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In-Depth Information
11.5 2-APB as an Effective Inhibitor of Ca
2+
Signaling and NO
Production in Endothelial Cells Freshly Isolated
from the Luminal Surface of the UA Ex Vivo
The development of imaging methods to detect NO production and [Ca
2+
]i simulta-
neously in freshly isolated UA endothelium provides a powerful means to monitor
cell function in the very cells that perform the vasodilation response in vivo. In
these studies, the pharmacological agonists ionomycin (calcium ionophore) and
Thapsigargin (an endoplasmic reticulum Ca
2+
pump inhibitor), were used to max-
imally elevate [Ca
2+
]
i
above the physiologic range, and so fully activate eNOS in
a receptor independent manner as a measure of total eNOS expression [36]. The
corresponding NO production stimulated by ionomycin (5
M)
were similar at 1.95 /2.05 fold respectively in endothelium from late term pregnant
ewes and 1.34/1.37 fold in endothelium from ewes synchronized to the follicular
phase when compared to control responses determined in endothelium from ewes
in the luteal phase. In contrast, the physiologic agonist, ATP (100
μ
M) vs TG (10
μ
M), stimulated
a 3.43 fold increase in NO in endothelium from pregnant ewes, and 1.90 fold in
endothelium from follicular phase ewes when expressed normalized to the response
observed in the luteal phase [36]. This once again suggested that during pregnancy
and in the follicular phase, eNOS activation is enhanced beyond simple changes in
expression of eNOS protein in vivo. Of further relevance here, however, is the obser-
vation that the extent to which [Ca
2+
]i rose in the sustained phase of the response
to ATP was a closer indicator of the extent to which NO was increased (Fig. 11.3).
In addition, studies performed in endothelium freshly isolated from pregnant ewes
showed that 2-APB could totally prevented the ATP-induced [Ca
2+
]
i
response, just
as it had in UAEC in culture, but could not completely inhibit NO production
(Fig. 11.3), consistent with findings in UAEC in primary culture. This is a par-
ticularly important observation since it strongly implies the observations made in
UAEC are directly relevant to the situation observed in the intact endothelium in
vivo.
μ
11.6 The Case for TRPC Channels as Mediators of CCE
in UAEC
Of further relevance to the actual mechanism of Ca
2+
influx from outside the cell,
a striking finding using 2-APB in P-UAEC is that the Ki for inhibition of the
[Ca
2+
]i plateau is almost a log unit lower than that for inhibiting the initial release
from the ER. Indeed the Ki for the inhibition of the initial peak is actually that
expected for IP3-R inhibition (6
M) while that for inhibition of [Ca
2+
]i plateau
−
7
μ
is closer to 1
M. Assuming the effects to be on the IP3-R itself this can be inter-
preted in two possible ways. The first is that following the initial peak the IP3-R
is altered in some way (phosphorylation, calmodulin binding, or binding another
protein) that raises its affinity for 2-APB. The second is 2-APB binds another
μ
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