Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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The
Cl secretory response of
colonic cells to Ca2+-mediated
agonists is transient despite a sustained elevation of intracellular Ca2+. We evaluated the effects of
second messengers proposed to limit Ca2+-mediated
Cl
secretion on the
basolateral membrane,
Ca2+-dependent
K+ channel
(KCa) in colonic secretory
cells, T84. Neither protein kinase C (PKC) nor inositol
tetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affected
KCa in excised inside-out patches.
In contrast, arachidonic acid (AA; 3 µM) potently inhibited
KCa, reducing
NPo, the product
of number of channels and channel open probability, by 95%. The
apparent inhibition constant for this AA effect was 425 nM. AA
inhibited KCa in the presence of
both indomethacin and nordihydroguaiaretic acid, blockers of the
cyclooxygenase and lipoxygenase pathways. In the presence of albumin,
the effect of AA on KCa was
reversed. A similar effect of AA was observed on
KCa during outside-out recording.
We determined also the effect of the
cis-unsaturated fatty acid linoleate,
the trans-unsaturated fatty acid
elaidate, and the saturated fatty acid myristate. At 3 µM, all of
these fatty acids inhibited KCa,
reducing NPo by 72-86%. Finally, the effect of the cytosolic phospholipase
A2 inhibitor
arachidonyltrifluoromethyl ketone
(AACOCF3) on the
carbachol-induced short-circuit current
(Isc) response
was determined. In the presence of
AACOCF3, the peak
carbachol-induced
Isc response was
increased ~2.5-fold. Our results suggest that AA generation induced
by Ca2+-mediated agonists may
contribute to the dissociation observed between the rise in
intracellular Ca2+ evoked by these
agonists and the associated
Cl
secretory response.
protein kinase C; inositol tetrakisphosphate; potassium channels; chloride secretion; intestine
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INTRODUCTION |
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CALCIUM-MEDIATED INTESTINAL
Cl secretion was proposed
by Dharmsathaphorn and Pandol (22) to be dependent on the opening of a
basolateral membrane K+
conductance
(GK) in the
absence of any change in apical membrane Cl
conductance
(GCl). Thus
activation of GK
would hyperpolarize the apical and basolateral membrane potentials,
increasing the electrochemical driving force for
Cl
exit from the cell
through apical membrane Cl
channels that were constitutively active (22). An increase in
intracellular Ca2+ alone is
sufficient to induce a Cl
secretory response in the T84 cell line (27). However, there is a
dissociation between both the time course and magnitude of the cellular
Ca2+ rise and the associated
Cl
secretory response,
measured as the agonist-induced short-circuit current
(Isc; Refs. 21,
51, 52). These results have led to speculation that second messengers
other than Ca2+ may produce a
secondary inhibition of
Isc that causes
the secretory response to
Ca2+-mediated agonists to be
transient. Numerous inhibitory second messengers have been postulated,
with the most prominent being protein kinase C (PKC; Refs. 10, 31, 37,
45, 46), inositol tetrakisphosphate
(InsP4; Refs. 26,
47), and arachidonic acid (AA; Refs. 5, 6, 31).
This model for Ca2+-mediated
Cl secretion was based
initially on isotopic flux assays, but more recent electrophysiological data have supported this concept. Measurements of membrane potential in
T84 cells (12, 16, 48) indicated that the resting potential of these
cells is between
30 and
40 mV. These values are very near
the predicted Cl
equilibrium potential, suggesting that the
GCl of these
cells is dominant under resting conditions. In the absence of secretory agonists, a GCl
was detected in ion replacement studies using both whole cell
current-clamp (16, 48) and intracellular microelectrode measurements
(48). Stimulation by muscarinic agonists resulted in hyperpolarization
of the membrane potential (7, 12, 16, 48, 50), due to an increase in
GK (7, 12, 16,
50). In contrast, muscarinic agonists failed to increase
Cl
current
(ICl) during
voltage-clamp recordings in T84 cells (16) or dissociated crypts (7).
