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the surprise was in the nature of the [Ca
2+
]i responses observed in groups of cells.
Previous studies had reported rapid [Ca
2+
]i oscillations in single cells but observa-
tions of groups of cells had not been made prior to that time. Since the prior imaging
technique was photometry (i.e. simply measures total light output from the view
field without actually 'imaging' pixel by pixel) then it was assumed any oscillations
of single cells in a crowd would be lost in the noise of unsynchronized responses.
However when groups of freshly isolated endothelial cells from P-UA comprising
patches of 12-25 endothelial cells at a time were imaged there was indeed evidence
of synchronous peaks of [Ca
2+
]i and a pregnancy related difference in their like-
lihood. At doses of ATP from 30 to 300
M almost half the observations of cells
from pregnant ewes showed synchronous and regular periodic oscillations similar to
those reported previously in P-UAEC, while in fresh cells from NP ewes only 1/22
recordings showed periodic oscillatory behavior [9]. This was consistent with obser-
vations of single cells at passage 4 and suggested that cells may communicate and
synchronize their oscillations in vivo. There are several profound physiologic impli-
cations of this phenomenon, including the recruitment of increasing numbers of
cells to provide vasodilator synthesis, the fact such events may be agonist-specific.
Furthermore it raised the intriguing possibility of 'digital' communication between
cells and further that distributing vessels of the uterus may be able to communicate
with resistance level vessels and vice versa, independently of the direction of blood
flow.
In order to further address the possibility of cell-cell communication and syn-
chronization, further studies using digital video imaging system to examine UAEC
responses to ATP at low (20%), intermediate (70%, our normal density in the past)
and high density (100%) have been performed, and recording times extended to
30 min [37]. Such studies have clearly shown that increased cell density is associ-
ated with a greater number of cells responding to ATP, and further that cells treated
at high density with ATP can oscillate synchronously in groups with P-UAEC >
NP-UAEC. A more subtle difference for the response to ATP was the ability of the
oscillatory response to show sharply localized [Ca
2+
]i bursts separated by a return
to basal level (Fig. 11.5a). The number of 'bursts' in a given 30 min observation
period in response to ATP was found to be greater in pregnancy. In particular, while
the number of cells responding with the initial spike of [Ca
2+
]i that is already depen-
dent on release of Ca
2+
from the ER is only marginally density dependent for ATP
in NP and P-UAEC, the ability to show higher numbers of subsequent bursts in the
CCE phase of the remaining 30 min is far greater in P- than in NP-UAEC. Indeed
cells from pregnant ewes have
twice
the likelihood of showing 5 bursts in 30 min
than the nonpregnants at the 100% density seen in vivo [37] (Fig. 11.5b). This is
perhaps the most physiologically relevant observation in this field made to date
since (1) it relates to cells at the most physiologic density, i.e. confluent at 100%,
(2) even if NO secretion per second was not greater in P than in NP UA endothe-
lium, a more sustained level of NO production would maintain chronic vasodilation
more effectively, and (3) a greater frequency of 'bursts' over a longer period
of time could sustain signal propagation between cells more effectively via Gap
junctions.
μ
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