Functional Properties of the Type-3 InsP3 Receptor in 16HBE14o- Bronchial Mucosal Cells*

Ludwig MissiaenDagger §, Jan B. ParysDagger , Ilse SienaertDagger par , Karlien MaesDagger , Karl Kunzelmann**, Masaaki TakahashiDagger Dagger , Kazuhiko TanzawaDagger Dagger , and Humbert De SmedtDagger

From the Dagger  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 Dagger Dagger  Biological Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140, Japan

    ABSTRACT
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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
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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
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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
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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 (bullet ) or 16HBE14o- cells (black-square) 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 (bullet ) or without (open circle ) 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 (open circle ) or with the indicated [thimerosal] (Thim. bullet ), 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 (bullet ) and 16HBE14o- cells (black-square) 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.).

par 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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Results & Discussion
References

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