Functional Properties of the Type-3 InsP3 Receptor
in 16HBE14o
Bronchial Mucosal Cells*
Ludwig
Missiaen
§,
Jan B.
Parys
¶,
Ilse
Sienaert
,
Karlien
Maes
,
Karl
Kunzelmann**,
Masaaki
Takahashi
,
Kazuhiko
Tanzawa
, and
Humbert
De Smedt
From the
Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium,
** the Physiologisches Institut der Albert-Ludwigs-Universität
Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany, and the

Biological Research Laboratories, Sankyo
Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140, Japan
 |
ABSTRACT |
The type-3 inositol 1,4,5-trisphosphate
(InsP3) receptor is the major isoform expressed in
16HBE14o
cells from bronchial mucosa, representing 93% at the
mRNA level as determined by ratio reverse transcription-polymerase
chain reaction and about 81% at the protein level as determined
with isoform-specific antibodies (Sienaert, I., Huyghe, S., Parys,
J. B., Malfait, M., Kunzelmann, K., De Smedt, H.,
Verleden, G. M., and Missiaen, L., Pflügers Arch. Eur. Y. Physiol., in press). The present 45Ca2+
efflux experiments indicate that these InsP3 receptors were
3 times less sensitive to InsP3 and 11 times less sensitive
to ATP than those in A7r5 cells, where the type-1 InsP3
receptor is the main isoform. ATP did not increase the cooperativity of
the InsP3-induced Ca2+ release in 16HBE14o
cells, in contrast to its effect in A7r5 cells. The sulfhydryl reagent
thimerosal also did not stimulate InsP3-induced
Ca2+ release in 16HBE14o
cells, again in contrast to its
effect in A7r5 cells. Adenophostin A was more potent than
InsP3 in stimulating the release in both cell types. The
biphasic activation of the InsP3 receptor by cytosolic
Ca2+ occurred in both cell types. We conclude that
Ca2+ release mediated by the type-3 InsP3
receptor mainly differs from that mediated by the type-1
InsP3 receptor by its lack of stimulation by sulfhydryl
oxidation and its lower ATP and InsP3 sensitivity. The
predominant expression of the type-3 InsP3 receptor in the
bronchial mucosa may be part of a mechanism coping with oxidative
stress in that tissue.
 |
INTRODUCTION |
Many cells use inositol 1,4,5-trisphosphate
(InsP3)1 as a
second messenger to release Ca2+ from their internal stores
(1). The InsP3 receptors (InsP3R) are encoded
by three different genes, resulting in the existence of an
InsP3R-1, -2, and -3 (2-4), which differ in their affinity for InsP3 (InsP3R-2 > InsP3R-1 > InsP3R-3) (5). Reports on isoform-specific effects of cytosolic Ca2+ on
InsP3 binding are conflicting (6, 7), and nothing is known
about the effect of other regulators. We recently observed that
16HBE14o
cells from bronchial mucosa express 93% type-3 InsP3R, as judged from the relative levels of steady-state
mRNA as determined by polymerase chain
reaction.2 Experiments using
isoform-specific antibodies revealed that the type-3 InsP3R
was also the main isoform expressed at the protein level (about 81%).
Two reasons may underlie the small difference in values obtained with
the two techniques. First, it should be emphasized that a quantitative
reverse transcription-polymerase chain reaction method was used for
determining the mRNA level, whereas the determination at the
protein level was semi-quantitative and based on a comparison between
cell types. Second, in any cell type, the mRNA level does not
necessarily reflect the protein level. Whatever the reason for the
small difference, both methods are in general agreement and indicate
that InsP3R-3 is in 16HBE14o
cells the major (>81%)
InsP3R-isoform expressed. We now compared the basic
properties of the InsP3-induced Ca2+ release in
16HBE14o
cells with those in A7r5 cells, where InsP3R-1 is the predominant isoform (9). The properties of the
InsP3R-3-expressing 16HBE14o
cells differed mainly from
those of InsP3R-1-expressing A7r5 cells by a lack of
stimulation by sulfhydryl oxidation, an 11 times lower ATP sensitivity
and a 3 times lower InsP3 sensitivity.
 |
EXPERIMENTAL PROCEDURES |
Adenophostin A was isolated as described previously (10).
