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J. Biol. Chem., Vol. 277, Issue 43, 40324-40334, October 25, 2002
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From the School of Biochemistry and Molecular Biology, Faculty of
Science, Australian National University,
Canberra ACT 0200, Australia
Exposure of HTC rat hepatoma cells to a 33%
decrease in extracellular osmolality caused the cytosolic
Ca2+ concentration ([Ca2+]i) to
increase transiently by ~90 nM. This rise in
[Ca2+]i was inhibited strongly by apyrase, grade
VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI
(which has a high ATP/ADPase ratio) or hexokinase, indicating that
extracellular ADP and/or ATP play a role in the
[Ca2+]i increase. The hypotonically induced rise
in [Ca2+]i was prevented by the prior discharge
of the intracellular Ca2+ store of the cells by
thapsigargin. Removal of extracellular Ca2+ or inhibition
of Ca2+ influx by 1-10 µM Gd3+
depleted the thapsigargin-sensitive Ca2+ stores and thereby
diminished the rise in [Ca2+]i. The hypotonically
induced rise in [Ca2+]i was prevented by
adenosine 2'-phosphate-5'-phosphate (A2P5P) and
pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of
purinergic P2Y1 receptors for which ADP is a major agonist.
Both inhibitors also blocked the rise in [Ca2+]i
elicited by addition of ADP to cells in isotonic medium, whereas A2P5P
had no effect on the rise in [Ca2+]i elicited by
the addition of the P2Y2 and P2Y4 receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat
P2Y1, P2Y2, and P2Y6 receptors.
Inhibition of the hypotonically induced rise in
[Ca2+]i blocked hypotonically induced
K+ (86Rb+) efflux, modulated the
hypotonically induced efflux of taurine, but had no significant effect
on Cl
The Role of P2Y1 Purinergic Receptors and Cytosolic
Ca2+ in Hypotonically Activated Osmolyte Efflux from a
Rat Hepatoma Cell Line*
,
(125I) efflux. The
interaction of extracellular ATP and/or ADP with P2Y1
purinergic receptors therefore plays a role in the response of HTC
cells to osmotic swelling but does not account for activation of all
the efflux pathways involved in the volume-regulatory response.
*
This work was supported by the Australian National Health
and Medical Research Council Grant F97082.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: School of Biochemistry
and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT 0200, Australia. Tel.: 61-2-6125-0640; Fax:
61-2-6125-0313; E-mail: pauline.junankar@anu.edu.au.
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