In contrast to these findings, muscarinic agonists were shown to
increase GCl in
T84 cells using the perforated whole cell patch-clamp technique (4,
12), and GCl was
stimulated by Ca2+ ionophores
during whole cell recordings (9). This
Ca2+-dependent
Cl conductance was
confirmed at the single-channel level in the HT-29 colonic cell line
(34). These results appear contradictory to the original model for
Ca2+-dependent
Cl
secretion, but it is now
apparent that expression of the
Ca2+-dependent
GCl is
differentiation dependent. In polarized epithelial monolayers,
increasing intracellular Ca2+
fails to increase apical
GCl. Several
lines of evidence support this conclusion.
1) The
Ca2+ ionophore A-23187 failed to
increase apical Cl
uptake
into T84 cells (32). 2) In
nystatin-permeabilized T84 monolayers,
Ca2+-dependent agonists failed to
increase apical
GCl (3, 18, 37,
53). 3) Using single-channel
recording techniques, Morris et al. (34) demonstrated a decrease in
Ca2+-dependent
Cl
channels after
polarization of HT-29 cells. 4) In
cystic fibrosis (CF),
Ca2+-dependent
Cl
secretion is lacking in
the intestine (49), yet this disease impairs only the adenosine
3',5'-cyclic monophosphate (cAMP)-activated GCl. In the CF
knockout mouse, disease severity is inversely correlated with the
magnitude of Ca2+-dependent
GCl in various
tissues (8). The mice die of intestinal obstruction because their
intestinal cells lack an apical
Ca2+-dependent
GCl; this can be
corrected by expression of the CF transmembrane conductance regulator
(CFTR) Cl
conductance in
the intestinal epithelium (54). The CF mouse does not suffer from
airway disease due to the presence of a significant apical
Ca2+-dependent
GCl (8). These
results support the concept that the basolateral membrane
Ca2+-dependent
K+ channel
(KCa) is the site at which
Ca2+-dependent agonists regulate
intestinal Cl
secretion.
We previously characterized the basolateral membrane
K+ channel activated by
Ca2+-dependent agonists
(KCa) in the T84 cell line using
single-channel patch-clamp techniques (14). Thus we can determine
directly whether the second messengers proposed to limit the
Ca2+-dependent secretory response
act on KCa. Our results
demonstrate that neither PKC nor
InsP4 has any
direct effect on KCa. In contrast, AA in particular, and fatty acids in general, are potent inhibitors of
KCa. Indeed, inhibition of
cytosolic phospholipase A2
(cPLA2) results in a potentiated
carbachol-dependent Cl
secretory response. These results suggest that an increase in fatty
acid liberation is responsible for the dissociation between an
agonist-induced secretory response and the rise in intracellular Ca2+.
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METHODS |
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Cell culture. T84 cells were grown in Dulbecco's modified Eagle's medium and Ham's F-12 (1:1) supplemented with 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 14 mM NaHCO3, and 10% fetal bovine serum. The cells were incubated in a humidified atmosphere containing 5% CO2 at 37°C. For measurements of Isc, T84 cells were seeded onto Costar Transwell cell culture inserts (0.33 cm2), and the culture media were changed every 48 h. Isc measurements were performed on filters after 14-21 days in culture. Patch-clamp experiments were performed on single cells plated onto glass coverslips 18-48 h before use.
Solutions.
For measurements of
Isc, the bath
solution contained (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4,
0.8 K2HPO4,
1.2 MgCl2, 1.2 CaCl2, and 10 glucose. The pH of
this solution was 7.4 when gassed with a mixture of 95%
O2-5%
CO2 at 37°C. The effects of
4-phorbol 12-myristate 13-acetate (PMA) and
1-ethyl-2-benzimidazolinone (1-EBIO) on apical membrane
ICl were assessed
after permeabilization of the serosal membrane with nystatin (360 µg/ml) and the establishment of a mucosa-to-serosa
Cl
concentration gradient.
Serosal NaCl was replaced by equimolar sodium gluconate, and
CaCl2 was increased to 4 mM to
compensate for the Ca2+ buffering
capacity of the gluconate anion. Nystatin was added to the serosal
membrane 15-30 min before the addition of drugs (17). Successful
permeabilization of the basolateral membrane was based on the recording
of a negative ICl
as described (17).
Isc measurements.
Costar Transwell cell culture inserts were mounted in an Ussing chamber
(Jim's Instruments, Iowa City, IA), and the monolayers were
continuously short circuited (model EC-825; Warner Instrument). Transepithelial resistance was measured by applying a 5-mV pulse at
60-s intervals, and the resistance was calculated using Ohm's law. The
T84 monolayers had resistances of 500-2,000
· cm2.
