Browsing by Subject "Plasmamembran-Calcium-ATPase"

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Zur Lokalisation und Funktion des Calciumtransporters im Innenohr von Knochenfischen (Oreochromis mossambicus)
(2014) Knie, Miriam; Hilbig, Reinhard
In fish the perception of gravity takes place in the otolithic organs of the inner ear. These structures are filled with endolymph fluid and posses a sensory macula epithelium on which the otolith, the so called inner ear stone, is attached. The otoliths mainly consist of calcium carbonate which is embedded in a protein matrix. The otolith growth is subjected to a diurnal rhythm. Up to now, it is not clear how the calcium ions, which are needed for the otolith minerlisation, are transported through the sensory epithelium into the endolymph. The aim of this study was to analyze the localization and function of the plasmamembrane-calcium ATPase (PMCA) as a candidate for the calcium transporting protein in the inner ear sensory epithelia of Oreochromis mossambicus with regard to the otolith biomineralization. This study was carried out using larval cichlids in different developmental stages, because in the course of the larval development, the otoliths are characterised by a rapid increase in size and dynamic rearrangements. In addition, further mineralised tissues like the teeth and the skull bones have been analysed. Moreover, the organs generally participating in the calcium metabolism like gills, intestine and kidney have been examined in the same developmental profiles. All analysed epithelia and tissues are charaterised by a co-expression of the PMCA-isoforms 1 to 4 throughout the larval development. Based on the results of this study it can be assumed that each of the four PMCA-isoforms fullfills a specialised function in the examined epithelia and tissues. In the inner ear sensory epitehlia PMCA 1 is involved in the transport of calcium ions into the endolymph, whereas isoform 2 rather performs the function of a "housekeeping gene" and is important for the maintenance of the basal calcium concentration. In the bones, the calcium export function is mainly performed by the isoforms 1 and 4, the basal calcium levels are kept stable by isoform 3. Thus the regulation of the calcium ions may be a complex and delicately regulated process. The results of the expression analysis - a specific reaction with the sense-, as well as the antisense-probe - led to the assumption that, in addition to the sense-RNA, also naturally occuring antisense-RNA is transcribed for the four plasmamembrane-calcium ATPase isoforms in Oreochromis mossambicus. These hypothesis was tested with the Northern Blot method, thereby further clues for the existence of natural antisense-RNA have been obtained. Naturally occuring antisense-transcripts can either directly serve as a template for transcription or influence the expression of the corresponding sense-RNA. To check if this is also true for Oreochromis mossambicus quantitative in-situ hybridizations in the context of the diurnal otolith growth rhythm have been performed. By the help of this method it was possible to observe changes in the expression levels of both transcripts, as well as changes in the ratio between sense- and antisense-RNA. Herefrom it was deducible, that the regulation of PMCA in the inner ear sensory epithelia may occur via the transcription rate, as well as by the expression of the antisense-transcripts themselves, in this case probably the ratio between the sense- and antisense-RNA matters. Which of the regulatory mechanisms known for naturally occuring antisense-RNA is acting in detail requires further research.