Inhibition of Ca2+-dependent Clminus secretion in T84 cells: membrane target(s) of inhibition is agonist specific

Kim E. Barrett, Jane Smitham, Alexis Traynor-Kaplan, and Jorge M. Uribe

Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous studies have indicated that Ca2+-dependent Cl- secretion across monolayers of T84 epithelial cells is subject to a variety of negative influences that serve to limit the overall extent of secretion. However, the downstream membrane target(s) of these inhibitory influences had not been elucidated. In this study, nuclide efflux techniques were used to determine whether inhibition of Ca2+-dependent Cl- secretion induced by carbachol, inositol 3,4,5,6-tetrakisphosphate, epidermal growth factor, or insulin reflected actions at an apical Cl- conductance, a basolateral K+ conductance, or both. Pretreatment of T84 cell monolayers with carbachol or a cell-permeant analog of inositol 3,4,5,6-tetrakisphosphate reduced the ability of subsequently added thapsigargin to stimulate apical 125I-, but not basolateral 86Rb+, efflux. These data suggested an effect on an apical Cl- channel. Conversely, epidermal growth factor reduced Ca2+-stimulated 86Rb+ but not 125I- efflux, suggesting an effect of the growth factor on a K+ channel. Finally, insulin inhibited 125I- and 86Rb+ effluxes. Thus effects of agents that inhibit transepithelial Cl- secretion are also manifest at the level of transmembrane transport pathways. However, the precise nature of the membrane conductances targeted are agonist specific.

chloride channels; potassium channels; 3-phosphorylated lipids; calcium

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE SECRETION OF CHLORIDE by a variety of epithelia subserves a number of key physiological processes (2). For example, Cl- secretion is a major driving force for hydration of the airways (4) and also drives water secretion into the intestinal lumen, providing for the fluidity of intestinal contents (19). It is to be expected, therefore, that both under- and overexpression of Cl- secretion can have significant pathophysiological consequences, such as in cystic fibrosis and secretory diarrhea, respectively. Given this physiological and pathophysiological significance, substantial effort has been directed to understanding the cellular and subcellular basis of Cl- secretion.

The transport pathways comprising the Cl- secretory mechanism have been reasonably well characterized at this point (2). Cl- is taken up across the basolateral pole of the cell via a Na+-K+-2Cl- cotransporter, which represents a secondary active transport driven by electrochemical gradients established by a basolateral Na+-K+-ATPase. K+ is then recycled across the basolateral membrane via K+ channel(s), and Cl- exits the cell through apically localized Cl- channel(s). The presence of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in Cl--secreting epithelial cells has been well established (12). Secretory epithelial cells may also express a second apical, Ca2+-activated Cl- conductance (CaCC), which may also be significant for the overall process of Cl- secretion (6, 13). The intracellular mechanisms controlling the level of secretion are dependent on the nature of the agonist and the resulting second messenger cascades that are evoked by agonist binding. Two broad classes of Cl- secretagogues have been described (2). The first of these acts via elevations in cyclic nucleotides and consequent opening of apical CFTR channels secondary to protein kinase A-dependent phosphorylation. The second class acts through elevations in intracellular Ca2+. Activation of Cl- secretion in this case is thought to be stimulated primarily via opening of Ca2+-activated K+ channels, although activation of apical CaCC by Ca2+ and calmodulin-dependent protein kinase may also contribute (5, 7, 8).

We have also identified several agonists that are capable of inhibiting Ca2+-dependent Cl- secretion (3). Some of these agents (such as carbachol) first exert stimulatory effects on secretion followed by a more prolonged inhibitory action (16), whereas others [such as epidermal growth factor (EGF)] act as inhibitors of secretion without themselves serving as secretagogues (22). For both carbachol and EGF, inhibition of subsequent secretory responses occurs without altering the rise in intracellular Ca2+ that occurs in response to the receptor-independent secretagogue, thapsigargin (16, 17, 22). Thus the inhibition results from the "uncoupling" of the increase in cytosolic Ca2+ from the downstream response of secretion. Some information has been obtained about the intracellular messengers that mediate the inhibitory effects of both carbachol and EGF on Cl- secretion. For carbachol, the inhibition appears to be largely ascribable to the effects of a specific inositol phosphate, inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4], which is elevated in cells after carbachol stimulation (24). For EGF, inhibition can be related to the ability of the growth factor to activate the enzyme phosphatidylinositol 3-kinase (PI3K) and thus may represent an effect of the 3-phosphorylated lipids that are the products of this enzyme (23). However, the points at which these putative inhibitory messengers alter the secretory mechanism was unknown. We hypothesized that Ins(3,4,5,6)P4 or 3-phosphorylated lipids might block an apical Cl- conductance and/or a basolateral K+ channel to account for the inhibitory effects of carbachol and EGF, respectively, on Cl- secretion. The present studies were designed to test this hypothesis.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. All secretagogues and inhibitors were added bilaterally. Carbachol, histamine, and wortmannin were obtained from Sigma Chemical (St. Louis, MO). Thapsigargin was purchased from LC Laboratories (Woburn, MA), and EGF was from Genzyme (Cambridge, MA). Cell culture membrane inserts (Millicell, 0.45-µm pore size mixed cellulose ester) were obtained from Millipore (Bedford, MA). 86Rb+ and 125I- were obtained from New England Nuclear (Boston, MA). The cell-permeant acetoxymethyl ester analog of Ins(3,4,5,6)P4 (24) was the generous gift of Drs. Carsten Schultz and Roger Tsien [Department of Pharmacology, University of California, San Diego (UCSD)]. All other chemicals used were obtained commercially and were of at least reagent grade.

