Ioncsatornák, modulációs mechanizmusok és szinaptikus kapcsolatok jelentősége a patkány nucleus cochlearis jelfeldolgozó működésében = Roles of ion channels, modulatory mechanisms and synaptic connections in the signal processing of the rat cochlear nucleus

A projekt célja a nucleus cochlearis jelfeldolgozó tevékenységében jelentős ioncsatornák, modulációs mechanizmusok és szinaptikus kapcsolatok elemzése volt. A munka legfontosabb eredményei a következők: (a) Azonosítottuk a nucleus cochlearisban expresszált HCN-csatornaalegységeket. Feltártuk a h-áram jelentőségét az óriássejtek elektromos sajátságaiban és szinaptikus kapcsolataik finomhangolásában. (b) Leírtuk a kolinerg ingerlés hatását az óriássejtek elektrofiziológiai sajátságaira és a rajtuk kialakuló posztszinaptikus áramokra. Azonosítottuk a hatást közvetítő receptor-altípusokat. (c) Elemeztük a szemcsesejtek aktivitásfüggő intracelluláris kalciumkoncentráció-változásait. Kifejlesztettünk egy olyan félautomatikus kiértékelési eljárást, ami alkalmas a neuronális kalciumtranziensek felismerésére és analízisére. (d) Feltártuk egyes muszkarinerg receptorok szerepét sejttenyészeti körülmények között fenntartott astrocyták intracelluláris kalciumkoncentráció-változásaiban. (e) Képalkotó és elektrofiziológiai módszereket alkalmazva leírtuk a nucleus cochlearisban található Purkinje-szerű neuronok sajátságait. (f) Kvantitatív morfometriai analízis segítségével azonosítottuk a pyramis- és az óriássejtek elkülönítésére legalkalmasabb paramétereket. (f) Leírtuk a tacrolimus hatását a pyramis-sejteken kialakuló posztszinaptikus áramok tulajdonságaira. (g) Feltártuk a protein-foszfatáz-1M és a Rho-kináz szerepét a bushy-sejtek és az acusticus rostok közötti szinapszis működésében. | Our aim was to assess the roles of ionic channels, modulatory mechanisms, and synaptic connections in regulating the activity of the cochlear nucleus (CN). The most important results are as follows. (a) We identified all HCN channel subunits expressed in the CN. We revealed the significance of the h-current in shaping the electrical properties and fine-tunig the synaptic connections of the giant cells. (b) We described the effects of cholinergic stimulation on the membrane properties and postsynaptic currents of the giant cells along with the identification of the receptor subtypes mediating the effects. (c) We investigated activity-related cytoplasmic calcium concentration changes of the granule neurones, and developed a semiautomatic method for the identification and evaluation of neuronal calcium transients. (d) We assessed the involvement of various muscarinergic receptors in the regulation of intracellular calcium concentration changes of cultured astrocytes. (e) Using imaging and electrophysiological techniques, we described the properties of Purkinje-like cells of the CN. (f) We identified morphological parameters that are suitable for the unambiguous discrimination between pyramidal and giant neurones. (f) We documented the effects of tacrolimus on the postsynaptic currents recorded from pyramidal neurones. (g) We revealed and described the roles of the protein-phosphatase-1M and Rho-kinase in the synaptic transmission between the bushy cells and acoustic fibers

Mitochondrial expression of TASK-3 channels in human malignant melanoma cell cultures and their role in cell survival and proliferation

