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

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 InsP3receptor 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 InsP3receptor 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.

Many cells use inositol 1,4,5-trisphosphate (InsP 3 ) 1 as a second messenger to release Ca 2ϩ from their internal stores (1). The InsP 3 receptors (InsP 3 R) are encoded by three different genes, resulting in the existence of an InsP 3 R-1, -2, and -3 (2)(3)(4), which differ in their affinity for InsP 3 (InsP 3 R-2 Ͼ InsP 3 R-1 Ͼ InsP 3 R-3) (5). Reports on isoform-specific effects of cytosolic Ca 2ϩ on InsP 3 binding are conflicting (6,7), and nothing is known about the effect of other regulators. We recently observed that 16HBE14oϪ cells from bronchial mucosa express 93% type-3 InsP 3 R, as judged from the relative levels of steadystate mRNA as determined by polymerase chain reaction. 2 Experiments using isoform-specific antibodies revealed that the type-3 InsP 3 R 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 InsP 3 R-3 is in 16HBE14oϪ cells the major (Ͼ81%) InsP 3 R-isoform expressed. We now compared the basic properties of the InsP 3 -induced Ca 2ϩ release in 16HBE14oϪ cells with those in A7r5 cells, where InsP 3 R-1 is the predominant isoform (9). The properties of the InsP 3 R-3-expressing 16HBE14oϪ cells differed mainly from those of InsP 3 R-1-expressing A7r5 cells by a lack of stimulation by sulfhydryl oxidation, an 11 times lower ATP sensitivity and a 3 times lower InsP 3 sensitivity.

EXPERIMENTAL PROCEDURES
Adenophostin A was isolated as described previously (10). 45 Ca 2ϩ fluxes on monolayers of saponin-permeabilized A7r5 cells from embryonic rat aorta and 16HBE14oϪ 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 MgCl 2 , 5 mM ATP, 0.44 mM EGTA, 10 mM NaN 3 , and 150 nM free Ca 2ϩ (23 Ci/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 M thapsigargin. Additions of InsP 3 , adenophostin A, ATP, thimerosal, or Ca 2ϩ are indicated in the figures. The free [ 40 Ca 2ϩ ] 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 45 Ca 2ϩ remaining in the stores was released by incubation with 1 ml of a 2% sodium dodecyl sulfate solution for 30 min. InsP 3 in 16HBE14oϪ cells (closed squares). This difference in EC 50 value probably represents the different affinity of the major InsP 3 R isoform expressed, which is InsP 3 R-1 in A7r5 cells (9) and InsP 3 R-3 in 16HBE14oϪ cells. 2 InsP 3 R-3 had a much lower affinity than InsP 3 R-1 if bacterial recombinant ligand binding domains of these two isoforms were compared under identical conditions (5).

RESULTS AND DISCUSSION
Adenophostin A is much more potent than InsP 3 in stimulating InsP 3 R-1 (10,12). These data were confirmed for A7r5 cells, i.e. a cell type where InsP 3 R-1 is the main isoform (open circles in Fig. 1). Also, 16HBE14oϪ cells responded to adenophostin A (open squares in Fig. 1), indicating that adenophostin A activated InsP 3 R-3 as well. The EC 50 was again lower than for InsP 3 .

Effect of ATP on InsP 3 -induced Ca 2ϩ
Release in Permeabilized A7r5 and 16HBE14oϪ Cells-Adenine nucleotides specifically interact with the InsP 3 R and potentiate the Ca 2ϩ release (13-16). Fig. 2 shows that the half-maximal [ATP] for stimulating the release induced by 3 M InsP 3 was 11 times higher in  16HBE14oϪ cells (341 M) than in A7r5 cells (32 M). 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 InsP 3 (17), was not observed in the present study, probably because of the higher [InsP 3 ] used in the present study.
ATP increases the cooperativity of the InsP 3 -induced Ca 2ϩ release in A7r5 cells (16) . Fig. 3 compares how the response of the stores of A7r5 and 16HBE14oϪ cells to a progressively increasing [InsP 3 ] is affected by 1 mM ATP. We confirmed that ATP increased the steepness of the response to InsP 3 in A7r5 cells, as judged by the clear shift of the maximum of the curve toward lower InsP 3 concentrations (arrowheads in Fig. 3A). This effect was, however, much less pronounced in 16HBE14oϪ cells (arrowheads in Fig. 3B).
A comparison of the sequences of the three InsP 3 R isoforms reveals that one predicted adenine-nucleotide binding site is conserved in all isoforms (18). InsP 3 R-1 in addition has, depending on the splice isoform, 1 or 2 other potential adeninenucleotide binding sites (18,19). The higher affinity for ATP of InsP 3 R-1 and/or its effect on the cooperativity of the Ca 2ϩ release may therefore represent the activity of one of the latter sites.
Effect of Thimerosal on InsP 3 -induced Ca 2ϩ Release in Permeabilized A7r5 and 16HBE14oϪ Cells-The sulfhydryl reagent thimerosal can, depending on its concentration, both stimulate and inhibit the InsP 3 R in many cell types (20 -26). In contrast, thimerosal does not stimulate the (as yet unidentified) InsP 3 R isoform in mouse lacrimal cells (24). Since it is not known which of the three InsP 3 R isoforms are stimulated by thimerosal, we have compared its effect on the response of permeabilized A7r5 and 16HBE14oϪ cells to a progressively increasing [InsP 3 ] (Fig. 4). In A7r5 cells, thimerosal dose-dependently shifted the threshold for initiating Ca 2ϩ release toward lower InsP 3 concentrations (closed circles in Fig. 4A). The sensitizing effect of thimerosal did not occur in 16HBE14oϪ 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).
Effect of Cytosolic Ca 2ϩ on InsP 3 -induced Ca 2ϩ Release in Permeabilized A7r5 and 16HBE14oϪ Cells- Fig. 5 illustrates how cytosolic Ca 2ϩ modified the Ca 2ϩ release induced by 3 M InsP 3 . The biphasic activation of the InsP 3 R by Ca 2ϩ (13,27,28,30) occurred both in A7r5 cells (circles) and in 16HBE14oϪ cells (squares). The almost complete inhibition of the release at 10 M free Ca 2ϩ in 16HBE14oϪ cells indicates that all the InsP 3 Rs expressed in this cell type were inhibited. As a consequence, the predominantly expressed isoform (InsP 3 R-3) was also inhibited by cytosolic Ca 2ϩ . The curve for 16HBE14oϪ cells reached its maximum at 0.1 M Ca 2ϩ , whereas A7r5 reached its maximum at 0.3 M Ca 2ϩ . This finding indicates that the activation by Ca 2ϩ of the type-1 InsP 3 R, which is a quantitatively unimportant isoform in 16HBE14oϪ cells, could not be responsible for the stimulatory part of the Ca 2ϩ response curve in 16HBE14oϪ cells.
Conclusions-We observed major differences in the regulation of the InsP 3 -induced Ca 2ϩ release between a cell type that predominantly expresses InsP 3 R-3 and a cell type that mainly expresses InsP 3 R-1. We confirmed at the functional level the known difference in InsP 3 affinity (5) and in addition observed that InsP 3 R-3 was 11 times less sensitive to ATP and was not activated by sulfhydryl oxidation. The predominant expression in the respiratory mucosa of InsP 3 R-3 and the predominant expression of the SERCA3 Ca 2ϩ 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.