45Ca2+ fluxes on monolayers of
saponin-permeabilized A7r5 cells from embryonic rat aorta and
16HBE14o
cells from a bronchial surface epithelium were done at
25 °C as described (11). The stores were loaded for 1 h in 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM MgCl2, 5 mM ATP, 0.44 mM EGTA, 10 mM NaN3, and 150 nM free Ca2+ (23 µCi/ml) and then washed
twice in an efflux medium containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 1 mM EGTA, and 2 µM thapsigargin. Additions of InsP3,
adenophostin A, ATP, thimerosal, or Ca2+ are indicated in
the figures. The free [40Ca2+] of the efflux
medium was calculated using MaxChelator (Dr. C. Patton, Stanford
University, CA). 1 ml of this medium was then added to the cells and
replaced every 6 s or 2 min. At the end of the experiment, the
45Ca2+ remaining in the stores was released by
incubation with 1 ml of a 2% sodium dodecyl sulfate solution for 30 min.
 |
RESULTS AND DISCUSSION |
InsP3- and Adenophostin A-induced Ca2+
Release in Permeabilized A7r5 and 16HBE14o
Cells--
The
non-mitochondrial Ca2+ stores were loaded with
45Ca2+ and then challenged with a progressively
increasing [InsP3] or [adenophostin A] in efflux
medium. The closed symbols in Fig.
1 illustrate the Ca2+ release
as a function of the [InsP3] for the two cell types. The
EC50 was 0.7 µM InsP3 in A7r5
cells (closed circles) and 1.9 µM
InsP3 in 16HBE14o
cells (closed squares). This
difference in EC50 value probably represents the different
affinity of the major InsP3R isoform expressed, which is
InsP3R-1 in A7r5 cells (9) and InsP3R-3 in
16HBE14o
cells.2 InsP3R-3 had a much lower
affinity than InsP3R-1 if bacterial recombinant ligand
binding domains of these two isoforms were compared under identical
conditions (5).

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Fig. 1.
InsP3- and adenophostin A-induced
Ca2+ release from the non-mitochondrial Ca2+
stores in permeabilized A7r5 and 16HBE14o cells. The
non-mitochondrial Ca2+ stores of A7r5 cells
(circles) or 16HBE14o cells (squares) were
loaded to steady state with 45Ca2+, then
incubated in efflux medium containing 1 mM ATP, and
challenged with a progressively increasing [InsP3]
(closed symbols) or [adenophostin A] (open
symbols) after 6 min of efflux. The [agonist] was increased in
70 steps, each lasting 6 s. The extent of Ca2+ release
was normalized to that induced by 10 µM agonist. Each
tracing is the mean of at least five independent
experiments.
|
|
Adenophostin A is much more potent than InsP3 in
stimulating InsP3R-1 (10, 12). These data were confirmed
for A7r5 cells, i.e. a cell type where InsP3R-1
is the main isoform (open circles in Fig. 1). Also,
16HBE14o
cells responded to adenophostin A (open squares
in Fig. 1), indicating that adenophostin A activated InsP3R-3 as well. The EC50 was again lower than
for InsP3.
Effect of ATP on InsP3-induced Ca2+ Release
in Permeabilized A7r5 and 16HBE14o
Cells--
Adenine nucleotides
specifically interact with the InsP3R and potentiate the
Ca2+ release (13-16). Fig. 2
shows that the half-maximal [ATP] for stimulating the release induced
by 3 µM InsP3 was 11 times higher in
16HBE14o
cells (341 µM) than in A7r5 cells (32 µM). ADP was as effective as ATP, whereas AMP, GTP, and
ITP were less effective in both cell types (Table
I). The inhibition in A7r5 cells by high
ATP concentrations (16), which is due to a competition with
InsP3 (17), was not observed in the present study, probably because of the higher [InsP3] used in the present
study.

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Fig. 2.