Cells. All studies were performed using monolayers of the T84 cell line and cells from passages 15-35 only. Procedures for the growth of these cells have been reported previously (10). In brief, cells were plated on permeable Millicell inserts (see Materials) and maintained for 7-10 days before experiments to develop confluent monolayers with stable transepithelial resistances. The cells were grown in DMEM/Ham's F-12 media (JRH Biosciences, Lenexa, KS) supplemented with 5% newborn calf serum (Hyclone, Logan, UT) and 50 U/ml each of penicillin/streptomycin (Core Cell Culture Facility, UCSD). Medium was replaced twice weekly.

Efflux studies. To monitor the opening of basolateral K+ channels or apical Cl- channels in response to agonists, radionuclide efflux techniques were employed. These were adapted from procedures published previously by Venglarik et al. (25). Essentially, the published method was modified for use with cells grown on permeable supports, and the efflux of 125I- or 86Rb+ was monitored as the rate of nuclide appearance in the appropriate reservoir (apical vs. basolateral, respectively). Cell monolayers, grown on permeable insert supports, were rinsed with Hanks' balanced salt solution (HBSS) containing (in mM) 137.6 Na+, 146.3 Cl-, 5.8 K+, 0.44 H2PO-4, 0.34 HPO2-4, 1 Ca2+, 1 Mg2+, 15 HEPES (pH 7.2), and 10 D-glucose. The cells were then loaded with either 125I- (20 µCi/insert, added apically) or 86Rb+ (10 µCi/insert, added bilaterally) for 30 min at 37°C. After this, the monolayers were subjected to four gentle 2-min rinses with HBSS to remove extracellular isotope. After the final rinse, the inserts were transferred to fresh HBSS and warmed to 37°C in individual wells of a cell culture plate, and HBSS was also added to the apical aspect. The buffer was maintained at 37°C by placing the culture plates on a thermostatic heating block. The inserts were sequentially transferred to new wells at 2-min intervals for the 86Rb+ efflux assay, whereas the apical buffer was sampled and replaced at the same time intervals for the 125I- efflux assay. At various times, as indicated by the experimental design, the buffer was switched to a solution of the appropriate agonist(s) in HBSS, as noted. The agonist(s) were then continuously present for the remainder of the assay. At the end of the experiment, the culture insert was retained to assess remaining cell-associated counts. All samples were then assessed for their content of either 86Rb+ or 125I- using open-channel readings from a liquid scintillation counter.

Data analysis. The data were analyzed as described by Venglarik et al. (25) to yield apparent rates of nuclide efflux averaged over successive 2-min periods through the course of the experiment. These rate constants were then plotted against time. Student's t-test or analysis of variance were used where appropriate to test for significant differences between group means. Values of P < 0.05 were considered to represent significant differences. Changes in efflux rates induced by the second agonist [i.e., thapsigargin, histamine, or carbachol (see Tables 1-3, respectively)] were calculated for individual experiments by subtracting the efflux rate immediately before second agonist addition from the peak efflux rate observed after addition. The percent inhibition of this change in efflux rate induced by carbachol (Tables 1 and 2) or EGF (plus or minus wortmannin, Table 3) was calculated by comparing the change in efflux rate in control and pretreated cells and by expressing the difference as a percentage of the control response.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of thapsigargin on efflux of 86Rb or 125I from T84 cells in the presence or absence of carbachol

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of histamine on efflux of 86Rb or 125I from T84 cells in the presence or absence of carbachol

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Effect of EGF on carbachol-stimulated 86Rb and 125I effluxes from T84 cells in the presence or absence of wortmannin

