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Fischer, Florian (2011): Klonierung und pharmakologische Charakterisierung des equinen Histamin H4 Rezeptors. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

In the present study, the equine histamine 4 receptor (eH4R) was cloned, sequenced and pharmacologically characterized. The findings were compared to those, obtained with the human H4R (hH4R). Due to its expression in cells of the immune system, the eH4R provides a promising target for the development of novel therapeutic strategies in allergic diseases, such as Recurrent Airway Obstruction (RAO) and allergic dermatitis in the horse. To clone the eH4R, mRNA was isolated from horse white blood cells and cDNA was synthesized by reverse transcription. Specific primers were used to amplify the eH4R sequence, which was then cloned into pJET1.2/blunt vectors. The open reading frame is 1185 bp long and codes for a 394 amino acid protein which shows 72,9 % homology to the human receptor. The cDNA sequence was published in the NCBI GenBank under the accession number HM015200. To pharmacologically and functionally characterize the eH4R and hH4R, their cDNAs were subcloned into the expression vector pcDNA3.1 and either transfected transiently into COS-7 cells or stably into HEK293 cells. Binding-characteristics were examined by homologous und heterologous competition experiments using the antagonist 3H-pyrilamine or the agonist 3H-histamine as radioligand. High affinity binding of histamine could only be detected in hH4R, but not in eH4R transfected COS-7 cells. Nevertheless, histamine was able to inhibit cAMP-production in stably transfected HEK293 cells via the eH4R and the hH4R. The eH4R expressed in HEK293 cells is coupled to the stimulation of ERK1/2, while the hH4R shows already high constitutive activity. The antagonists JNJ7777120, Thioperamide, Pyrilamine and Diphenhydramine display considerable species-specific differences concerning the affinities between eH4R and hH4R and also vary in their intrinsic activities. Thioperamide, known for its inverse agonism at the hH4R showed agonist behaviour in ERK1/2 regulation. In contrast, the non-selective antagonist Diphenhydramin showed inverse agonist behaviour, which was more pronounced at the hH4R than the eH4R. These findings suggest that there are considerable pharmacological and functional differences between the cloned eH4R and hH4R.