Effect of ATP on InsP3-induced
Ca2+ release in permeabilized A7r5 and 16HBE14o
cells. Ca2+ stores of A7r5 ( ) or 16HBE14o cells
( ) loaded to steady state with 45Ca2+ were
incubated in efflux medium for 10 min, at which time 3 µM
InsP3 plus the indicated [ATP] was added for 2 min. The
efflux medium was collected each 2 min. The stimulation of the
Ca2+ release by ATP is expressed as the percentage increase
of the Ca2+ release above the control value obtained in the
absence of ATP. The data points are the means of four independent
experiments. The S.E. was always less than 10%.
|
|
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Table I
Effect of nucleotides on InsP3-induced Ca2+ release
in permeabilized A7r5 and 16HBE14o cells
The stimulation of the Ca2+ release induced by 3 µM InsP3 by the indicated compounds (1 mM) was determined as in Fig. 2. The stimulation was
normalized to that observed in the presence of 1 mM ATP.
The values are expressed as mean ± S.E. for three independent
experiments.
|
|
ATP increases the cooperativity of the InsP3-induced
Ca2+ release in A7r5 cells (16). Fig.
3 compares how the response of the stores
of A7r5 and 16HBE14o
cells to a progressively increasing [InsP3] is affected by 1 mM ATP. We confirmed
that ATP increased the steepness of the response to InsP3
in A7r5 cells, as judged by the clear shift of the maximum of the curve
toward lower InsP3 concentrations (arrowheads in
Fig. 3A). This effect was, however, much less
pronounced in 16HBE14o
cells (arrowheads in Fig.
3B).

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Fig. 3.
Effect of ATP on the cooperativity of the
InsP3-induced Ca2+ release in permeabilized
A7r5 and 16HBE14o cells. The non-mitochondrial Ca2+
stores of A7r5 cells (A) and 16HBE14o cells (B)
were loaded to steady state with 45Ca2+. After
a 7-min incubation in efflux medium with ( ) or without ( ) 1 mM ATP, 3 nM InsP3 was added. The
[InsP3] was then increased from 3 nM to 3 µM in 60 steps, each lasting 6 s, as indicated below
the tracings. The results are plotted as fractional loss,
i.e. the rate of Ca2+ release over a 6-s period
divided by the Ca2+ content of the stores at that time. The
arrowheads, which point to the maximum of the curves, are
discussed in the text. Each curve is the mean of five independent
experiments.
|
|
A comparison of the sequences of the three InsP3R isoforms
reveals that one predicted adenine-nucleotide binding site is conserved in all isoforms (18). InsP3R-1 in addition has, depending
on the splice isoform, 1 or 2 other potential adenine-nucleotide binding sites (18, 19). The higher affinity for ATP of
InsP3R-1 and/or its effect on the cooperativity of the
Ca2+ release may therefore represent the activity of one of
the latter sites.
Effect of Thimerosal on InsP3-induced Ca2+
Release in Permeabilized A7r5 and 16HBE14o
Cells--
The sulfhydryl
reagent thimerosal can, depending on its concentration, both stimulate
and inhibit the InsP3R in many cell types (20-26). In
contrast, thimerosal does not stimulate the (as yet unidentified)
InsP3R isoform in mouse lacrimal cells (24). Since it is
not known which of the three InsP3R isoforms are stimulated by thimerosal, we have compared its effect on the response of permeabilized A7r5 and 16HBE14o
cells to a progressively increasing [InsP3] (Fig. 4). In A7r5
cells, thimerosal dose-dependently shifted the threshold
for initiating Ca2+ release toward lower InsP3
concentrations (closed circles in Fig. 4A). The
sensitizing effect of thimerosal did not occur in 16HBE14o
cells
(closed circles in Fig. 4B). Thimerosal also
lowered the maximum of the curves, which represents the inhibitory
effect. This inhibition occurred in both cell types. The inhibition in A7r5 cells occurred at a lower [thimerosal] than reported before (22). This is most likely due to the absence of ATP in the present experiments (data not shown).

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Fig. 4.
