G5 Doctoral dissertation (article)

α2B-ADRENOCEPTORS IN THE REGULATION OF VASCULAR SMOOTH MUSCLE CELL CONTRACTION AND PROLIFERATION




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

URL: http://urn.fi/URN:ISBN:978-951-29-6918-0


Abstract

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.


Last updated on 2021-24-06 at 08:44