1-EBIO, forskolin, thapsigargin, PMA, glibenclamide,
arachidonyltrifluoromethyl ketone
(AACOCF3), and luffariellolide
were added to both sides of the monolayers at the indicated
concentrations. Bumetanide and charybdotoxin (CTX) were added only to
the serosal bathing solution. Changes in
Isc are
calculated as a difference current between the sustained phase of the
response and their respective baseline values.
Single-channel recordings.
Single-channel currents were recorded using a List EPC-7 amplifier
(Medical Systems) and recorded on videotape for later analysis as
described previously (14). All recordings were performed during
continuous perfusion of the patch-clamp chamber. Pipettes were
fabricated from KG-12 glass (Willmad Glass). All recordings were done
at a holding voltage of 100 mV. The voltage is referenced to the
extracellular compartment, as is standard for membrane potentials.
Inward currents are defined as the movement of positive charge from the
extracellular compartment to the intracellular compartment and are
presented as downward deflections from baseline in all recording
configurations.
Chemicals.
Nystatin was a generous gift from Dr. S. Lucania (Bristol
Meyers-Squibb). 1-EBIO was obtained from Aldrich.
Glibenclamide, bumetanide, PMA, and the fatty acids were
obtained from Sigma Chemical. Inositol 1,4,5-trisphosphate
(InsP3),
inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4],
inositol 3,4,5,6-tetrakisphosphate
[Ins(3,4,5,6)P4], and sn-1,2-dioctanoyl-glycerol
(DiC8) were obtained from
Calbiochem. The inositol polyphosphates were made as 3 mM stock
solutions in water buffered with HEPES to pH 7.2. CTX was obtained from Accurate Chemical and Scientific Corporation and made as a 10 µM
stock solution in standard bath solution. Thapsigargin was obtained
from Research Biochemicals.
AACOCF3, luffariellolide, indomethacin, and nordihydroguaiaretic acid (NDGA) were obtained from
Biomol. 1-EBIO, thapsigargin,
DiC8, PMA, glibenclamide,
AACOCF3, and luffariellolide were
made as 1,000-fold stock solutions in dimethyl sulfoxide (DMSO).
Nystatin was made as a 180 mg/ml stock solution in DMSO and sonicated
for 30 s just before use. Bumetanide was made as a 1,000-fold stock
solution in ethanol. All fatty acids were made as
1,000-fold stock
solutions in DMSO and stored under
N2 at
80°C. The fatty
acids were dissolved to the final working concentration just before
use, and all solutions were continuously bubbled with
N2 during perfusion through the
patch-clamp chamber. Cell culture medium was obtained from GIBCO.
Data analysis. All data are presented as means ± SE, where n indicates the number of experiments. Statistical analysis was performed using the Student's t-test. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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Effect of PKC activation on Cl
secretory responses that involve KCa.
Initially, we wished to determine whether activation of PKC would
affect Cl
secretory
responses in which the current flow across the basolateral membrane is
due to activation of KCa. PKC has
been proposed to be an inhibitory modulator of
Ca2+-mediated
Cl
secretion (27, 37, 46,
53). This experiment is complicated by the fact that
Ca2+-dependent agonists such as
carbachol induce only a transient Cl
secretory response, and
nonreceptor agonists such as thapsigargin or
Ca2+ ionophores only weakly
stimulate Cl
secretion in
T84 cells. However, we recently identified a novel pharmacological
opener of KCa in both intestinal
and airway epithelia, 1-EBIO, which stimulates a sustained
transepithelial Cl
secretory response (17, 19). We took advantage of this sustained response to determine whether PKC activation would inhibit
KCa and hence the transepithelial
Cl
secretory response. The
results of one experiment are shown in Fig.
1A.
1-EBIO (300 µM) stimulated a sustained
Isc response as previously described (17, 19). Rather than inhibiting
Isc, addition of
PMA (100 nM) stimulated an additional increase in Isc that was
sensitive to block by bumetanide. PMA alone had no effect on basal
Isc (data not
shown), as described by others (28). In eight experiments, 1-EBIO
increased Isc an
average of 49 ± 9 µA/cm2
from a baseline of 2.6 ± 0.7 µA/cm2. In five of these
experiments, the subsequent addition of PMA further increased
Isc by 65 ± 8 µA/cm2. In three additional
experiments, the inactive phorbol ester 4
-PMA failed to stimulate an
increase in Isc
subsequent to 1-EBIO (data not shown).
