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Fig. 10.7
ATP release after mechanical stimulation in control and in the presence of histamine,
thrombin or TRAP-6. ATP release in basal conditions and after mechanical stimulation (MS) of
BCEC (
N
=
63) (
a
). Effect of histamine (100
μ
M for 10 min;
N
=
8) (
b
), thrombin (2 U/ml for
5min;
N
=
55) (
c
),andTRAP-6(10
μ
M for 30 min;
N
=
16) (
d
) on ATP release after mechanical
stimulation.
∗
p
<0.05
(Fig. 10.6), but did not significantly reduce the fluorescence recovery in FRAP
experiments, indicating that the inhibition of Ca
2+
wave propagation by histamine is
not via an effect on the GJIC pathway. In cells pretreated with
43
Gap26 (Fig. 10.6c)
or apyrases (Fig. 10.6d), activation of histamine receptors did not cause a signifi-
cant further reduction of Ca
2+
wave propagation, providing evidence that histamine
exerts its effect by inhibiting PIC. In consistence with this conclusion, histamine
precluded enhancement of Ca
2+
wave propagation by the ecto-nucleotidase inhibitor
ARL-67156 (Fig. 10.6e). Exposure of BCEC to histamine in Ca
2+
-free medium led
to complete inhibition of lucifer yellow uptake. Histamine also markedly reduced
ATP release upon mechanical stimulation (Fig. 10.7b). These experiments provide
evidence that histamine inhibits PIC by inhibiting the hemichannel-mediated ATP
release.
To investigate whether thrombin also affects IC, we first examined expression
of PAR receptors. RT-PCR showed transcripts for the PAR receptors PAR-1 and
PAR-2, but not for PAR-4 (Table 10.1). Immunocytochemistry showed thrombin-
sensitive PAR receptors as well as trypsin-sensitive PAR-2 receptors. Both thrombin
and the selective PAR-1 agonist TRAP-6 strongly reduced the active area of the Ca
2+
wave (Fig. 10.8) [26]. The effect of thrombin and TRAP-6 on the Ca
2+
wave was
inhibited by a peptide antagonist of PAR-1, but not by the indazole derivative YD-3,
a selective PAR-4 antagonist. While thrombin and TRAP-6 reduced the fluorescence
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