Pflugers Arch - Eur J Physiol (2004) 448: 592–595
DOI 10.1007/s00424-004-1295-0
CELL AND MOLECULAR PHYSIOLOGY
Jie Wu
.
Teruko Takeo
.
Sechiko Suga
.
Takahiro Kanno
.
Tomohiro Osanai
.
Katsuhiko Mikoshiba
.
Makoto Wakui
2-Aminoethoxydiphenyl borate inhibits agonist-induced Ca
2+
signals by blocking inositol trisphosphate formation in acutely
dissociated mouse pancreatic acinar cells
Received: 23 February 2004 / Accepted: 29 April 2004 / Published online: 12 June 2004
# Springer-Verlag 2004
Abstract Evidence suggests that 2-aminoethoxydiphenyl
borate (2-APB) modulates intracellular Ca
2+
signals in a
complex manner. 2-APB inhibits or potentiates intracellu-
lar Ca
2+
signals in different cell types, perhaps through
different mechanisms. Here, we report a novel mechanism
underlying 2-APB-induced inhibition of agonist-activated
Ca
2+
oscillations in mouse pancreatic acinar cells, using
patch-clamp and biochemical techniques. Pre-treatment of
the cells with 100 µM 2-APB completely abolished ACh-
but not inositol trisphosphate (InsP
3
)-induced Ca
2+
oscillations, suggesting that the mechanism of inhibition
occurs between cytoplasmic receptors and InsP
3
receptor
activation. In addition, 100 µM 2-APB significantly
inhibited ACh-induced phospholipase C (PLC) activation.
These findings indicate that, in mouse pancreatic acinar
cells, in addition to modulating InsP
3
receptors and
blocking the store-operated Ca
2+
pathway, high concentra-
tions of 2-APB also inhibit agonist-induced Ca
2+
signals
by reducing InsP
3
formation.
Keywords 2-Aminoethoxydiphenyl borate (2-APB)
.
Inositol 1,4,5-trisphosphate
.
Phospholipase C (PLC)
.
Calcium stores
.
Calcium oscillation
.
Patch-clamp
.
Pancreatic acinar cells
.
Mouse
Introduction
Since the initial report that the membrane-permeable
compound 2-aminoethoxydiphenyl borate (2-APB) might
serve as a novel InsP
3
receptor antagonist [1, 2], numerous
studies have addressed the actions of this compound. The
evidence now shows the pharmacological effects of 2-
APB on intracellular Ca
2+
signals to be more complex than
previously thought. 2-APB inhibits inositol trisphosphate
(InsP
3
)-induced Ca
2+
release (IICR), although its potency
differs in different cell types [1, 2, 3]. In addition, 2-APB
inhibits the sarco-endoplasmic reticulum Ca
2+
-ATPase
(SERCA) [4], blocks the store-operated Ca
2+
pathway
[5] and may interfere with mitochondrial function [6]. On
the other hand, our previous work has indicated that 2-
APB potentiates, rather than inhibits, IICR in mouse
pancreatic acinar cells [7]. In the present study, we
investigated the inhibitory effect of 2-APB on agonist-
induced Ca
2+
oscillations. 2-APB inhibited agonist-in-
duced phospholipase C (PLC) activation, which may be
responsible for the observed inhibition of acetylcholine
(ACh)-induced Ca
2+
oscillations in mouse pancreatic
acinar cells.
Materials and methods
Single, isolated, mouse pancreatic cells were prepared according to
[7]. Briefly, adult ICR mice were anesthetized using ether. The
pancreatic glands were removed quickly, fragments of tissue minced
and digested by collagenase (150–200 U/ml, 20–25 min, 37 °C) in
J. Wu (*)
Division of Neurology, Barrow Neurological Institute, St.
Joseph’s Hospital and Medical Center,
350 West Thomas Road,
Phoenix, AZ 85013-4496, USA
e-mail: jwu2@chw.edu
Tel.: +1-602-4066376
Fax: +1-602-4067172
T. Takeo
Department of Medical Technology, Hirosaki University
School of Health Science,
036-8564 Hirosaki, Japan
S. Suga
.
T. Kanno
.
M. Wakui
Department of Physiology, Hirosaki University School of
Medicine,
036-8562 Hirosaki, Japan
T. Osanai
Second Department of Internal Medicine, Hirosaki University
School of Medicine,
036-562 Hirosaki, Japan
K. Mikoshiba
Department of Molecular Neurobiology, Institute of Medical
Science, Tokyo University,
108-8639 Tokyo, Japan