Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103
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ABSTRACT |
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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
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INTRODUCTION |
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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
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.
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MATERIALS AND METHODS |
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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
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 · 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.
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RESULTS |
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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
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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We thank Glenda Wheeler for assistance with manuscript preparation and Dr. Stephen Keely for perceptive review of the manuscript.
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FOOTNOTES |
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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.
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