Elektrophysiologische Charakterisierung der Effekte von Menthol auf die hypothalamische Mauszelllinie GT1 mit der Patch-Clamp-Technik

Menthol bewirkt eine Kälteempfindung wenn es mit der Haut oder Schleimhaut in Berührung kommt. Der zugrunde liegende molekulare Mechanismus ist jedoch noch nicht vollständig geklärt. Die Inhibition von Ca2+-Kanälen mit der Folge einer Reduktion von Ca2+ abhängigen K+-Strömen wurde diskutiert. Auß...

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Bibliographic Details
Main Author: Wegner, Sven
Contributors: Voigt, Karl-Heinz (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2009
Online Access:PDF Full Text
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It is well known that menthol evokes a sensation of cold when applied to skin or mucosa. The underlying ionic mechanisms, however, are still under debate. Blockade of Ca2+ currents with reduction of Ca2+ dependent K+ currents have been discussed. Recordings from DRG neurons clearly demonstrated menthol induced activation of TRPM8 (CMR1) ion channels and depolarisation of the cell membrane caused by Ca2+ influx. Moreover, recent studies suggest multiple actions of menthol including, for example, excitation of TRPV3 and inhibition of TRPA1 (ANKTM1) channels. Here menthol was used as an agent to examine desensitization effects on a hypothalamic GT1-7 neuronal cell line by using PCR, Ca2+-Imaging and whole cell patch clamp techniques. PCR studies verified the expression of TRPM8 mRNA, but Ca2+-Imaging does not show an increase of intracellular Ca2+ during menthol application. The patch-clamp studies demonstrated a pronounced reduction of an outward current which cannot be explained by the conventional assumptions about the action of menthol via TRP channels. The data point to a dose dependent inhibition of voltage-dependent K+ channels which is completely reversible. Recordings made under extracellular K+ concentration of 140 mM support this concept. Menthol effects occurred also in Ca2+ free bath solution. To examine whether menthol affects further currents besides K+, Na+ currents were isolated and are not affected by menthol. The two major voltage dependent K+-currents (delayed rectifier of A- and DR-current) were identified according to their different activation characteristics. Menthol blocks the majority of DR-currents, while the A-currents persist. In a dose response curve the EC50 value of all blocked outward-currents is 380 μM. As menthol blocks the DR-type K+ current, the shape of APs might be changed. Functionally, this could lead to different firing rates and patterns, which is thought to influence hormone release.