Differential effects of apical and basolateral uridine triphosphate on intestinal epithelial chloride secretion

Jane E. Smitham and Kim E. Barrett

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our goal was to examine the sidedness of effects of the purinergic agonist, uridine 5'-triphosphate (UTP), on Cl- secretion in intestinal epithelial cells. We hypothesized that UTP might exert both stimulatory and inhibitory effects. All studies were conducted with T84 intestinal epithelial cells. UTP induced Cl- secretion in a concentration-dependent fashion. Responses to serosally added UTP were smaller and more transient than those evoked by mucosal addition, but there was no evidence that mucosal responses involved cAMP-dependent mechanisms. Pretreatment with serosal UTP inhibited subsequent Ca2+-dependent Cl- secretion induced by carbachol or thapsigargin, or secretion induced by mucosal UTP, in a manner that was reversed by a tyrosine kinase inhibitor. The inhibitory effect of serosal UTP on Cl- secretion was not additive with that of carbachol, known to exert its inhibitory effects through the tyrosine kinase-dependent generation of inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4]. Moreover, responses to both serosal and mucosal UTP were reduced by prior treatment of T84 cells with carbachol. Finally, serosal, but not mucosal, UTP evoked an increase in Ins(3,4,5,6)P4. We conclude that different signaling mechanisms lie downstream of apical and basolateral UTP receptors in epithelial cells, at least in the intestine. These differences may be relevant to the use of UTP as a therapy in cystic fibrosis.

intestine; calcium; uridine 5'-triphosphate; purinergic agonists


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SECRETION OF CHLORIDE into the lumen of various epithelial organs is coordinated by a polarized network of transporters, energy-dependent pumps, and channel proteins. These are sorted and selectively expressed in the apical and basolateral aspects of the epithelium. If the function and/or expression of any of these transport proteins is compromised, the ion transport capability of the epithelium can be altered, with pathophysiological consequences. For example, >800 different mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene product have been documented, many of which cause deleterious effects on the function, expression, or localization of this Cl- channel (43). In normal cells, intracellular ATP, cAMP-dependent protein kinase (PKA), and protein kinase C activate the wild-type channel by interaction with and/or phosphorylation of the cytosolic portion of the protein. However, mutations in CFTR can lead to its mislocalization and/or inability to function as a cAMP-regulated Cl- channel, resulting in the disease cystic fibrosis (CF) (51).

One therapeutic strategy proposed to treat CF has been to utilize Ca2+-dependent Cl- secretagogues, such as the nucleotides adenosine and uridine triphosphate (ATP and UTP) (38, 44). Hypothetically, these agents could bypass the defect in cAMP-mediated Cl- secretion by activating an alternative Cl- conductance pathway, as has been demonstrated in a variety of cell types (10-12, 18, 21, 29, 30). This strategy depends on the presence of the alternative pathway for Cl- exit across the apical membrane. Indeed, Ca2+-dependent Cl- channels (CLCAs) are present in the apical membranes of many secretory epithelial cells (17, 22, 25, 26, 41, 42). Moreover, Ca2+-regulated Cl- transport is apparently intact in the airways and in cells derived from the airway, nose, and pancreas of human patients with CF, suggesting that these cells express functional CLCAs (6, 11, 56, 59). However, intestinal tissues derived from CF patients or animal models of the disease do not consistently conduct Cl- in response to agonists that elevate intracellular Ca2+, in addition to their expected defect in cAMP-regulated transport (5, 23, 24, 40).

The failure of intestinal epithelial cells with the CF defect to respond consistently to agonists that elevate intracellular Ca2+ has long been considered to reflect an absence of apically localized CLCAs (40, 45, 48). However, there is increasing support for the existence of distinct Cl- conductances regulated by cAMP and Ca2+ in intestinal epithelial cell lines (13, 42, 57, 62) as well as evidence that the expression of intestinal CLCAs may ameliorate severity in mouse models of CF (61). In addition, an apically located protein immunoreactive with antibodies raised against a bovine CLCA has been detected in epithelial cells of intestinal origin (both wild-type and Delta F508 CF mouse intestine and T84 cells) (15). Furthermore, recent studies using the intestinal epithelial cell line, T84, and the CF pancreatic cell line, CFPAC-1, indicate not only the functional existence of an apically located CLCA, but also that this channel is inhibited by the intracellular messenger, inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] (3, 27, 32, 53, 63, 64). This messenger is endogenously generated in T84 cells in response to the Ca2+-dependent Cl- secretagogue, carbachol (CCh) (53). Similarly, recent data suggest that UTP can increase Ins(3,4,5,6)P4 in CFPAC-1 cells (8). Together, these data suggest that the failure of some epithelial cells to exhibit Ca2+-dependent Cl- secretion may be due to negative signaling events rather than simply a lack of CLCAs. Moreover, they imply that the efficacy of UTP and related agonists in CF might be limited by the existence of negative signaling pathways, particularly in the intestine.