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of carbachol on thapsigargin-stimulated 86Rb+ and 125I- efflux. We hypothesized that the ability of carbachol, and thus Ins(3,4,5,6)P4, to inhibit transepithelial Cl- secretion could reflect an inhibitory effect of the inositol phosphate directed at either a basolateral K+ conductance and/or an apical Cl- conductance. We first examined whether carbachol had any effect on Ca2+-stimulated K+ channel opening, as monitored by efflux of 86Rb+ across the basolateral membrane, since a basolateral K+ channel has been thought to be the primary control point for Ca2+-dependent Cl- secretion (8). Thus cells were pretreated either with carbachol or with buffer alone, and then subsequent Ca2+-dependent effluxes were stimulated with the microsomal Ca2+-ATPase inhibitor, thapsigargin (2 µM). As shown in Fig. 1A, addition of thapsigargin alone to T84 monolayers at 22 min evoked a significant increase in the rate of 86Rb+ efflux across the basolateral membrane. As expected from previous studies, the addition of carbachol evoked a prompt yet transient increase in the rate of 86Rb+ efflux. However, when thapsigargin was added, it induced an equivalent efflux response for at least 20 min after addition whether or not carbachol pretreatment had been applied. Likewise, pretreatment with carbachol did not affect the maximal increment in the rate of 86Rb+ efflux that was attributable to thapsigargin (Table 1). However, at later time points, 86Rb+ efflux induced by thapsigargin was significantly lower in carbachol-treated cells than in control cells. However, these differences were apparent at times considerably delayed from those when a significant inhibitory effect of carbachol on thapsigargin-induced transepithelial transport can be appreciated (16). In Ussing chambers, the inhibitory effect of carbachol on thapsigargin-stimulated Cl- secretion can be appreciated almost immediately upon thapsigargin addition and is certainly obvious within 10 min, the time point when the effect of thapsigargin on Cl- secretion is maximal (16). Thus it is unlikely that the delayed effect of carbachol on thapsigargin-stimulated 86Rb+ efflux can account for the inhibitory effect of carbachol on thapsigargin-induced Cl- secretion. It was concluded that the inhibitory effect of carbachol on thapsigargin-induced Cl- secretion is unlikely to be due to an effect directed at a basolateral K+ channel.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of thapsigargin (TG) on efflux of 86Rb+ (A) and 125I- (B) from preloaded T84 cells in presence (bullet ) or absence (open circle ) of carbachol (Carb). Carbachol (100 µM) and thapsigargin (2 µM) were added to both sides of the monolayers at times indicated by arrows, and efflux of 86Rb+ and 125I- was monitored into the basolateral or apical reservoir, respectively. Values are means ± SE for 4 experiments in each case. * P < 0.05 and ** P < 0.01 by Student's t-test: values for thapsigargin-stimulated nuclide efflux (i.e., after 22 min) that differ significantly from those observed in absence of carbachol pretreatment.

We therefore next examined whether carbachol pretreatment had any effect on the ability of thapsigargin to induce 125I- efflux across the apical membrane of T84 cells. Cells were pretreated with carbachol or buffer and subsequently with thapsigargin. As shown in Fig. 1B, addition of thapsigargin alone at 24 min to T84 monolayers resulted in a significant increase in 125I- efflux, occurring with considerably more rapid kinetics than the effect of thapsigargin on 86Rb+ efflux (Table 1). Moreover, pretreatment with carbachol also stimulated 125I- efflux and significantly attenuated the efflux response to thapsigargin. In contrast to the effect of carbachol on thapsigargin-stimulated 86Rb+ efflux, the inhibitory effect of carbachol on 125I- efflux was apparent immediately, was statistically significant within 8 min after the addition of thapsigargin, and was also associated with a significant decrease (~59%) in the maximal increment in the rate of 125I- efflux that was attributable to thapsigargin (Table 1). The extent and pattern of inhibition of the thapsigargin-stimulated 125I- efflux are highly reminiscent of that observed when monolayers are pretreated with carbachol in Ussing chambers, with Cl- secretion then induced by the addition of thapsigargin (16).

We also tested the effect of a cell-permeant analog of Ins(3,4,5,6)P4, the putative mediator of the inhibitory effect of carbachol on Ca2+-dependent Cl- secretion (24). Cells were preincubated with the acetoxymethyl ester of the inositol phosphate (100 µM), and then the ability of thapsigargin to elicit 125I- efflux across the basolateral membrane was examined. As shown for carbachol, pretreatment with cell-permeant Ins(3,4,5,6)P4 significantly reduced the effect of thapsigargin on 125I- efflux (%inhibition 37.5 ± 7.3, n = 6, P < 0.05 by Student's t-test). Because of the limited availability of the inositol tetrakisphosphate analog, its effect on thapsigargin-stimulated 86Rb+ efflux was not assessed.

Effect of carbachol on histamine-stimulated 86Rb+ and 125I- efflux. The ability of carbachol to reduce thapsigargin-stimulated 125I- but not 86Rb+ efflux from T84 cells was surprising, since the Ca2+-dependent, transepithelial process of Cl- secretion in T84 cells has previously been thought to be dependent primarily on Ca2+-stimulated K+ channel opening (2, 8). Thus, to test whether it would be possible to detect a decrease in 86Rb+ efflux if one was indeed occurring, we examined the effect of carbachol on the efflux of both 125I- and 86Rb+ stimulated by histamine. Carbachol pretreatment significantly reduces Cl- secretory responses to subsequently added histamine in Ussing chamber experiments. However, unlike the inhibition of the response to thapsigargin, this inhibitory effect is not wholly reflective of an uncoupling phenomenon. Rather, the ability of histamine to mobilize intracellular Ca2+ stores is significantly impaired in carbachol-treated cells, probably because both agonists mobilize the same Ca2+ pool via their effects on phospholipase C and the consequent generation of inositol 1,4,5-trisphosphate (17). Thus, in the experiment described, we predicted that carbachol should reduce histamine-stimulated 86Rb+ efflux secondary to a reduction in the signal for K+ channel opening (i.e., cytoplasmic Ca2+).