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Effect of PKC activation on transepithelial IK and ICl. The above results suggest that activation of PKC increases either a basolateral membrane GK or apical membrane GCl subsequent to addition of 1-EBIO. To resolve the conductance pathways activated by PMA, the pore-forming antibiotic nystatin was used to permeabilize either the apical or basolateral membrane, and the appropriate transepithelial ion gradients were established to measure IK or ICl, respectively (see METHODS). The effect of PMA on IK after 1-EBIO stimulation is shown in Fig. 1C. After nystatin permeabilization, 1-EBIO (300 µM) induced a sustained increase in IK as previously described (17, 19). The subsequent addition of PMA (100 nM) had no effect on the 1-EBIO-induced IK; CTX (100 nM) inhibited this current, confirming activation of KCa by 1-EBIO (17, 19). Similar results were obtained in six experiments. 1-EBIO increased IK an average of 205 ± 59 µA/cm2, and this was not affected by PMA. The subsequent addition of CTX inhibited IK an average of 60 ± 6%.
It is possible that an inhibitory effect of PMA on KCa requires a Ca2+-dependent PKC isoform, and 1-EBIO does not reproduce this aspect of the response to a Ca2+-mediated agonist. Therefore, we determined the effect of PMA on a thapsigargin-induced IK (Fig. 1D). In contrast to thapsigargin's effect on Isc, it induces a sustained increase in IK (Fig. 1D). Similar to what we observed with 1-EBIO, PMA had no effect on IK after activation of KCa by thapsigargin. Again, CTX inhibited the thapsigargin-induced current, confirming activation of KCa. Similar results were obtained in four experiments. Thapsigargin (1 µM) increased IK by 229 ± 34 µA/cm2, and this was not affected by PMA. The subsequent addition of CTX reduced IK an average of 87 ± 3%. These results further indicate that activation of PKC does not modulate the activity of KCa in T84 cells. Because PMA did not affect IK, these results suggest that PMA is increasing Isc subsequent to 1-EBIO by increasing an apical GCl. Therefore, we determined the effect of PMA on ICl after stimulation by 1-EBIO (Fig. 1B). After nystatin permeabilization, 1-EBIO increased ICl as described previously (17). The addition of PMA (100 nM) stimulated an additional transient increase in ICl that was sensitive to block by glibenclamide (300 µM); this is consistent with activation of CFTR (13, 39, 41). In five experiments, 1-EBIO increased ICl by 51 ± 5 µA/cm2, and PMA transiently increased ICl an additional 17 ± 7 µA/cm2.Effect of PKC on KCa in excised patches. We next determined whether PKC activation would modulate the activity of KCa in excised patch-clamp recordings. Initially, we attempted to use the lipid analog of 1,2-diacylglycerol (DAG), DiC8, as an activator of PKC in our patch-clamp experiments. However, as illustrated in Fig. 2, DiC8 (3 µM) alone potently inhibited KCa during inside-out recording; the bath contained no ATP. In six patches, DiC8 reduced NPo by 88 ± 4%, from 1.48 ± 0.64 to 0.14 ± 0.04.
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Effect of InsP3 and InsP4 on KCa. Traynor-Kaplan and colleagues (26, 47) have shown that stimulation by carbachol induces a prolonged rise in InsP4. They speculated that the generation of InsP4 may be an inhibitory modulator of KCa (26). We directly assessed the effect of two isoforms of InsP4 (1,3,4,5 and 3,4,5,6), as well as InsP3, on the NPo of KCa in excised inside-out patches. Neither Ins(1,3,4,5)P4 (control, 1.07 ± 0.28; InsP4, 0.91 ± 0.24; n = 3) nor Ins(3,4,5,6)P4 (control, 0.79 ± 0.39; InsP4, 0.74 ± 0.39; n = 5) affected KCa NPo when added directly to the bathing solution at concentrations previously shown to be produced by carbachol in T84 cells (i.e., 6-12 µM; Ref. 47). Similarly, InsP3 (6-12 µM) had no effect on the NPo of KCa (control, 0.92 ± 0.69; InsP3, 0.76 ± 0.53; n = 4). These results demonstrate that inositol polyphosphates do not directly modulate the activity of KCa in the T84 cell line.