Despite intense study, the mechanism(s) by which purinergic agonists modulate epithelial Cl- secretion remain controversial. There are sound data to indicate that the effects of such agonists on airway epithelial cells are mediated predominantly by P2Y2 receptors, which are activated equally by ATP and UTP (29, 37, 38, 49). However, in intestinal tissues or cell lines, the situation is more complex. For ATP at least, and in T84 cells, Stutts et al. (50) concluded that responses were not mediated by P2Y receptors at all, but rather reflected the breakdown of ATP to adenosine. However, since that time, at least five human P2Y receptors have been cloned, several of which are sensitive to UTP (37, 46). These additional receptors are candidates to mediate secretory effects of ATP and/or UTP. Moreover, Cressman et al. (16) concluded, from studies on P2Y2 knockout mice, that this receptor is the major determinant of nucleotide-stimulated Cl- secretion in the trachea, but only a partial contributor to gallbladder responses and unimportant in the jejunum. These studies also did not address the sidedness of the evoked responses. It follows that additional information is needed regarding the relative roles of P2Y receptor subtypes in modulating Cl- secretion, particularly in the intestine. Overall, we sought to define further the mechanisms by which nucleotides activate and/or modify Cl- secretion in epithelial cells, with a view to optimizing the use of such agents in CF therapy.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. DMEM/F-12 medium (JRH Biosciences, Lenexa, KS), newborn calf serum (Hyclone Laboratories, Logan, UT), UTP, carbachol (Sigma Chemical, St. Louis, MO), thapsigargin (TG; Sigma Chemical or Alexis, San Diego, CA), dibutyryl adenosine 3',5'-cyclic monophosphate, acetoxymethyl ester (Bt2cAMP/AM), H-89 dihydrochloride (Calbiochem, San Diego, CA), 12-mm 0.45 µm-pore size mixed cellulose ester Millicell-HA tissue culture plate well inserts (Millipore, Bedford, MA), enzyme immunoassay system for detection of cAMP (Amersham Life Science, Arlington Heights, IL), and a DC protein determination kit (Bio-Rad Life Sciences, Hercules, CA) were purchased from the sources indicated. All other chemicals were of at least analytical grade and were obtained commercially. Ringer solution contained (in mM) 140 Na+, 5.2 K+, 1.2 Ca2+, 1.2 Mg2+, 119.8 Cl-, 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 2.4 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 0.4 HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and 10 glucose.

Cell culture. Monolayers of the human colonic epithelial cell line, T84, were grown as previously described (19) except that these studies were conducted using cells plated on commercial permeable inserts consisting of uncoated hydroxyapatite (see above) rather than the rat tail collagen coated-polycarbonate filters used previously. At the time of study, cells grown on these supports were functionally indistinguishable from those grown on collagen, with transepithelial resistances in excess of 1,000 Omega  · cm2. Monolayers were grown on these inserts in a DMEM/F-12 medium supplemented with 5% newborn calf serum for 10-15 days before study. All cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C.

Cl- secretion. Cl- secretion was assayed in Ussing chambers adapted for use with cultured monolayers and using methods that have been described previously (19). Both apical and basolateral reservoirs contained Ringer solution at 37°C and equilibrated with 95% O2-5% CO2. Monolayers were voltage clamped to zero potential difference by the continuous application of short-circuit current (Isc). Under these conditions, changes in Isc have been shown to be wholly reflective of Cl- secretion in T84 cells (19, 58).

cAMP measurements. T84 cell monolayers (grown on 12-mm inserts) were rinsed and equilibrated with Ringer solution in a 37°C, 5% CO2 humidified incubator. Drugs or buffer alone were added to appropriate wells and the reaction was stopped by the addition of ice-cold ethanol/Ringer solution (1:1, vol/vol). Samples were dried down under nitrogen, reconstituted in sample buffer, and the cAMP content was then assessed using an enzyme immunoassay system (Amersham). Results were standardized relative to the average protein content of representative monolayers from each experiment, measured using the Bio-Rad assay.

