Biology Reference
In-Depth Information
while pregnancy adaptation of signaling must occur at a
post
receptor level, it is
not apparently due to simple changes in expression of individual G proteins or
PLC itself.
11.3 IP3 as a Regulator of the Sustained Phase [Ca
2+
]i Response
to ATP
In a preliminary screen, the IP3 receptor antagonist 2-APB was found to not only
block the initial [Ca
2+
]i response that was not dependent on extracellular Ca
2+
,but
also the sustained [Ca
2+
]i response to ATP which was dependent on extracellular
Ca
2+
. Similar results were seen using the PLC inhibitor U73122, a compound struc-
turally distinct from 2-APB which prevents IP3 generation at the source rather than
at its site of action. Given the complete dependence of all aspects of the [Ca
2+
]i
response to IP3 (or more specifically Ins(1,4,5)P3), P2X involvement in the sus-
tained [Ca
2+
]i phase could be eliminated altogether [30], and the conclusion had
to be made that either IP3 receptor was in some way directly involved in control-
ling the sustained Ca
2+
influx or, more likely, the influx from outside was being
channeled in a manner sensitive to the IP3-dependent emptying of the ER, possibly
through a Store Operated Channel Entry (SOCE)/Capacitative Ca
2+
Entry (CCE)
type mechanism. Further examination of NP-and P-UAEC [Ca
2+
]i responses to both
Thapsigargin [11] and to ATP [12] in the absence of extracellular Ca
2+
and then with
subsequent re-addition of Ca
2+
back to the extracellular medium confirmed that NP-
and P-UAEC were both capable of CCE; removal of Ca
2+
from the medium before
stimulation with ATP resulted in the complete loss of the sustained phase in UAEC,
and subsequent replacement of the Ca
2+
only after the initial peak had gone and
[Ca
2+
]i was down to basal resulted in both a recovery and even some overshoot in
the sustained phase [12]. Of note the magnitude of the CCE response to ATP in P-
UAEC and NP-UAEC was similar under these non-physiologic conditions [12], but
the experiment using Thapsigargin [11] revealed P-UAEC had a greater capacity to
respond than NP-UAEC. Thus responses to ATP only utilize a part of the capacity
of the cell to respond through CCE type mechanisms. The ability of Thapsigargin to
trigger CCE in UAEC also shows that the opening of the channels mediating CCE
can occur independently of P2Y2 receptor occupancy and so are directly respon-
sive to emptying of the ER Ca
2+
pool (SOCE, or CCE), such as could occur when
IP3 opens IP3-R for any length of time. In addition, studies with either ATP or
Thapsigargin revealed that the CCE mediated influx of Ca
2+
(on reapplication of
Ca
2+
to the medium) in each case could be prevented if 2-APB (an inhibitor of IP3
binding to its receptor) was already present, and could also be arrested and even
reversed if 2-APB was added after the extracellular Ca
2+
[11, 12]. The speed of this
action was pretty much instantaneous and subsequent studies (see below) confirmed
that indeed 2-APB may be working on a plasma membrane channel mediating CCE
or, more likely, interacting with IP3-R that had docked with plasma membrane Ca
2+
channels responsible for CCE, consistent with the findings emerging from other
laboratories at that time (reviewed in [4]).
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