As shown in Fig. 2A, addition of histamine alone to monolayers preloaded with 86Rb+ led to a significant, though transient increase in the rate of efflux of the nuclide across the basolateral membrane. The kinetics of the efflux response to histamine are comparable with that induced by carbachol, although the absolute magnitude is somewhat less. This is in keeping with the fact that the maximal effect of histamine on transepithelial Cl- secretion is also somewhat less than that seen with carbachol (11, 26). When monolayers were pretreated with carbachol then stimulated with histamine, the ability of histamine to induce 86Rb+ efflux was significantly inhibited. A similar time course of the response to histamine was seen in the presence or absence of carbachol. However, the maximal increment in the rate of 86Rb+ efflux attributable to histamine was decreased by 80% (Table 2). Thus it is certainly possible to detect decreases in 86Rb+ efflux that are induced by carbachol, if they occur.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of histamine (Hist) on efflux of 86Rb+ (A) and 125I- (B) from preloaded T84 cells in presence (bullet ) or absence (open circle ) of carbachol. Carbachol (100 µM) and histamine (100 µM) were added to both sides of the monolayers at times indicated by arrows, and efflux of 86Rb+ and 125I- was monitored into the basolateral or apical reservoir, respectively. Values are means ± SE for 4-6 experiments. * P < 0.05 and ** P < 0.01 by Student's t-test: values for histamine-stimulated nuclide efflux (i.e., after 22 or 20 min, for A and B, respectively) that differ significantly from those observed in absence of carbachol pretreatment.

Figure 2B depicts the effect of histamine on apical 125I- efflux, with or without carbachol pretreatment. Histamine alone evoked a rapid and transient increase in the rate of 125I- efflux across the apical membrane. Moreover, in keeping with the data shown in Fig. 2A, carbachol pretreatment significantly reduced the ability of histamine to increase 125I- efflux. As shown in Table 2, the inhibition of histamine-stimulated 125I- efflux induced by carbachol exceeded 80%.

Effect of EGF on carbachol-stimulated 86Rb+ and 125I- efflux. EGF has been shown to inhibit Ca2+-dependent Cl- secretion without itself acting as a secretagogue (22). Moreover, in contrast to the inhibitory effect of carbachol on Cl- secretion, which appears to be due primarily to the actions of Ins(3,4,5,6)P4, the inhibitory effect of EGF appears to be largely independent of this inositol phosphate (Uribe, Traynor-Kaplan, and Barrett, unpublished observations). Instead, the ability of EGF to inhibit Cl- secretion, as assessed in Ussing chambers, can be reversed by wortmannin, an inhibitor of the enzyme PI3K (23). Moreover, the inhibitory effect of EGF corresponds to time points when there is an increase in two products of this enzyme within the cells: phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (23). Thus, because EGF and carbachol appear to use different signal transduction pathways and messengers to mediate their inhibitory effects on Cl- secretion, it was of interest to determine whether the same or different ion conductances were targeted by the two agents.

In these experiments, carbachol was used as the second stimulus to evoke nuclide efflux, rather than thapsigargin. The rationale for this experimental design came from our observation that thapsigargin-stimulated Cl- secretion in Ussing chambers seems to be tonically inhibited by PI3K and is potentiated by pretreatment with wortmannin (23). However, wortmannin at the dose studied here has no effect on Cl- secretion induced by carbachol (nor on the inhibitory effect of carbachol on subsequent Ca2+-dependent Cl- secretion) (23). The use of carbachol as the second stimulus also simplified data analysis, given that this agent induces a more pronounced yet more transient effect on nuclide efflux than the effect of thapsigargin. Figure 3A shows the effect of carbachol on 86Rb+ efflux across the basolateral membrane of T84 cells, in the presence or absence of EGF. EGF alone had no effect on basal rates of 86Rb+ efflux, in keeping with its failure to induce Cl- secretion in Ussing chamber experiments (22, 23). However, and in contrast to the lack of an immediate effect of carbachol on thapsigargin-stimulated 86Rb+ efflux, pretreatment with EGF promptly and significantly inhibited the effect of carbachol on 86Rb+ efflux rates. EGF inhibited the maximal increment in efflux rate attributable to carbachol by an average of ~30%, an effect that was statistically significant (Table 3). These data are in contrast to those shown in Fig. 3B. Figure 3 depicts the effect of carbachol on 125I- efflux across the apical membrane of T84 cells in the presence or absence of EGF. Again, EGF had no effect on basal rates of nuclide efflux. It also failed to alter significantly the ability of carbachol to stimulate 125I- efflux from the cells.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of carbachol on efflux of 86Rb+ (A) and 125I- (B) from preloaded T84 cells in presence (bullet ) or absence (open circle ) of epidermal growth factor (EGF). EGF (16.3 nM) and carbachol (100 µM) were added to both sides of the monolayers at times indicated by arrows, and efflux of 86Rb+ and 125I- was monitored into the basolateral or apical reservoir, respectively. Values are means ± SE for 7-9 experiments. ** P < 0.01 by Student's t-test: values for carbachol-stimulated nuclide efflux (i.e., after 20 min) that differ significantly from those observed in absence of EGF pretreatment.