Effect of AA on KCa in excised,
inside-out patches.
Ca2+-dependent agonists are known
to increase AA subsequent to the cellular
Ca2+ rise in a wide variety of
tissues (33, 36). Therefore, we determined the effect of AA on
KCa in excised, inside-out
patches. The results of one experiment are shown in Fig.
3A. AA (3 µM) dramatically inhibited the activity of
KCa at a holding potential of
100 mV. In 22 patches, AA (3 µM) inhibited
NPo by 95 ± 1%, from 1.36 ± 0.21 to 0.07 ± 0.02. An average
concentration-response curve is shown in Fig.
3B. AA inhibited
KCa with a predicted inhibition constant (Ki)
of 425 nM. Although we did not routinely voltage clamp to positive
holding potentials, a similar inhibition was observed at +100 mV (data
not shown), indicating this inhibition is voltage independent.
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Effect of elevated Ca2+ on the AA-induced inhibition of KCa. We determined whether the inhibition of KCa by AA could be overcome by increasing Ca2+ at the cytoplasmic face of the channel. For these experiments, the channel was first inhibited by AA (3 µM) in the presence of 400 nM free Ca2+. Then, in the continued presence of AA, free Ca2+ was increased to 10 µM. The results of one experiment are illustrated in Fig. 4. Increasing the free Ca2+ concentration after inhibition by AA failed to induce recovery of channel activity. In six experiments, AA reduced NPo from 1.22 ± 0.27 to 0.09 ± 0.04, and the subsequent increase in Ca2+ failed to induce a recovery of NPo (0.07 ± 0.03). These results suggest that AA is not simply displacing Ca2+ from its binding site to cause the reduction in NPo.
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The unbound form of AA is required for inhibition of KCa. AA is normally transported in the bloodstream bound to albumin. Therefore, we determined the effect of AA in the presence of albumin. In the presence of 2.5 g/l albumin, AA (3 µM) reduced the NPo of KCa from 1.09 ± 0.27 to 0.62 ± 0.09 (n = 5; P < 0.001). This 37% inhibition of NPo is significantly less than the 95% inhibition seen in the absence of albumin (P < 0.001). The subsequent removal of albumin in the continued presence of AA (3 µM) resulted in a further reduction of NPo to 0.03 ± 0.01 (n = 5). This suggests that AA must be free in solution to inhibit KCa.
After inhibition of KCa by AA, removal of AA results in only a modest recovery of channel activity, suggesting that AA remains bound to its inhibitory site. Based on the above results, we determined whether albumin would induce a further recovery of channel activity. The results of one experiment are shown in Fig. 5. AA (3 µM) nearly abolished channel activity, as shown above. After removal of AA from the bathing solution, a small increase in channel activity is apparent. However, the subsequent addition of albumin in the continued absence of AA results in a significant increase in channel activity. In five experiments, AA reduced NPo from 1.30 ± 0.56 to 0.02 ± 0.01, and NPo increased to 0.15 ± 0.08 upon removal of AA. The subsequent addition of albumin resulted in NPo, increasing to 65% of control (0.98 ± 0.56). This result suggests that albumin competes with membrane-associated AA, thus removing the block of KCa.
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Effect of AA from the external side of KCa. We determined whether AA would inhibit KCa from the extracellular side of the channel using the excised, outside-out recording technique. The results of one experiment are shown in Fig. 6. AA (3 µM) inhibited KCa with an apparently similar affinity to that seen in inside-out recordings. Similar results were observed in four additional patches.
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Specificity of the AA effect on KCa.
We next determined whether the observed inhibitory effect of AA on
KCa was specific for AA or whether
additional fatty acids would also modulate
KCa activity. For these
experiments, we used an additional
cis-unsaturated fatty acid, linoleic
acid (C18; cis,cis-9,
12),
the trans-unsaturated fatty acid
elaidic acid (C18;
trans-
9),
and a saturated fatty acid, myristic acid
(C14). The results of these
experiments are shown in Fig. 7. At 3 µM,
all of these fatty acids significantly inhibited
KCa. Both linoleic acid
(n = 11) and elaidic acid
(n = 6) reduced
NPo by 86 ± 2%. This inhibition is significantly less than that observed in the
presence of AA (P < 0.01). Myristic
acid reduced NPo
by 72 ± 2% (n = 11). This effect
is less than that caused by any of the unsaturated fatty acids
(P < 0.001). These results
demonstrate that fatty acids in general are potent inhibitors of the
basolateral membrane KCa in T84
cells, although some structural specificity is apparent.