Measurement of inositol tetrakisphosphate. The ability of UTP to increase levels of InsP4 in T84 cells was assessed using methods that have been detailed previously (33, 52). Briefly, cells grown to confluence on Millicell-HA filters were labeled by incubation with inositol-free tissue culture medium supplemented with myo-[2-3H]inositol (12.8 Ci/mmol, 50 µCi/ml) for 72 h total. After labeling, cells were washed four times with Ringer solution and then treated with the agonists under study (or with Ringer solution alone for coincubated controls). Inositol phosphates were extracted by a slight modification of the method of Berridge et al. (4). HPLC separation employed an Alltech Absorbosphere SAX column (4.6 × 250 mm, 5 µm packing) (28). Radiolabel in eluates was monitored continuously by a 171 radioisotope detector (Beckman), and identification of inositol phosphates was based on a comparison of their elution times with those of authentic radiolabeled standards.

Data analysis. Data are expressed as means ± SE. The data were analyzed for statistical significance by either Student's t-test or analysis of variance with Tukey-Kramer's post hoc test, as appropriate.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of UTP-dependent Cl- secretion in T84 cells. We first examined the ability of UTP to induce Cl- secretion, assessed as changes in Isc, across T84 cell monolayers mounted in Ussing chambers. UTP induced an increase in Isc when added to either the mucosal (apical) or serosal (basolateral) aspects of T84 cells (Fig. 1), although with differing kinetics (Fig. 2). The response to serosal addition was very transient, similar to findings described with Ca2+-dependent secretagogues, such as CCh (20). The response to mucosal addition was slightly more prolonged, typically with a rapid-onset phase followed by a plateau phase (Fig. 2), although even mucosal addition did not produce the sustained response characteristic of cAMP-mediated secretagogues in this system (1). The ability of both serosal and mucosal UTP to induce Cl- secretion was concentration dependent (Fig. 1). UTP (1 mM) induced maximal Delta Isc responses irrespective of the side of addition. The dose response depicted in Fig. 1, however, indicates that mucosal UTP was a more efficacious Cl- secretagogue than serosal UTP at all concentrations tested. Further, simultaneous addition of serosal and mucosal UTP to the monolayer induced a response that was not significantly different from that evoked by mucosal UTP alone (Fig. 1, inset), suggesting that the signaling events utilized by mucosal and serosal UTP to induce Cl- secretion are at least partially overlapping. However, the increased efficacy and different kinetics of Cl- secretion evoked by mucosal UTP compared with serosal UTP indicate that additional signaling pathway(s) (either positive or negative) may be generated by activation of receptors for this agonist that are asymmetrically localized in T84 cells.


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Fig. 1.   Concentration dependence of the effect of UTP on Cl- secretion across T84 colonic epithelial cells. Results are shown for both mucosal () and serosal (open circle ) addition. The data are presented as the maximal changes in short-circuit current (Delta Isc) evoked by a given concentration applied to a single monolayer and are means ± SE for a minimum of 3 experiments at each concentration. The inset shows maximal Delta Isc responses evoked by mucosal (M), serosal (S), or bilateral (M+S) addition of 1 mM UTP as means ± SE for 6-7 experiments. The response to serosal addition differed significantly from that evoked by either mucosal or bilateral addition (*P < 0.05 by ANOVA).



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Fig. 2.   Isc responses of T84 cells to UTP and carbachol (CCh). Time course of Cl- secretory responses evoked by either mucosal (A) or serosal (B) UTP (1 mM) and the effect of UTP on subsequent responsiveness of T84 cells to serosal CCh (100 µM). In A and B, the response to CCh alone is shown with filled symbols. Insets in A and B summarize the maximal Isc response evoked by CCh addition in the absence (open bar) or presence (solid bar) of UTP pretreatment. Values are means ± SE for 4-7 experiments. Asterisks denote responses to CCh in the presence of UTP that differed significantly from those seen in the absence of UTP (*P < 0.05; ***P < 0.001 by Student's t-test). Please note that different scales have been used in each panel for clarity.