To determine whether the effects of EGF on nuclide efflux were related to the ability of the growth factor to activate PI3K, we also conducted experiments with wortmannin. Wortmannin had no effect on basal rates of 86Rb+ or 125I- efflux (Table 3). By itself, it also had no effect on the ability of carbachol to evoke either 86Rb+ or 125I- efflux (data not shown). However, it partially reversed the inhibitory effect of EGF on carbachol-stimulated 86Rb+ efflux. Thus, in the presence of EGF plus wortmannin, the maximal increment in 86Rb+ efflux rate attributable to carbachol did not differ significantly from that induced by carbachol alone.

Effect of insulin on carbachol-stimulated 86Rb+ and 125I- efflux. As noted above, the inhibitory effect of EGF on transepithelial Ca2+-dependent Cl- secretion appears to be largely dependent on the activity of PI3K. However, EGF has also been shown to increase levels of Ins(3,4,5,6)P4 in T84 cells, albeit to a lesser degree than seen with carbachol. Thus we questioned whether the effects of EGF on 86Rb+ efflux, as described above, might represent a synergism or other interaction between Ins(3,4,5,6)P4 and another inhibitory signal generated as a result of PI3K activity. To examine this question, we tested whether pretreatment with insulin could modify carbachol-stimulated 86Rb+ or 125I- effluxes. We have recently reported that insulin inhibits Ca2+-dependent Cl- secretion in Ussing chambers, likely via (at least in part) a PI3K-dependent pathway, but the hormone does not measurably alter levels of Ins(3,4,5,6)P4 (N. Chang, J. M. Uribe, S. J. Keely, and K. E. Barrett, unpublished observations).

The effect of carbachol on 86Rb+ effluxes in the presence and absence of insulin in shown in Fig. 4A. As seen for EGF, insulin had no effect on basal rates of 86Rb+ efflux but slightly modified the time course of the effect of carbachol on the efflux rate of this nuclide (in a statistically significant fashion). Thus, while the peak response to carbachol was unchanged by insulin, the overall effect of carbachol was slower in onset and more rapidly resolved in the presence of insulin. Moreover, and in contrast to the effect of EGF, insulin also significantly inhibited the ability of carbachol to increase the 125I- efflux rate, again without itself altering basal rates of efflux of the nuclide (Fig. 4B). Thus, while both EGF and insulin inhibit transepithelial Cl- secretion via a mechanism associated with the activation of PI3K, the target(s) of the inhibitory effects of these modulators may be different.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of carbachol on efflux of 86Rb+ (A) and 125I- (B) from preloaded T84 cells in presence (bullet ) or absence (open circle ) of insulin. Insulin (33 nM) and carbachol (100 µM) were added to both sides of the monolayers at times indicated by arrows, and efflux of 86Rb+ and 125I- was monitored into the basolateral or apical reservoir, respectively. Values are means ± SE for 9 (A) or 3 (B) experiments. * P < 0.05 by Student's t-test: values for carbachol-stimulated nuclide efflux (i.e., after 19 min) that differ significantly from those observed in absence of insulin pretreatment.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

As noted above, Cl- secretion is a process of substantial physiological and pathophysiological significance. Thus significant attention has been paid to the precise intracellular mechanisms that result in the stimulation of this process. Rather less attention has been paid to the intracellular mechanisms that limit or terminate secretion, although it is clear that such inhibitory effects do occur, particularly in the setting of Ca2+-dependent Cl- secretion (3). Thus Ca2+-dependent secretory responses are transient, even in the face of prolonged elevations in intracellular Ca2+ concentrations (3, 9). Likewise, the extent of Ca2+-dependent Cl- secretory responses is poorly correlated with the magnitude of the rise in intracellular Ca2+, when responses to a range of agonists are compared (9). These findings suggest that the actions of Ca2+ within the epithelial cell are modified, amplified, or antagonized by additional second messengers that are produced in response to various agonists. We have reported that Ins(3,4,5,6)P4 and products of PI3K activity, among others, likely serve as such modulatory intracellular messengers for the overall process of Ca2+-dependent Cl- secretion (3). However, the precise intracellular targets of these putative inhibitory messengers were unclear.