A TASK-3-csatornák számos sejttípusban jelen vannak, egészséges és daganatos elfajulást mutató sejtekben egyaránt. A csatornafehérje tumorképződést serkentő funkcióját már számos daganattípusban felvetették. Jelen munkánkban immunjelölési módszerekkel vizsgáltuk a TASK-3-csatornafehérje megoszlási mintázatát tenyészetben fenntartott melanoma malignum sejtekben, valamint a daganatos elváltozást nem mutató HaCaT humán keratinocyta sejtvonalban. Az immuncitokémiai kísérletek elvégzése előtt a melanoma sejtek TASK-3-expresszióját mRNS-szinten is igazoltuk. A csatornafehérje sejten belüli megoszlása elsősorban intracelluláris mintázatot mutatott, míg a sejtfelszíni membrán jelölődése nem volt szembetűnő. Kettős jelölés alkalmazásával megállapítottuk, hogy a TASK-3-csatornák és a mitokondriumok jelölődési mintázata mindkét vizsgált sejttípusban nagymértékű átfedést mutat. Ezen eredményt megerősítették a humán bőrből készített szövettani metszeteken végzett immunhisztokémiai kísérleteink, ahol hasonló festődési mintázatot tapasztaltunk a melanocytákban és keratinocytákban egyaránt. Eredményeink alapján feltételezhető, hogy a TASK-3-csatornák a melanoma sejtek mitokondriális membránjában helyezkednek el, és szerepet játszanak a mitokondriális funkcióban. Egyes felvetések szerint a hypoxia iránti tolerancia fokozása révén hozzájárulnak a sejtek életképességének fenntartásához, támogatva a daganatos sejtek életképességét és a tumornövekedést, ennek pontos mechanizmusa azonban nem ismert. A mitokondriumok által expresszált TASK-3-csatornának a sejtek életképességében betöltött szerepét munkánk során a csatornafehérje expressziójának csökkentése révén vizsgáltuk. A melanoma sejteken shRNS-kazetták alkalmazásával tranziens transzfekciót végeztünk, és TASK-3 géncsendesített sejttenyészeteket hoztunk létre. A csökkent TASK-3-expresszió eredményeképpen a sejteken jellegzetes morfológiai változásokat figyelhettünk meg, csökkent a sejtek DNS-tartalma és metabolikus aktivitása, valamint a mitokondriális funkció is károsodást szenvedett. Ezen változások arra engednek következtetni, hogy a mitokondriumokban expresszált TASK-3-csatornák kulcsfontosságú szerepet játszanak a melanoma sejtek életképességének fenntartásában. További vizsgálatokat igényel annak tisztázása, hogy a csatornafunkció és a sejtek életképessége közötti összefüggés valamilyen módon kiaknázható-e a daganatellenes terápiában. TASK-3 channels are expressed in several cell types including both healthy and malignantly transformed cells. The role of channel function in tumourigenesis has been proposed in different human cancers. In this work the distribution pattern of TASK-3 channel protein was investigated in cultured melanoma malignum cells and in non-malignant HaCaT keratinocytes using immunochemical methods. Before these experiments, the TASK-3 expression of melanoma cells was confirmed on mRNA level as well. The subcellular localization of the channel protein was found to be mainly intracellular, while the labelling of cell surface membrane was not significant. Double-labelling experiments revealed that the distribution of TASK-3 channels shows strong co-localisation with mitochondria in both investigated cell types. These results were confirmed by immunohistochemical staining of wax-embedded human skin tissue sections, where the same labelling pattern could be observed in melanocytes and keratinocytes as well. Our findings suggested that TASK-3 channels are present in the mitochondrial membrane of melanoma cells, where they might contribute to mitochondrial function. It has been proposed earlier that they might provide greater hypoxia tolerance for tumour cells, which might contribute to cell survival and tumour growth, but the exact mechanism by which these effects are exerted is still unknown. We investigated the significance of TASK-3 mitochondrial TASK-3 channels in cell viability by interfering with the function of the channel protein. Melanoma cells were transiently transfected with shRNA cassettes, and TASK-3 knockdown cell cultures were generated. The reduced expression of TASK-3 resulted in characteristic changes of the cell morphology, reduction of DNA content, decreased metabolic activity and impaired mitochondrial function. These changes indicate that TASK-3 channels expressed in the mitochondrial membrane may have crucial roles in maintaining the viability of melanoma cells. However, further experiments are needed to clarify whether the inhibition of TASK-3 channel function could be exploited in anti-cancer therapy

Cytoplasmic Ca²⁺ concentration changes evoked by muscarinic cholinergic stimulation in primary and metastatic melanoma cell lines

Experiments were performed to explore differences between cultured primary and metastatic melanoma cell lines in their muscarinic acetylcholine receptor-mediated intracellular Ca²⁺ signalization. The expression of type 1 and type 3 muscarinic receptors was detected and compared at the protein level using both immunocytochemistry and semiquantitative western blotting. The functionality of muscarinic receptors was tested by applying carbamylcholine (CCh; 1 mmol/l) and by recording the associated increases in cytoplasmic Ca²⁺ using Ca²⁺ imaging with the application of the Ca²⁺ indicator dye, fluo-4. These data indicate that the expression levels of the receptor proteins were not significantly different in the metastatic (HT199, HT168-M1) and the primary (WM35) cell lines. Although Ca²⁺ transients were evoked in all the three cell lines by CCh, the proportion of the CCh-positive cells was smaller amongst the WM35 cells. The Ca²⁺ transients could be effectively blocked by atropine (0.1 mmol/l). The time courses of the Ca²⁺ transients were highly variable, and in some instances they showed a late (plateau-like) component whose presence crucially depended on the influx of extracellular Ca²⁺. When the extracellular Ca²⁺ concentration was reduced, the duration of the CCh-evoked transients was considerably decreased; a phenomenon that was more pronounced in the metastatic cell lines. Although there are no fundamental differences in the muscarinic receptor-mediated Ca²⁺ signalization of the primary and metastatic cell lines, the quantitative differences showed in this study may partially explain the increased malignancy and migratory potential of the metastatic cells.12 page(s