In an attempt to identify additional positive second messenger(s) responsible for the larger responses to mucosal UTP, Cl- secretory responses to mucosal UTP were examined in the absence and presence of the PKA inhibitor, H-89 (Fig. 3). However, H-89 had no significant effect on responses to mucosal UTP, implying that cAMP is not involved in the response. In two control experiments, 25 µM H-89 markedly suppressed Cl- secretory responses evoked by Bt2cAMP/AM (Delta Isc: 53.4 ± 6.7 and 14.2 ± 0.9 µA/cm2 in the absence and presence of H-89, respectively, means ± SE). Moreover, neither mucosal nor serosal UTP (irrespective of the time of UTP incubation) were capable of increasing cAMP levels compared with unstimulated monolayers (data not shown). Finally, in other epithelial cells, UTP stimulates production of prostaglandins, which might also contribute to stimulating Cl- secretory responses (31, 60, 66). However, various concentrations of the prostaglandin synthesis inhibitor, indomethacin, were also without effect on Cl- secretory responses to mucosal UTP (data not shown).


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Fig. 3.   Role of protein kinase A in Isc responses to UTP. The effect of mucosal UTP (1 mM) on Cl- secretion by T84 cells in the presence (open circle ) or absence () of the protein kinase A inhibitor, H-89 (25 µM). Where present, H-89 was added 15 min before UTP. Values are means ± SE for 3 experiments.

Effect of UTP on subsequent Cl- secretory responses in T84 cells. Based on its kinetics and data in the literature, at least part of the Cl- secretory response to UTP, particularly after serosal addition, is likely attributable to the receptor-dependent mobilization of intracellular Ca2+ (7, 12, 36). We have previously reported that other receptor-dependent Ca2+-mobilizing agonists, such as CCh, induce the production of negative signals that then limit subsequent Ca2+-dependent Cl- secretion (33, 53). We therefore proceeded to examine whether either mucosal or serosal addition of UTP could alter subsequent Cl- secretory responses to CCh. As shown in Fig. 2A, pretreatment with mucosal UTP had no effect on subsequent responses to CCh. In contrast, pretreatment with serosal UTP, while having a lesser effect on Cl- secretion by itself, inhibited the Cl- secretory responses to CCh by >60% (Fig. 2B). Similarly, pretreatment with serosal UTP significantly reduced Cl- secretory responses to subsequently added mucosal UTP (Fig. 4), whereas mucosal UTP had no effect on either the magnitude or kinetics of Isc responses to serosal UTP (data not shown). These data further support the concept that UTP activates different signaling pathways depending on the side of addition and that some of these pathways might be inhibitory.


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Fig. 4.   Interactions between Isc responses to mucosal and serosal UTP. The effect of mucosal UTP (1 mM) on Cl- secretion by T84 cells in the presence (open circle ) or absence () of pretreatment with serosal UTP (1 mM). Values are means ± SE for 3 experiments. Asterisks denote responses to mucosal UTP that were significantly inhibited after serosal UTP pretreatment compared with controls (*P < 0.05; **P < 0.01; ***P < 0.001 by Student's t-test).

We previously reported in T84 cells that CCh generates the endogenous second messenger, Ins(3,4,5,6)P4, which is responsible for inhibiting subsequent Ca2+-dependent Cl- secretory responses (53). Because the data presented here and in other systems suggest that at least part of the response to UTP is Ca2+ dependent (7, 8, 12, 36), we tested whether CCh was able to modify subsequent Cl- secretory responses to UTP. As shown in Fig. 5, when T84 cells were pretreated with CCh, secretory responses to both mucosal and serosal UTP were significantly inhibited. Of interest, the smaller and more transient responses to serosal UTP were more sensitive to inhibition by CCh than those induced by mucosal UTP (Fig. 5). This latter point underscores our previous suggestion that multiple signaling pathways are generated in response to UTP depending on the side of addition.


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Fig. 5.   CCh inhibits subsequent Isc responses to UTP. Time course of Cl- secretory responses evoked across T84 cells by either mucosal (A) or serosal (B) UTP (1 mM) at zero minutes in the presence or absence of pretreatment at -15 min with serosal CCh (100 µM). In A and B, the response to UTP alone is shown with filled symbols, whereas the open symbols depict responses to sequential agonists. Values are means ± SE for 4-7 experiments. * Significantly different from UTP alone, P < 0.05.

