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the 3HA-adenosine A
1
receptor (3HA-A
1
R) and/or HA-TP
plasmids 24 h post
seeding. After 48 h, the mediumwas changed to Eagle's minimum essential medium,
20 mMHEPES, and preincubated for 20 min at 37
C. The cells were incubated with
100
a
M of Ro 20-1724 as a phosphodiesterase inhibitor for 15 min with or without
receptor antagonists. The cells were then stimulated with a receptor agonist and
100
m
M forskolin as an adenylyl cyclase activator and were incubated for another
10 min. Reactions were terminated by adding 2.5% perchloric acid. Acid extracts
were mixed with a 1/10 volume of 4.2 N KOH to neutralize the acid, which resulted
in the formation of potassium perchlorate as the precipitate. The cAMP in the super-
natant was succinylated and determined using a radioimmunoassay kit (Yamasa, To-
kyo, Japan) according to the manufacturer's directions. Briefly, the mixture of
succinyl cAMP and fixed amount of [
125
I]cAMP was reacted with anti-cAMP anti-
bodies. After the removal of unbound cAMP by adsorption on dextran-coated char-
coal radioactivity, the supernatant was measured with a gamma spectrometer.
m
12.2.2
Extracellular signal-regulated kinase1/2 assay
One of the key components in GPCR-induced signaling is mitogen-activated protein
kinase (MAPK) cascades. It has been reported that extracellular signal-regulated ki-
nase1/2 (ERK1/2), a major kinase of MAPK cascades, is phosphorylated by the in-
dividual activation of both the adenosine A
1
and TP
receptors (
Dickenson, Blank, &
Hill, 1998; Miggin & Kinsella, 2001
). We examined whether the coexpression of
adenosine A
1
and TP
a
receptors affected ERK1/2 phosphorylation.
The protocol for the activation of the ERK1/2 assay was as follows. HEK293T
cells were seeded onto 12-well plates at a density of 1
a
10
5
cells/well. The cells were
transfected with the 3HA-A
1
R and HA-TP
plasmids 24 h post seeding. The cells
were washed with a Tyrode-HEPES solution 48 h after transfection (
Table 12.1
)
and were preincubated for 20 min at 37
C. Stimulation with CPA and/or
U46619, which are adenosine A
1
and TP
a
receptors agonists, respectively, for
10 min at 37
C, was terminated by aspiration of the medium, and cells were lysed
in 150
a
m
l of ice-cold Laemmli sample buffer.
12.2.3
Effect of the coexpression of receptors on the signaling
pathway
The coexpression of adenosine A
1
and TP
receptors affected receptor signal trans-
duction. The accumulation of cAMP and activation of ERK1/2 were enhanced by the
costimulation of both receptor agonists (
Mizuno et al., 2012
). These enhancements
were only observed in the coexpressing cells and not in the cells expressing either of
these receptors alone, which indicated that ligand-receptor-selective intracellular re-
sponses were modulated by another ligand-receptor system via receptor oligomer-
ization and/or crosstalk between signals.
a