Carbachol was shown here to inhibit the maximal rates of subsequent Ca2+-stimulated 125I- but not 86Rb+ efflux. We can conclude from this finding that the presumed downstream inhibitory signal generated by carbachol, Ins(3,4,5,6)P4, predominantly targets an apical Cl- conductance to exert its inhibitory effect on Ca2+-stimulated Cl- secretion. This is also in keeping with recently reported patch-clamp studies, where Ins(3,4,5,6)P4 was dialyzed into the interior of T84 cells in the whole cell recording mode (27). This resulted in the blockade of a Ca2+ conductance activated in response to thapsigargin or by introduction of calmodulin kinase II into the cell. It cannot be determined from either this study or the patch-clamp studies of others (27) whether Ins(3,4,5,6)P4 interacts directly with a Cl- channel to block Cl- secretion. However, we also recently observed that Ins(3,4,5,6)P4 was able to block the function of a cloned CaCC from bovine trachea, which was inserted in planar lipid bilayers (15). These latter data would tend to support the concept of a direct interaction, without the need for additional, intermediary signaling components.

The data presented here also raise interesting points about the relative importance of various Cl- channels in Ca2+-activated Cl- secretion in T84 cells. While a CaCC has been described in airway epithelial cells, it had previously been concluded that this channel was absent from native intestinal epithelium and from intestinal cell lines, such as T84 (1, 14). The prevailing dogma, therefore, regarding the mechanism of Ca2+-dependent Cl- secretion held that the response was driven by the opening of basolateral K+ channels and subsequent movement of Cl- across a small proportion of apical Cl- channels (likely CFTR) that were constitutively open (1, 2). However, we show here that carbachol was able to inhibit 125I- efflux from T84 cells at time points before those where the agonist had an inhibitory effect on thapsigargin-stimulated 86Rb+ efflux. Likewise, the maximal rate of 125I- efflux was inhibited by carbachol in the absence of an effect on maximal 86Rb+ efflux. Carbachol did eventually cause an inhibition of thapsigargin-stimulated 86Rb+ efflux, but this occurred at times that were too late to account for the inhibitory effect of carbachol on Ca2+-dependent Cl- secretion as examined in Ussing chambers (16). The late inhibition of 86Rb+ efflux seen in response to carbachol probably reflects an eventual rundown of the driving force for K+ exit, when Cl- exit is reduced. In total, therefore, these data imply that carbachol targets an apical Cl- conductance to exert its inhibitory effects on Cl- secretion. Moreover, the findings additionally suggest a greater role for a CaCC in mediating T84 secretory responses to Ca2+-mobilizing agonists than had hitherto been proposed (1).

The ability of carbachol to inhibit 125I- efflux, in the absence of a simultaneous effect on 86Rb+ efflux, was specific for the uncoupling phenomenon, where inhibition of Ca2+-dependent Cl- secretion occurs without an alteration in the rise in intracellular Ca2+ produced by thapsigargin. When histamine was used as the second stimulus, 125I- and 86Rb+ effluxes were inhibited by carbachol pretreatment to an approximately equal extent. Under these circumstances, we have previously shown that the Ca2+ mobilization response to histamine is also markedly reduced (17). This further emphasizes that a rise in intracellular Ca2+ likely targets both K+ and Cl- channels in producing an increase in the rate of transepithelial Cl- secretion.

In contrast to the findings with carbachol, the inhibitory effect of EGF was targeted to a basolateral K+ conductance. This inhibitory effect appeared to involve the activity of PI3K, in that it was partially reversed by wortmannin. The precise details of the inhibitory pathway have yet to be determined. Because the EGF receptor is localized to the basolateral surface in T84 cells (22), ligand binding would likely also recruit PI3K to that site, where it could then act on inositol phospholipids in the basolateral membrane itself. It is possible that the resulting generation of 3-phosphorylated lipids in the vicinity of basolateral K+ channels might alter their activity via an effect on the bulk properties of the membrane. Alternatively, or in addition, products of PI3K have been shown to activate various novel and atypical isoforms of protein kinase C, and activity of basolateral K+ channels in T84 cells has been shown to be negatively regulated by protein kinase C phosphorylation (18, 20, 21). Additional experiments will be required to fully define the precise mechanisms whereby EGF acts on K+ channels. We have shown, however, that the ability of EGF to reduce 86Rb+ efflux likely is not codependent on an increase in Ins(3,4,5,6)P4. Thus insulin, which has no measurable effect on cellular levels of Ins(3,4,5,6)P4 but does inhibit Cl- secretion in a manner apparently dependent on PI3K, was able to inhibit 86Rb+ efflux to some extent. However, unlike EGF, insulin failed to inhibit the peak 86Rb+ efflux response induced by carbachol, but rather altered the kinetics of the response. This may suggest that the precise mechanism whereby insulin alters K+ channel activity may be subtly different from that used by EGF. Moreover, insulin also inhibited 125I- efflux across the apical membrane, an action not shared by EGF. This effect could also contribute to the overall effect of insulin on Cl- secretion. The mechanism of this latter effect is currently unknown. Our observations of differences between EGF and insulin are in keeping, however, with preliminary observations (Smitham and Barrett, unpublished observations) that maximally inhibitory doses of EGF and insulin have additive effects on Cl- secretion in Ussing chambers, suggesting that the signaling pathways utilized by these two agonists are not wholly overlapping.