To determine whether the mechanism whereby serosal UTP inhibits Cl- secretion is similar to that utilized by CCh, we examined the effect of adding combinations of CCh and serosal UTP on subsequent Ca2+-dependent Cl- secretory responses to TG. TG is a receptor-independent agonist that activates Cl- secretion by elevating Ca2+ secondary to inhibition of the ATP-dependent, Ca2+-reuptake pump on the surface of the endoplasmic reticulum (34). TG does not increase Ins(3,4,5,6)P4 (33). Figure 6 indicates that either CCh or serosal UTP alone could inhibit subsequent Cl- secretory responses to TG. However, when CCh and serosal UTP were added together, the extent of inhibition of TG-stimulated Cl- secretion did not exceed that achieved with CCh alone. These data suggest that CCh and serosal UTP use similar or at least partially overlapping intracellular pathways to inhibit subsequent Ca2+-dependent Cl- secretion.


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Fig. 6.   CCh and UTP inhibit Isc responses to thapsigargin. Maximal increases in Cl- secretion (as Delta Isc) across T84 cells evoked by bilateral thapsigargin (5 µM) alone or after pretreatment with either serosal UTP (1 mM), serosal CCh (100 µM), or the combination of serosal UTP + serosal CCh. Values are expressed as a percentage of the control response to thapsigargin occurring in the absence of pretreatment and are means ± SE for 7 experiments. Asterisks denote significant inhibitory effects of pretreatments compared with the control response (***P < 0.001 by ANOVA).

Previously, we had shown that the tyrosine kinase inhibitor, genistein, could partially reverse the increase in Ins(3,4,5,6)P4 levels induced by CCh in T84 cells (55). In parallel, genistein was able to partially reverse the inhibitory effect of CCh on subsequent Ca2+-dependent Cl- secretion (55). Therefore, we examined whether genistein could modify the inhibitory effect of serosal UTP on subsequent TG-mediated Cl- secretion. Genistein [0.1 µM (not shown) and 1 µM (Fig. 7)], at concentrations shown to be effective in reversing the inhibitory effect of CCh on Cl- secretion (55), was also able to reverse the inhibitory effect of serosal UTP on subsequent TG-stimulated Cl- secretion. Thus responses to TG in the presence of 1 µM genistein alone or 1 µM genistein plus serosal UTP did not differ significantly from those induced by TG alone (Fig. 7), whereas the response to TG in the presence of serosal UTP alone was significantly inhibited, as expected. Moreover, while genistein by itself had no effect on Isc at a concentration of 1 µM (consistent with findings in other studies, e.g., Ref. 39), it significantly potentiated responses to serosal UTP. Thus Delta Isc responses to UTP were 10.0 ± 3.2 vs. 21.4 ± 2.5 µA/cm2 in the absence or presence of genistein, respectively (means ± SE, n = 6, P < 0.05). This implies that the signaling pathway utilized by serosal UTP to inhibit Cl- secretion requires tyrosine kinase activity. These data are also consistent with the hypothesis that serosal UTP and CCh inhibit Cl- secretion via similar mechanisms.


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Fig. 7.   Genistein reverses the inhibitory effect of serosal UTP on Ca2+-mediated Cl- secretion. Ability of genistein to reverse the inhibitory effect of pretreatment with serosal UTP on thapsigargin-induced Cl- secretion across T84 cells. Cells were stimulated with thapsigargin (5 µM, bilateral) alone or after pretreatment with either genistein (1 µM, bilateral), serosal UTP (1 mM), or the combination of bilateral genistein + serosal UTP. Values are expressed as a percentage of the control response to thapsigargin occurring in the absence of pretreatment and are means ± SE for 4 experiments. Asterisk denotes a significant inhibitory effect of UTP pretreatment compared with the control response (*P < 0.05 by ANOVA). Other responses did not differ significantly from the control.

Effect of UTP on InsP4 in T84 cells. Finally, to examine directly whether UTP acts to inhibit Cl- secretion (at least in part) via an increase in Ins(3,4,5,6)P4, we treated T84 cell monolayers with UTP (1 mM) from either the mucosal or serosal side and measured InsP4 production. Cells treated with CCh (100 µM, serosal) served as a positive control. As shown in Fig. 8, both serosal UTP and serosal CCh produced a significant increase in InsP4 levels in T84 cells, whereas the increase attributable to mucosal UTP did not achieve statistical significance. The ability of serosal UTP, at least, to elevate InsP4 is in keeping with findings reported recently by Carew et al. (8), working in CFPAC-1 cells (a line of pancreatic epithelial cells displaying a CF phenotype). It should be noted that it was not possible to definitively identify the InsP4 peak as Ins(3,4,5,6)P4 in these studies due to the lack of a standard for this specific isomer and the fact that peaks for other InsP4 isomers were only poorly resolved under the HPLC conditions used. Thus the results presented are representative of total InsP4. However, because the elution pattern of samples incubated with UTP and CCh were similar, and because CCh has been shown previously to elevate Ins(3,4,5,6)P4 specifically (53), it is reasonable to assume that the increase in InsP4 that occurs in response to UTP treatment is largely attributable to this latter isomer.


