ATP Release Via Connexin Hemichannels Controls Intercellular Propagation of Ca Waves in Corneal Endothelial Cells - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 169

Since P2Y4 and P2Y6 are pyrimidine receptors with very low sensitivity to
ATP, these receptors are unlikely to have an important contribution to the Ca 2+
wave propagation. To investigate the involvement of P2Y1 receptors (which can
be activated by ATP and ADP) and P2Y2 receptors (which are activated by ATP
but only very weakly sensitive to ADP), we also examined the effect of nucleotide
hydrolysis by exogenous apyrase VI (which has a high ATPase/ADPase ratio) and
apyrase VII (which preferentially hydrolyses ADP). Apyrase VII caused a some-
what stronger inhibition in the outermost cell layers when compared to apyrase
VI. The combination of 5 U/ml apyrase VI and 5 U/ml apyrase VII had a cumu-
lative effect, causing a more pronounced inhibition of the Ca 2+ wave than either
10 U/ml apyrase VI or 10 U/ml apyrase VII (Fig. 10.3). These experiments pro-
vide evidence that both ATP and ADP are involved [44], and suggest that P2Y1 and
P2Y2 receptors are involved in PIC in BCEC. Furthermore, these results indicate
that ATP is released in response to mechanical stimulation. Direct evidence for the
involvement of ATP release in the intercellular Ca 2+ wave propagation was obtained
by ATP-dependent luciferin-luciferase bioluminescence measurements, showing
that mechanical stimulation increases the amount of ATP in the extracellular
medium [43].
10.3.2.2 Mechanism of ATP Release
Around the time of our findings, Stout et al. [108] and Braet et al. [15] provided
evidence for the involvement of hemichannels in ATP release during Ca 2+ wave
propagation in astrocytes. We therefore examined Cx hemichannels as candidate
pathways for ATP release involved in Ca 2+ wave propagation in BCECs.
Using RT-PCR, we showed the presence of mRNA for Cx26, Cx43, Cx45 and
Cx50 in BCEC (Table 10.1) and we demonstrated expression of Cx43 protein in the
membrane, using Western blot and immunofluorescence (Fig. 10.4).
Next, we investigated the contribution of Cx hemichannels to PIC in
BCECs using the hemichannel blockers flufenamic acid (FFA) and 43 Gap26
(VCYDKSFPISHVR) [37]. 43 Gap26 is a Cx mimetic peptide with a sequence iden-
tical to a part of the EL1 sub-domain (first extracellular loop sequence) of Cx43,
which constitutes a significant portion of the pore-lining. This peptide has been
shown to inhibit Cx43-mediated hemichannel opening [15]. Application of FFA or
43 Gap26 resulted in a pronounced inhibition of the propagation of the Ca 2+ wave
evoked by mechanical stimulation (Fig. 10.5).
Since Stout et al. [108] have shown that Cx hemichannels open upon expo-
sure to low extracellular Ca 2+ and show permeability for a number of hydrophilic
dyes such as lucifer yellow, we performed dye uptake experiments in Ca 2+ -free
solution using lucifer yellow as a membrane-impermeable hemichannel-permeable
reporter dye. Removal of extracellular Ca 2+ -induced uptake of lucifer yellow, which
was completely inhibited by FFA and 43 Gap26 [43]. Our observations provide evi-
dence that hemichannels are present in BCECs and can be blocked by FFA and
43 Gap26.