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related protein with higher affinity and this protein is involved in Ca
2+
influx. Of
direct relevance, a newly described family of proteins (TRPC) are now increas-
ingly implicated as mediators of Ca
2+
influx in many cells [38] including endothelial
cells (Reviewed in [24]), and interactions of IP3-R with TRPC have more recently
been described in several cells [5, 17, 23, 25]. Further, a recent report proposed
evidence that 2-APB binds TRPC with a higher affinity than IP3-R and inhibits
Ca
2+
influx to cells [4]; it remains unclear, however, if 2-APB binds directly on
TRPC itself or, more likely, that 2-APB binds to IP3-R with higher affinity once
TRPC binds IP3-R and so has in turn altered IP3-R conformation. Either mech-
anism would explain how ATP stimulated Ca
2+
influx during the sustained phase
was immediately and completely blocked by 2-APB. Nonetheless, studies on the
effects of Thapsigargin on [Ca
2+
]i in UAEC [11] are at least supportive of the acti-
vation of TRPC by emptying of the ER stores, an event known to be sensed by
IP3-R.
Thapsigargin is known to elevate [Ca
2+
]i by promoting leakage of Ca
2+
from the
ER through the inhibition of reuptake. The effects of Thapsigargin to empty the ER
should be complete within just a few minutes yet the effect on [Ca
2+
]i in UAEC
are particularly long lasting - as long as 60 min elevation of [Ca
2+
]i before a return
to basal levels [11]. Nonetheless in the absence of extracellular Ca
2+
this is dra-
matically blunted to a smaller (
20 min. Thus
the capacity of the ER itself is not unusually excessive, and the prolonged [Ca
2+
]i
response in normal medium must involve a mechanism that is greatly dependent
upon prolonged Ca
2+
influx. Whether emptying of the ER promotes IP3-R binding
to TRPC to allow direct Ca
2+
entry to the cytosol or alternatively the two form a
pipe to allow direct refilling of the ER, so sustaining the ER capacity to function, is
unclear from these studies alone. Either way, however, the behavior of the plasma
membrane Ca
2+
channels activated in response to emptying of the ER in UAEC, and
mediating a CCE type response in a manner sensitive to 2-APB inhibition, strongly
implicates TRPC channel involvement.
∼
30% maximum) response lasting
∼
11.7 Evidence for Enhanced TRPC3-IP3R-2 Functional
Interaction in P-UAEC
The proposal that TRPC type channels are involved in the sustained CCE phase
of [Ca
2+
]i response to ATP in UAEC is given some further support by the obser-
vation that transfection of TRPC3 into bovine pulmonary artery endothelial cells
allowed the induction of a more sustained Ca
2+
entry in response to ATP, bradykinin
and intracellular IP3 where no such response occurred before [16]. A subsequent
survey of NP- and P-UAEC using antisera specific to different TRPC subtypes
has clearly identified the presence of TRPC3 and TRPC6 [12]. In addition the
presence of IP3-R1, IP3-R2 and IP3-R3 in UAEC were also confirmed [11]. Of
interest, however, there were no apparent differences in the level of expression of
any of these proteins in NP- vs P-UAEC. Nonetheless it was intriguing to note that
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