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Fig. 8.   Effect of UTP on InsP4 levels in T84 cells. Cells were treated for 15 min with UTP (1 mM) added to either the serosal (s) or mucosal (m) side or with CCh (100 µM) added to the serosal side, and labeled inositol phosphates were extracted and quantitated by HPLC. Values are expressed as the increase in InsP4 above levels seen in untreated cells, expressed as a percentage of total [3H] disintegrations per minute, and are means ± SE for 4 experiments. Values that represent a significant increase above control values (0.531 ± 0.147) are designated by asterisks (*P < 0.05; **P < 0.01 by repeated-measures ANOVA).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously shown that at least some Ca2+-dependent agonists, such as CCh, have a dual effect on Cl- secretion in T84 cells. Thus after stimulating a transient increase in secretion, CCh then induces the production of negative signals that limit subsequent Ca2+-dependent responses (2). These data are of interest in light of the knowledge that Ca2+-dependent secretion is functionally not appreciable in CF intestine (5, 23, 24, 40). The apparent failure of CF intestine to respond normally to Ca2+-dependent agonists, therefore, might be explained by the presence of such negative signaling cascades. In other words, in the setting of CF, where transepithelial Cl- secretion would be rendered dependent on alternative Cl- channels, any intracellular messenger that inhibited such channels would mask their function. Furthermore, because generation of second messengers capable of abrogating Ca2+-dependent Cl- secretion might limit the efficacy of therapies designed to utilize this alternative Cl- secretory mechanism, we undertook a detailed analysis of the mechanism whereby UTP induces Cl- secretion across intestinal epithelial cells. These studies were also prompted by evidence from knockout animals that purinergic agonists must use different mechanisms to evoke Cl- secretion in the intestine vs. the lung. Secretory responses to such agonists in the lung were essentially abolished by genetic targeting of the P2Y2 receptor, whereas responses in the intestine were normal (16). These data implied that additional information was needed with respect to the pathways whereby UTP modulates Cl- secretion in intestinal epithelia.

UTP, added to either side of T84 monolayers, induced concentration-dependent increases in Cl- secretion. When added simultaneously to both the mucosal and serosal aspects, UTP evoked Cl- secretory responses that were no greater than those induced by either mucosal or serosal UTP added alone. This suggests that purinergic receptors on both sides of the monolayer utilize overlapping signal pathways and/or membrane targets to activate Cl- secretion. However, divergent signaling pathways do exist, since mucosal UTP induced Cl- secretory responses that were of greater magnitude than those evoked by serosal UTP. Similarly, serosal UTP evoked transient, monophasic responses suggestive of Ca2+-dependent Cl- secretion, unlike the somewhat more prolonged mucosal responses. Responses to cAMP-dependent agonists have sustained kinetics, and simultaneous addition of Ca2+ and cAMP-dependent agonists is known to result in synergistic Cl- secretory responses in T84 cells (9, 54). Moreover, it is interesting that these combined responses, while initially greater than predicted from summation of the Ca2+- and cAMP-mediated components, show more rapid decay than those seen with cAMP alone (54). This pattern, therefore, approximates that seen with mucosal UTP. Therefore, we wondered whether the more prolonged secretory responses evoked by mucosal UTP were reflective of both Ca2+- and cAMP-mediated components, perhaps mediated by more than one P2Y receptor type or a single receptor linked to more than one signaling pathway (14, 37, 54). However, we could find no evidence that cAMP contributes to Cl- secretion induced by mucosal UTP. As an alternative explanation, we therefore considered that the smaller and transient responses to serosal UTP compared with mucosal UTP might reflect the generation of negative signals that limited Cl- secretion following serosal addition of UTP.

