Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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.
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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+).
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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.
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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).
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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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-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.
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