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Fig. 10.2
Example of intercellular Ca
2+
wave propagation elicited by point mechanical stimula-
tion of a single BCEC cell in cells loaded with Fluo-4 AM. (
Panel A
) Fluorescence images at
different time points following mechanical stimulation of a single cell. The intensity scale at the
top left
represents the fluorescence changes induced by the [Ca
2+
]
i
changes. The first image shows
the fluorescence intensities before stimulation. The arrow in the second image (time
8.5 s) iden-
tifies the mechanically stimulated (MS) cell. The time course of [Ca
2+
]
i
-sensitive fluorescence of
MS and a typical cell from different neighboring (NB) cell layers is shown at the lower right of the
panel. (
Panel B
) The bar graph at the left of the panel shows the maximal normalized fluorescence
(NF) in the MS cell and in responsive NB cells. The bar graph of the lower right panel shows the
percentage of responsive cells (%RC) for each of the NB cell layers. From [44] with permission.
Copyright:
Association for Research in Vision and Ophthalmology
=
We first examined the presence of purinergic receptors in BCEC. RT-PCR
demonstrated expression in BCEC of mRNA for P2Y1, P2Y2, P2Y4, P2Y6, P2Y12
and for P2X4, P2X5 and P2X7 (Table 10.1). It was previously shown that exogenous
application of purinergic agonist results in Ca
2+
mobilization in BCEC [107]. Using
the fluorescent Ca
2+
indicator Fluo-4-AM, or measuring intracellular Ca
2+
using a
high throughput Fura-2 assay, we demonstrated that application of extracellular ATP
induces a Ca
2+
rise in BCEC, with an EC
50
for ATP of 1.5
M [44].
To examine whether ATP is the messenger in the intercellular propagation of the
Ca
2+
wave in BCEC, we used P2 receptor antagonists. Pretreatment of cells with
the non-selective purinergic receptor antagonist suramin (200
μ
M) indeed inhibited
Ca
2+
wave propagation. Also, exogenous application of apyrases (10 U/ml), which
hydrolyze extracellular nucleotides, decreased the active area of the Ca
2+
wave,
which is the total area of cells that show a rise in [Ca
2+
]
i
[44] (Fig. 10.3). Application
μ
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