Abstract
It has been suggested that A(3) adenosine receptors (ARs) play a role
in the pathophysiology of cerebral ischemia with dual and opposite
neuroprotective and neurodegenerative effects. This could be due
to a receptor regulation mediated by rapid phosphorylation and desensitization
carried out by intracellular kinases. In this study, we investigated
the involvement of extracellular regulated kinase (ERK 1 and 2),
members of the mitogen-activated protein kinase (MAPK) family, in
A(3) AR phosphorylation. A(3) AR mediated the activation of ERK 1/2
with a typical transient monophasic kinetics (5 min). The activation
was not affected by hypertonic sucrose cell pre-treatment, suggesting
that this effect occurred independently of receptor internalization.
The involvement of MAPK cascade in the A(3) AR regulation process
was evaluated using two well-characterized MAPK kinase inhibitors,
PD98059 (2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one) and U0126
(1,4-diamino-2,3-dicyano-1,4-bis (aminophenylthio) butadiene). The
exposure of cells to PD98059 prevented MAPK activation and inhibited
homologous A(3) AR desensitization and internalization, impairing
agonist-mediated receptor phosphorylation. PD98059 inhibited the
membrane translocation of G protein-coupled receptor kinase (GRK(2)),
which is involved in A(3) AR homologous phosphorylation, suggesting
this kinase as a target for the MAPK cascade. On the contrary, the
chemically unrelated inhibitor of the MAPK cascade, U0126, did not
significantly affect GRK(2) membrane translocation or receptor internalization.
Nevertheless, the inhibitor induced a significant impairment of receptor
phosphorylation and desensitization. These results suggested that
the MAPK cascade is involved in A(3) AR regulation by a feedback
mechanism which controls GRK(2) activity and probably involves a
direct receptor phosphorylation.
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