G5 Doctoral dissertation (article)

List of Authors: Huhtinen. Anna
Publisher: University of Turku
Place: Turku
Publication year: 2017
ISBN: 978-951-29-6917-3
eISBN: 978-951-29-6918-0


α2-Adrenoceptors (α2-ARs) belong to the large superfamily of G protein-coupled receptors 
(GPCRs). They mediate important actions of the endogenous catecholamines adrenaline and 
noradrenaline. All three α2-AR subtypes are involved in the regulation of blood pressure and 
vascular tone. α2-ARs can regulate both vascular smooth muscle contraction and remodeling 
of the blood vessel wall, but the intracellular signaling mechanisms involved in these functions 
have remained largely unknown. The aim of this thesis was to investigate the involvement of 
the α2B-AR subtype in the regulation of the contraction and proliferation of vascular smooth 
muscle cells (VSMC), and to clarify the related cellular signaling mechanisms. 
In order to characterize the effects of α2B-AR activation on VSMC contraction and proliferation 
and to investigate whether these functions could be altered by drug treatment, a VSMC 
line stably expressing the human α2B-AR was generated by transfection of rat A7r5 cells.
Characterization of the novel A7r5-α2B cell line indicated that the localization and ligand 
binding properties of the expressed α2B-ARs were in line with earlier studies of α2B-ARs in 
different host cell environments, and that the receptors had the expected pharmacological
characteristics. Therefore, the generated A7r5-α2B cell line was regarded as a useful tool for 
investigating the functions and regulation of α2B-ARs in VSMCs. α2B-ARs were demonstrated 
to be capable of mediating VSMC contraction by using a functional assay measuring myosin 
light chain phosphorylation, which is a biochemical readout of VSMC contraction. The 
network of signaling pathways involved in α2B-AR-mediated contraction of A7r5 VSMCs 
appeared to be complex and seemed to involve many mediators, such as Gi proteins, Gβγ 
subunits, phospholipase C (PLC), protein kinase C (PKC) and L-type Ca2+ channels. Different 
screening assays, namely DNA microarray, small inhibitor compound library screening and 
kinase activity profiling, were used to investigate the genetic regulation and intracellular 
signaling mechanisms involved in α2BAR-evoked proliferation of A7r5 VSMCs. The cellular 
mechanisms and signal transduction pathways participating in this response appeared to 
be complex and included redundancy. The employed screening assays and their respective 
data analysis approaches were found to be useful as tools to map the activation of cellular 
signaling networks in a situation where the exact mechanisms still remain unknown. 
These screening tools were considered suitable for hypothesis generation, but additional 
approaches will be required for further hypothesis testing.

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Last updated on 2019-29-01 at 10:17