As mentioned previously, CCh induces the production of Ins(3,4,5,6)P4 in T84 cells, which is responsible for attenuating subsequent Ca2+-dependent Cl- secretory responses (53). In the presence of Ca2+/calmodulin-dependent kinase and elevated intracellular Ca2+, Ins(3,4,5,6)P4 acts on a Ca2+-activated Cl- channel to reduce its open probability (32). Ins(3,4,5,6)P4 generation in T84 cells is dependent on tyrosine kinase activity (or at least on a cellular process susceptible to inhibition by genistein), since genistein has been shown to reverse the inhibitory effect of CCh on subsequent Cl- secretion and to block the CCh-stimulated increase in Ins(3,4,5,6)P4 (55). The inhibitory effect of serosal UTP on subsequent Ca2+-dependent Cl- secretion was also reversed by genistein and was not additive with the inhibitory effect of CCh, implying that a similar mechanism may be at work in the case of serosal UTP. Of interest, the respective muscarinic and purinergic receptors that are responsible for the inhibitory effects of CCh and UTP are both localized exclusively on the basolateral aspect of T84 cells. CCh stimulates and inhibits Cl- secretion through a G protein-linked receptor. By extension, since P2Y purinergic receptors are also G protein linked and thought to mediate UTP-dependent Cl- secretion in many epithelia (12, 29, 37, 38, 47, 49), and since serosal UTP-induced signaling events appear to overlap with those of CCh, serosal UTP may inhibit as well as stimulate secretion through activation of a single receptor of this class. It also remains possible that the stimulatory and inhibitory effects of serosal UTP are mediated through distinct P2Y receptors, since preliminary data suggest that mRNAs for several members of this family are expressed in T84 cells (J. E. Smitham, A. Zambon, P. A. Insel, and K. E. Barrett, unpublished observations). In any event, these data support the hypothesis that CCh and serosal UTP utilize similar, if not identical, signaling mechanisms to inhibit Cl- secretion in T84 cells. In fact, both CCh and serosal UTP evoked increases in InsP4 in T84 cells, although the increase evoked by CCh was more pronounced. However, it remains to be determined why mucosal UTP fails to propagate inhibitory signaling given that it likely also activates phospholipase C, a known initiator of Ins(3,4,5,6)P4 generation (65). We can speculate that this may reflect the spatial segregation of downstream effectors required to mediate the inhibitory effect of CCh and UTP. For example, the epidermal growth factor receptor that is transactivated by CCh and serves to limit secretion is found only at the basolateral pole of T84 cells (35). However, it is notable that mucosal UTP did increase InsP4 levels in some studies, although the effect was more variable than that seen with serosal UTP and did not achieve statistical significance. We can speculate, therefore, that the ability of serosal UTP (and CCh) to elicit inhibitory signaling in T84 cells may additionally be dependent on signals that are only generated in response to the ligation of receptors localized to the basolateral pole of the cell. However, the nature of such additional signals (or the cellular context in which InsP4 acts after various treatments) will require additional investigation.

Overall, in terms of second messenger signaling, purinergic receptor pharmacology, and CF treatment, our current findings illuminate Cl- secretory responses to the therapeutically relevant agent, UTP. They are also of interest with regard to mechanisms that underlie signaling specificity in polarized epithelial cells, given that UTP had divergent effects on Cl- secretion depending on the side of addition. On a positive note, if the findings with mucosal UTP can be extrapolated to airway epithelial cells, our data imply that inhibitory signal(s) are unlikely to limit the efficacy of UTP administered to the lumen of the airway in CF. However, the utility of systemically administered purinergic agonists in the intestine of CF patients may indeed be restricted by negative signals generated in response to UTP itself, or the cholinergic tone. Indeed, previous studies on intact CF intestinal tissues that have failed to detect Ca2+-dependent Cl- secretion could conceivably have involved artifactual activation of cholinergic nerve endings during tissue procurement/preparation. Understanding of negative signaling pathways may, therefore, be of value in the design of treatments to ameliorate gastrointestinal symptoms of CF.


    ACKNOWLEDGEMENTS

We thank Glenda Wheeler for assistance with manuscript preparation and Dr. Stephen Keely for perceptive review of the manuscript.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53480.

A preliminary account of these findings was presented at the annual meeting of the American Gastroenterological Association and has been published in abstract form (Gastroenterology 114: A417, 1998).

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

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.

Received 20 January 2000; accepted in final form 9 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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