CCK in this group of patients, given a pre-digested meal, strongly supports that the defective CCK release could be related to suppressive factors released by the inflammatory infiltrate. We therefore studied the effects of supernatants obtained from IEL and lamina propria lymphocyte cultures and various cytokines produced by mucosal lymphocytes on short-term organ cultures of duodenal biopsy specimens obtained from normal controls and on the CCK secreting murine neuroendocrine tumour cell line STC-1. We demonstrated a significant inhibitory effect of supernatants obtained from phytohaemagglutinin A-stimulated cultured lamina propria lymphocytes and from phytohaemagglutinin A-stimulated co-cultured lamina propria and intraepithelial lymphocytes, and of interleukin-1 and tumour necrosing factor- on CCK release. All together these data show that the defective CCK release is already present at the initial infiltrative type of mucosal lesion seen in celiac disease and that this defective release may be related to inflammation and suppressive factors associated with the inflammatory cells. Modulation of CCK release in the gut should be considered as a complex process implying co-regulation of nutrients, releasing peptides, neurotransmitters and soluble factors including cytokines. These results may be of help in finding new directions to improve the treatment of patients with celiac disease who continue to present with abdominal complaints in spite of a strict gluten-free diet.

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