Voltage-gated Potassium Channel (Kv) Subunits Expressed in the Rat Cochlear Nucleus

Because the neuronal membrane properties and firing characteristics are crucially affected by the depolarization-activated K+ channel (Kv) subunits, data about the Kv distribution may provide useful information regarding the functionality of the neurons situated in the cochlear nucleus (CN). Using immunohistochemistry in free-floating slices, the distribution of seven Kv subunits was described in the rat CN. Positive labeling was observed for Kv1.1, 1.2, 1.6, 3.1, 3.4, 4.2, and 4.3 subunits. Giant and octopus neurons showed particularly strong immunopositivity for Kv3.1; octopus neurons showed intense Kv1.1- and 1.2-specific reactions also. In the latter case, an age-dependent change of the expression pattern was also documented; although both young and older animals produced definite labeling for Kv1.2, the intensity of the reaction increased in older animals and was accompanied with the translocation of the Kv1.2 subunits to the cell surface membrane. The granule cell layer exhibited strong Kv4.2-specific immunopositivity, and markedly Kv4.2-positive glomerular synapses were also seen. It was found that neither giant nor pyramidal cells were uniform in terms of their Kv expression patterns. Our data provide new information about the Kv expression of the CN and also suggest potential functional heterogeneity of the giant and pyramidal cells. (J Histochem Cytochem 56:443–465, 2008

Expression of anti-Mullerian hormone receptor on the appendix testis in connection with urological disorders

The female internal sex organs develop from the paramesonephric (Mullerian) duct. In male embryos, the regression of the Mullerian duct is caused by the anti-Mullerian hormone (AMH), which plays an important role in the process of testicular descent. The physiological remnant of the Mullerian duct in males is the appendix testis (AT). In our previous study, we presented evidence for the decreased incidence of AT in cryptorchidism with intraoperative surgery. In this report, the expression of the anti-Mullerian hormone receptor type 2 (AMHR2), the specific receptor of AMH, on the AT was investigated in connection with different urological disorders, such as hernia inguinalis, torsion of AT, cysta epididymis, varicocele, hydrocele testis and various forms of undescended testis. The correlation between the age of the patients and the expression of the AMHR2 was also examined. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect the receptor's mRNA and protein levels, respectively. We demonstrate that AMHR2 is expressed in the ATs. Additionally, the presence of this receptor was proven at the mRNA and protein levels. The expression pattern of the receptor correlated with neither the examined urological disorders nor the age of the patients; therefore, the function of the AT remains obscure

Inhibition of TASK-3 (KCNK9) channel biosynthesis changes cell morphology and decreases both DNA content and mitochondrial function of melanoma cells maintained in cell culture

TASK-3 channel overexpression was shown to facilitate the survival of malignantly transformed cells, possibly by providing greater hypoxia tolerance through a still unknown mechanism. Although it has been suggested previously that TASK-3 channels are expressed in the mitochondrial membranes, their role here remains elusive. In this study, a transient transfection of TASK-3 knockdown melanoma cell cultures was produced to show the significance of TASK-3 expression. Reduction of the TASK-3 protein biosynthesis induced characteristic changes in cell morphology, reduced the amount of DNA and decreased metabolic activity and mitochondrial function of melanoma cells when compared with control. These findings indicate that TASK-3 channel expression and function is indispensable for the proliferation and/or survival of the melanoma cells, as they seem to contribute to their mitochondrial functions. The significance is that, in this study, we have shown that TASK-3 channels are expressed in the mitochondria of melanoma malignum cells, and they are essential for maintaining cellular integrity and viability. The TASK-3 knockdown melanoma cell line had altered morphology, reduced DNA content, decreased metabolic activity and impaired mitochondrial function. These data indicate that TASK-3 channels are functionally present in the mitochondria of the melanoma cells, and their function is essential for the survival of these cells, thus TASK-3 channels may be the possible targets of future anticancer therapy.15 page(s