In summary, we have demonstrated that the effects of agonists that inhibit transepithelial Cl- secretion are also manifest at the level of transmembrane transport pathways. However, the precise details of the transport pathways that are targeted are dependent on the specific agonist. This likely reflects divergence in the signaling pathways utilized. Carbachol, acting through Ins(3,4,5,6)P4, predominantly targets an apical Cl- conductance to inhibit secretion. Conversely, EGF, acting through PI3K, predominantly targets a basolateral K+ conductance. Finally, our data imply a greater role than has previously been appreciated for a CaCC in the Ca2+-dependent Cl- secretory process in T84 cells and perhaps in the native intestine.

    ACKNOWLEDGEMENTS

We are grateful to Glenda Wheeler for assistance with manuscript preparation and to Drs. C. Schultz and R. Tsien for their gift of the reagent used.

    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28305 to K. E. Barrett. J. M. Uribe was the recipient of a Predoctoral Fellowship from an Institutional Training Grant in Digestive Diseases (DK-07202).

Portions of this study were presented at the 94th and 96th Annual Meetings of the American Gastroenterological Association (in 1994 in Boston, MA, and in 1996 in San Francisco, CA, respectively) and have appeared in abstract form (A. E. Traynor-Kaplan, C. Schultz, R. Tsien, and K. E. Barrett, Gastroenterology 106: A277, 1994; J. Smitham, J. Uribe, and K. E. Barrett, Gastroenterology 110: A362, 1996).

Address for reprint requests: K. E. Barrett, Univ. of California, San Diego Medical Center, 8414, 200 West Arbor Dr., San Diego, CA 92103-8414 (E-mail: kbarrett{at}ucsd.edu).

Received 17 July 1997; accepted in final form 24 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Anderson, M. P., and M. J. Welsh. Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc. Natl. Acad. Sci. USA 88: 6003-6007, 1991[Abstract].

2.   Barrett, K. E. Positive and negative regulation of chloride secretion in T84 cells. Am. J. Physiol. 265 (Cell Physiol. 34): C859-C868, 1993[Abstract/Free Full Text].

3.   Barrett, K. E. Integrated regulation of intestinal epithelial transport: intercellular and intracellular pathways. Am. J. Physiol. 272 (Cell Physiol. 41): C1069-C1076, 1997[Abstract/Free Full Text].

4.   Clarke, L. L., and R. C. Boucher. Ion and water transport across airway epithelia. In: Pharmacology of the Respiratory Tract, edited by K. F. Chung, and P. J. Barnes. New York: Dekker, 1993, p. 505-550.

5.   Cliff, W. H., and R. A. Frizzell. Separate Cl- conductances activated by cAMP and Ca2+ in Cl--secreting epithelial cells. Proc. Natl. Acad. Sci. USA 87: 4956-4960, 1990[Abstract].

6.   Cunningham, S. A., M. S. Awayda, J. K. Bubein, I. I. Ismailov, M. P. Arrate, B. K. Berdiev, D. J. Benos, and C. M. Fuller. Cloning of an epithelial chloride channel from bovine trachea. J. Biol. Chem. 270: 31016-31026, 1995[Abstract/Free Full Text].

7.   Devor, D. C., and M. E. Duffey. Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study. Am. J. Physiol. 263 (Cell Physiol. 32): C780-C787, 1992[Abstract/Free Full Text].

8.   Devor, D. C., S. M. Simasko, and M. E. Duffey. Carbachol induces oscillations of membrane potassium conductance in a colonic cell line, T84. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990[Abstract/Free Full Text].

9.   Dharmsathaphorn, K., J. Cohn, and G. Beuerlein. Multiple calcium-mediated effector mechanisms regulate chloride secretory responses in T84 cells. Am. J. Physiol. 256 (Cell Physiol. 25): C1224-C1230, 1989[Abstract/Free Full Text].

10.   Dharmsathaphorn, K., J. A. McRoberts, K. G. Mandel, L. D. Tisdale, and H. Masui. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G204-G208, 1984[Abstract/Free Full Text].

11.   Dharmsathaphorn, K., and S. J. Pandol. Mechanisms of chloride secretion induced by carbachol in a colonic epithelial cell line. J. Clin. Invest. 77: 348-354, 1986[Medline].

