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argued that the hemichannels involved in ATP release in different preparations are
Panx hemichannels (see [95]). There is, however, compelling evidence for the pres-
ence and opening of Cx hemichannels in a number of different conditions. Direct
electrophysiological evidence for Cx43 hemichannel opening was demonstrated in
Cx43 overexpressing HeLa cells and C6 cells [23, 22, 53]. Romanov et al. [89] pro-
vided evidence that Cx hemichannels are responsible for ATP release in type II taste
cells, based on sensitivity to Cx- and Panx-mimetic peptides, effect of carbenox-
olone and kinetics of voltage-dependence. Anselmi et al. [3] provided evidence
for propagation of Ca
2+
signals via Cx hemichannel-mediated ATP release in the
inner ear. Single channel experiments in astrocytes have also demonstrated Cx43-
hemichannel-mediated ATP release [53]. Cx32 hemichannel-mediated ATP release
upon rise in intracellular Ca
2+
concentration has been established in a number of
different cell types [29, 30, 3].
Since Panx hemichannels do not open in Ca
2+
-free conditions [16], our exper-
iments provide evidence that the ATP release in BCEC is mediated via Cx43
hemichannels. Furthermore, Gap26 almost completely blocked Ca
2+
wave prop-
agation, lucifer yellow uptake and ATP release [43]. In addition, the Ca
2+
wave
propagation was not significantly inhibited by 10
M carbenoxolone, which is
known to inhibit Panx hemichannels with an IC
50
of
μ
M
[16]. Furthermore,
the Ca
2+
wave propagation in BCEC was almost completely inhibited by 50
∼
5
μ
M
flufenamic acid [43], which has only limited effect on Panx hemichannels [16].
In addition, preliminary evidence from experiments in our laboratory shows
that cell-permeable TAT-peptides corresponding to the L2 sequence of Cx43
(DGANVDMHLKQIEIKKFKYGIEEHGK) significantly inhibit the Ca
2+
wave
propagation in BCEC by more than 50%, while the H126K/I130N mutant used
as control peptide [100, 101] only causes a limited reduction of the active area,
which was, however, not significant (unpublished findings). These experiments pro-
vide evidence that the ATP release in BCEC is mediated by hemichannels formed
by Cx43.
μ
10.4.2 Effect of Histamine and Thrombin on PIC in BCEC
Our experiments demonstrate that histamine and thrombin, two agents that decrease
integrity of the endothelial barrier [94, 106], markedly reduce the purinergic sig-
naling by inhibiting ATP release via Cx43 hemichannels. We demonstrated that
thrombin enhances MLC phosphorylation through activation of PAR-1 receptors
[26]. As in smooth muscle cells, thrombin regulates the level of MLC phosphory-
lation in the endothelial cells by two opposing pathways: activation of myosin light
chain kinase (MLCK)-driven phosphorylation, and myosin light chain phosphatase
(MLCP)-driven dephosphorylation of MLC. Specifically, by increasing [Ca
2+
]
i
,
thrombin activates MLCK via Ca
2+
-Calmodulin, while it causes inactivation of
MLC phosphatase by mobilizing the RhoA-Rho kinase axis (Fig. 10.9). Since phos-
phorylation of MLC regulates myosin II activity in BCEC, we investigated the role
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