Effect of thimerosal on
InsP3-induced Ca2+ release in permeabilized
A7r5 and 16HBE14o cells. The non-mitochondrial Ca2+
stores of A7r5 cells (A) and 16HBE14o cells (B)
were loaded to steady state with 45Ca2+. After
a 7-min incubation in efflux medium without ( ) or with the indicated
[thimerosal] (Thim. ), 3 nM
InsP3 was added. The [InsP3] was then
increased from 3 nM to 3 µM in 60 steps, each
lasting 6 s, as indicated below the tracings. The results are
plotted as fractional loss, i.e. the rate of
Ca2+ release over a 6-s period divided by the
Ca2+ content of the stores at that time. The data points
are the means of five (A) and four (B)
independent experiments.
|
|
Effect of Cytosolic Ca2+ on InsP3-induced
Ca2+ Release in Permeabilized A7r5 and 16HBE14o
Cells--
Fig. 5 illustrates how
cytosolic Ca2+ modified the Ca2+ release
induced by 3 µM InsP3. The biphasic
activation of the InsP3R by Ca2+ (13, 27, 28,
30) occurred both in A7r5 cells (circles) and in 16HBE14o
cells (squares). The almost complete inhibition of the
release at 10 µM free Ca2+ in 16HBE14o
cells indicates that all the InsP3Rs expressed in this cell
type were inhibited. As a consequence, the predominantly expressed
isoform (InsP3R-3) was also inhibited by cytosolic
Ca2+. The curve for 16HBE14o
cells reached its maximum at
0.1 µM Ca2+, whereas A7r5 reached its maximum
at 0.3 µM Ca2+. This finding indicates that
the activation by Ca2+ of the type-1 InsP3R,
which is a quantitatively unimportant isoform in 16HBE14o
cells,
could not be responsible for the stimulatory part of the
Ca2+ response curve in 16HBE14o
cells.

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Fig. 5.
Biphasic activation of the
InsP3Rs in permeabilized A7r5 and 16HBE14o cells by
cytosolic Ca2+. The non-mitochondrial Ca2+
stores of A7r5 cells ( ) and 16HBE14o cells ( ) were loaded to
steady state with 45Ca2+ and incubated in
non-labeled efflux medium containing 2 µM thapsigargin
and 5 nM free 40Ca2+ for 10 min.
The stores were then challenged for 2 min with 3 µM
InsP3 in the presence of the indicated free
[40Ca2+], which was added at the time of
InsP3 addition. The Ca2+ release at the
indicated [Ca2+] is expressed as a percentage of the
Ca2+ release at 5 nM free Ca2+.
Mean ± S.E. is shown for five independent experiments.
|
|
Conclusions--
We observed major differences in the regulation
of the InsP3-induced Ca2+ release between a
cell type that predominantly expresses InsP3R-3 and a cell
type that mainly expresses InsP3R-1. We confirmed at the
functional level the known difference in InsP3 affinity (5) and in addition observed that InsP3R-3 was 11 times less
sensitive to ATP and was not activated by sulfhydryl oxidation. The
predominant expression in the respiratory mucosa of
InsP3R-3 and the predominant expression of the SERCA3
Ca2+ pump isoform (29), which is very resistant to reactive
oxygen species (8), may be part of a mechanism coping with oxidative stress in that tissue.
 |
ACKNOWLEDGEMENTS |
We thank Dr. D. C. Gruenert
(Cardiovascular Research Institute, Department of Laboratory Medicine,
Gene Therapy Core Center, University of California, San Francisco, CA)
for the supply of 16HBE14o
cells.
 |
FOOTNOTES |
*
This work was supported by Levenslijn Grant 7.0025.94 of the
Foundation for Scientific Research-Flanders (F.W.O.).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: Tel.: 32-16-345720;
Fax: 32-16-345991; E-mail: Ludwig.Missiaen{at}med.kuleuven.ac.be.
¶
Research associate of the Foundation for Scientific
Research-Flanders (F. W. O.).
Research assistant of the Foundation for Scientific
Research-Flanders (F.W.O.).
1
The abbreviations used are: InsP3,
inositol 1,4,5-trisphosphate; InsP3R, InsP3
receptor.
2
I. Sienaert, S. Huyghe, J. B. Parys, M. Malfait, K. Kunzelmann, H.
De Smedt, G. M. Verleden, and L. Missiaen, Pflügers
Arch. Eur. J. Physiol., in press.
 |
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