Klonierung und pharmakologische Charakterisierung des equinen Histamin H4 Rezeptors

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.