Regulation of Ca2?release through inositol 1,4,5-trisphosphate receptors
by adenine nucleotides in parotid acinar cells
Hyung Seo Park,* Matthew J. Betzenhauser,* Yu Zhang,* and David I. Yule
University of Rochester Medical Center, Department of Pharmacology and Physiology, University of Rochester, Rochester,
Submitted 18 August 2011; accepted in final form 27 September 2011
Park HS, Betzenhauser MJ, Zhang Y, Yule DI. Regulation of
Ca2?release through inositol 1,4,5-trisphosphate receptors by ade-
nine nucleotides in parotid acinar cells. Am J Physiol Gastrointest
Liver Physiol 302: G97–G104, 2012. First published September 29,
2011; doi:10.1152/ajpgi.00328.2011.—Secretagogue-stimulated in-
tracellular Ca2?signals are fundamentally important for initiating the
secretion of the fluid and ion component of saliva from parotid acinar
cells. The Ca2?signals have characteristic spatial and temporal
characteristics, which are defined by the specific properties of Ca2?
release mediated by inositol 1,4,5-trisphosphate receptors (InsP3R). In
this study we have investigated the role of adenine nucleotides in
modulating Ca2?release in mouse parotid acinar cells. In permeabil-
ized cells, the Ca2?release rate induced by submaximal [InsP3] was
increased by 5 mM ATP. Enhanced Ca2?release was not observed at
saturating [InsP3]. The EC50for the augmented Ca2?release was ?8
?M ATP. The effect was mimicked by nonhydrolysable ATP analogs.
ADP and AMP also potentiated Ca2?release but were less potent than
ATP. In acini isolated from InsP3R-2-null transgenic animals, the rate
of Ca2?release was decreased under all conditions but now enhanced
by ATP at all [InsP3]. In addition the EC50 for ATP potentiation
increased to ?500 ?M. These characteristics are consistent with the
properties of the InsP3R-2 dominating the overall features of InsP3R-
induced Ca2?release despite the expression of all isoforms. Finally,
Ca2?signals were measured in intact parotid lobules by multiphoton
microscopy. Consistent with the release data, carbachol-stimulated
Ca2?signals were reduced in lobules exposed to experimental hyp-
oxia compared with control lobules only at submaximal concentra-
tions. Adenine nucleotide modulation of InsP3R in parotid acinar cells
likely contributes to the properties of Ca2?signals in physiological
and pathological conditions.
ATP; multiphoton microscopy; intracellular Ca2?
AGONIST-INDUCED ELEVATIONS in the cytosolic calcium concen-
tration ([Ca2?]i) are intimately involved in initiating saliva
secretion, the primary physiological function of parotid gland
acinar cells (36). Activation of Ca2?-dependent effectors is
important for both the fluid and protein component of saliva.
The primary aqueous component of primary saliva is generated
as an increase in Ca2?acts initially on a Cl?-dependent
conductance expressed exclusively on the apical plasma mem-
brane (35, 41). Recently, a promising candidate for the molec-
ular identity of this conductance is a member of the
Anoctamin-1 (ANO-1/TMEM16A) family of Cl?channels
(41). Additionally, the hyperpolarized membrane potential nec-
essary for continued Cl?secretion is maintained by the action
of Ca2?on K?channels of the IK and BK families (42–44).
The increase in luminal Cl?and negative potential pulls Na?
through the paracellular pathway. The subsequent increase in
osmotic potential draws water into the lumen to create an
isoosmotic NaCl-rich solution. Exocytosis of secretory gran-
ules containing the protein components of saliva can also be
triggered by increases in cytosolic Ca2?, presumably in anal-
ogy to neurosecretion, by acting through synaptotagmin ho-
mologs expressed in the acinar cells (13, 46).
The most important physiological activator of Ca2?signal-
ing in salivary glands is acetylcholine released from parasym-
pathetic neurons following olfactory and gustatory stimulation
(2, 3, 36). Activation of muscarinic receptors on the acinar
cells results in the G?q-coupled activation of phospholipase C
and the production of inositol 1,4,5-trisphosphate (InsP3).
InsP3 then acts on inositol 1,4,5 trisphosphate receptors
(InsP3R) expressed on the endoplasmic reticulum (ER). InsP3R
are particularly abundant on ER immediately juxtaposed to the
apical plasma membrane (28), and rapid Ca2?release is
invariably initiated at the apical Ca2?release sites followed by
the subsequent globalization of the Ca2?signal (23, 28, 47).
The temporal and spatial information in this signal are ideally
suited both for initially activating the apical Cl?conductance
and exocytotic machinery followed by subsequent opening of
basolaterally localized K?channels. All three isoforms of the
InsP3R are expressed in mouse parotid acinar cells, but only
the type-2 and type-3 isoforms are essential for function (22,
23, 28). This fact is demonstrated by a study that revealed that
a transgenic animal null for both the type-2 and type-3 InsP3R
is refractory to muscarinic receptor stimulation because of
failure to mount a Ca2?signal on stimulation (22). Indeed, the
central role of these particular InsP3R isoforms in exocrine
secretion is exemplified by the observation that the animal dies
soon after weaning primarily through a failure to produce
saliva, resulting in the inability to consume food (22).
Given the importance of the specific temporal and spatial
characteristics of the signal, it is probable that modulation of
Ca2?release through InsP3 impacts Ca2?signaling and thus
gland function in physiological and pathological situations.
Indeed, InsP3Rs serve as signal integrators, receiving multiple
inputs that ultimately impact the activity of the channel (38, 39,
50). For example, InsP3Rs are regulated by numerous factors
including most importantly Ca2?itself but also by cellular
ATP levels, phosphorylation by various kinases, and through a
multitude of protein-protein interactions (20, 38, 39). As a
function of distinct primary sequence of individual InsP3R
isoforms, regulation can occur in a subtype-specific manner,
and thus the overall effect of a particular route of regulation of
Ca2?release will reflect the net effect on the complement of
receptor subtypes expressed in an individual cell. For example,
in parotid acinar cells, Ca2?release through InsP3R is mark-
edly potentiated following activation of protein kinase A (10,
* H. Park, M. Betzenhauser, and Y. Zhang contributed equally to the work.
Address for reprint requests and other correspondence: D. Yule, Univ. of
Rochester, 601 Elmwood Ave., Rochester, NY 14642 (e-mail: David_Yule
Am J Physiol Gastrointest Liver Physiol 302: G97–G104, 2012.
First published September 29, 2011; doi:10.1152/ajpgi.00328.2011.
0193-1857/12 Copyright © 2012 the American Physiological Societyhttp://www.ajpgi.orgG97
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NUCLEOTIDE REGULATION OF INSP3R IN PAROTID ACINI
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00328.2011 • www.ajpgi.org