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 170

Fig. 10.4 Presence of Cx43 in BCEC. A : Western blotting of Cx43 (43 kDa) in
BCEC, with knock-down of endogenous Cx43 by RNA silencing (100 nM duplex).
TR represents treatment with transfection reagent only. Scrambled siRNA duplex of
Cx43-1 was used as negative control. GAPDH (37 kDa) levels are visualized as a
loading control. Cx43-1 (region 322-344): 5'-GAAGGAGGAGGAACUCAAAdTdT Cx43-
2 (region 883-905): 5'-CAAUUCUUCCUGCCGCAAUdTdT Scramble (region 322-344):
5'-GGUAAACGGAACGAGAAGAdTdT B : Immunofluorescence staining of Cx43 in BCEC. A
monoclonal mouse anti-Cx43 antibody (Sigma, Clone CXN-6; Catalog number C8093) was used
for Cx43-detection in Western blot and immunofluorescence
This conclusion is further supported by experiments demonstrating that applica-
tion of Ca 2+ -free solution resulted in immediate marked increase in the rate of ATP
release. Similar to their effects on the dye uptake, 43 Gap26 and FFA also inhibited
the enhancement of ATP release in the absence of extracellular Ca 2+ , indicating that
both agents inhibit the release by the same mechanism [43].
Direct measurement of ATP in samples of the bathing fluid immediately after
mechanical stimulation demonstrated enhanced ATP release after mechanical stim-
ulation (Fig. 10.7A). 43 Gap26 and FFA inhibited this ATP release [43]. Our
experiments therefore demonstrated that functional hemichannels are present in
BCECs, which are opened by low extracellular Ca 2+ and in response to mechanical
stimulation, and that ATP release through hemichannels contributes to mechanical
stimulation-induced intercellular Ca 2+ wave propagation.