12.   Fuller, C. M., and D. J. Benos. CFTR! Am. J. Physiol. 263 (Cell Physiol. 32): C267-C286, 1992[Abstract/Free Full Text].

13.   Fuller, C. M., I. I. Ismailov, D. A. Keeton, and D. J. Benos. Phosphorylation and activation of a bovine tracheal ion channel by Ca2+/calmodulin-dependent protein kinase II. J. Biol. Chem. 269: 26642-26650, 1994[Abstract/Free Full Text].

14.   Grubb, B. R. Ion transport across the jejunum in normal and cystic fibrosis mice. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G505-G513, 1995[Abstract/Free Full Text].

15.   Ismailov, I. I., C. M. Fuller, B. K. Berdiev, V. G. Shlyonsky, D. J. Benos, and K. E. Barrett. A biologic function for an "orphan" messenger: D-myo-inositol (3,4,5,6) tetrakisphosphate selectively blocks epithelial calcium-activated chloride channels. Proc. Natl. Acad. Sci. USA 93: 10505-10509, 1996[Abstract/Free Full Text].

16.   Kachintorn, U., M. Vajanaphanich, K. E. Barrett, and A. E. Traynor-Kaplan. Elevation of inositol tetrakisphosphate parallels inhibition of calcium-dependent chloride secretion in T84 colonic epithelial cells. Am. J. Physiol. 264 (Cell Physiol. 33): C671-C676, 1993[Abstract/Free Full Text].

17.   Kachintorn, U., M. Vajanaphanich, A. E. Traynor-Kaplan, K. Dharmsathaphorn, and K. E. Barrett. Activation by calcium alone of chloride secretion in T84 epithelial cells. Br. J. Pharmacol. 109: 510-517, 1993[Abstract].

18.   Kachintorn, U., P. Vongkovit, M. Vajanaphanich, S. Dinh, K. E. Barrett, and K. Dharmsathaphorn. Dual effects of a phorbol ester on calcium-dependent chloride secretion by T84 human colonic epithelial cells. Am. J. Physiol. 262 (Cell Physiol. 31): C15-C22, 1992[Abstract/Free Full Text].

19.  Montrose, M. H., S. J. Keely, and K. E. Barrett. Electrolyte secretion and absorption: small intestine and colon. In: Textbook of Gastroenterology (3rd ed.), edited by T. Yamada, D. Alpers, L. Laine, D. Powell, and C. Owyang. Philadelphia, PA: Lippincott. In press.

20.   Reenstra, W. W. Inhibition of cAMP- and Ca-dependent Cl- secretion by phorbol esters: inhibition of basolateral K+ channels. Am. J. Physiol. 264 (Cell Physiol. 33): C161-C168, 1993[Abstract/Free Full Text].

21.   Toker, A., and L. C. Cantley. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 387: 673-676, 1997[Medline].

22.   Uribe, J. M., C. M. Gelbmann, A. E. Traynor-Kaplan, and K. E. Barrett. Epidermal growth factor inhibits calcium-dependent chloride secretion in T84 human colonic epithelial cells. Am. J. Physiol. 271 (Cell Physiol. 40): C914-C922, 1996[Abstract/Free Full Text].

23.   Uribe, J. M., S. J. Keely, A. E. Traynor-Kaplan, and K. E. Barrett. Phosphatidylinositol 3-kinase mediates the inhibitory effect of epidermal growth factor on calcium-dependent chloride secretion. J. Biol. Chem. 271: 26588-26595, 1996[Abstract/Free Full Text].

24.   Vajanaphanich, M., C. Schultz, M. T. Rudolf, M. Wasserman, P. Enyedi, A. Craxton, S. B. Shears, R. Y. Tsien, K. E. Barrett, and A. Traynor-Kaplan. Long-term uncoupling of chloride secretion from intracellular calcium levels by Ins(3,4,5,6)P4. Nature 371: 711-714, 1994[Medline].

25.   Venglarik, C. J., R. J. Bridges, and R. A. Frizzell. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells. Am. J. Physiol. 259 (Cell Physiol. 28): C358-C364, 1990[Abstract/Free Full Text].

26.   Wasserman, S. I., K. E. Barrett, P. A. Huott, G. Beuerlein, M. Kagnoff, and K. Dharmsathaphorn. Immune-related intestinal Cl- secretion. I. Effect of histamine on the T84 cell line. Am. J. Physiol. 254 (Cell Physiol. 23): C53-C62, 1988[Abstract/Free Full Text].

27.   Xie, W., M. A. Kaetzel, K. S. Bruzik, J. R. Dedman, S. B. Shears, and D. J. Nelson. Inositol 3,4,5,6 tetrakisphosphate inhibits the calmodulin-dependent protein kinase II-activated chloride conductance in T84 cells. J. Biol. Chem. 271: 14092-14097, 1996[Abstract/Free Full Text].


AJP Cell Physiol 274(4):C958-C965
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society