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Effect of PLA2 inhibition on the
carbachol-induced Isc response.
Our results demonstrate that AA is a potent inhibitor of
KCa in T84 cells. Therefore, we
wished to determine whether inhibition of
PLA2 would induce a potentiation
of the carbachol-induced Cl
secretory response. Initially, we evaluated the effect of the cPLA2 inhibitor
AACOCF3. In 14 control filters,
carbachol (100 µM) increased
Isc from a
baseline of 1.1 ± 0.2 µA/cm2
to a peak of 32.6 ± 2.7 µA/cm2 (Fig.
8). Although
AACOCF3 (100 µM)
increased baseline
Isc by only 1.0 ± 0.2 µA/cm2
(n = 10), the subsequent response to
carbachol was dramatically potentiated (82.4 ± 7.6 µA/cm2;
n = 10;
P < 0.001; Fig. 8). In
contrast, the secreted PLA2 inhibitor luffariellolide (2 µM) failed to potentiate the carbachol-mediated Cl
secretory response (37.1 ± 4.7 µA/cm2;
n = 6; Fig. 8).
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DISCUSSION |
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A clear dissociation exists between the agonist-induced rise in
intracellular Ca2+ and the
concomitant Cl secretory
response in the colonic cell line T84 (21, 51, 52). It has been
proposed that additional second messengers modulate the effects of
Ca2+ on secretory mechanisms to
produce the characteristic transient response to
Ca2+-dependent agonists. The
candidate messengers that have received the greatest attention in this
regard are PKC,
InsP4, and AA. In
the present study, we evaluated the effects of PKC,
InsP4, and AA on
KCa in both excised patches and
Ussing chamber experiments.
PKC does not acutely regulate KCa.
Ca2+-mediated agonists, such as
carbachol, histamine, and taurodeoxycholic acid, are known to activate
phospholipase C, resulting in the hydrolysis of phosphatidylinositol
4,5-bisphosphate and subsequent increases in cell
Ca2+ and PKC activity in the T84
cell line (10, 15, 23, 52, 53). Although the
InsP3-mediated
release of Ca2+ from intracellular
stores has been convincingly linked to the Cl secretory current evoked
by these agonists (15, 16, 21, 22, 53), it has been speculated that PKC
likely contributes to the secondary downregulation of the secretory
response that follows this initial stimulation, despite a continuing
elevation of cell Ca2+ (10, 27,
31, 37, 46). As summarized in the introduction, any negative regulation
of Cl
secretion by PKC
likely occurs at the level of KCa.
Because Ca2+-mediated
Isc responses in
T84 cells are virtually complete within 5-10 min, we focused our
experiments on determining the acute effect of PKC activation on
KCa activity.
Inositol polyphosphates do not acutely regulate
KCa.
When carbachol stimulates
Cl secretion in T84 cells,
it inhibits the subsequent secretory responses to other
Ca2+-dependent agonists (26). This
inhibitory effect correlated with a sustained, carbachol-induced
elevation of
InsP4 that was not observed with histamine (26). The use of cell-permeant analogs of
InsP4 suggested
that
Ins(3,4,5,6)P4
was the isoform most likely involved in inhibiting
Isc (47). It was
postulated that
InsP4 may
attenuate Cl
secretion by
inhibiting KCa (26). Based on
these observations, we determined the effect of inositol polyphosphates
(InsP3,
InsP4) on
KCa in excised, inside-out
patches. Our results demonstrate that neither
InsP4 (1,3,4,5 or
3,4,5,6) nor
InsP3 modulates
the activity of KCa in excised
patches from T84 cells, demonstrating that this is not a site for
inhibitory modulation by
InsP4. Consistent with this notion is the recent report demonstrating inhibition of a
Ca2+-activated
Cl
channel from bovine
trachea by InsP4
(25). These results suggest an apical
GCl may be the
inhibitory site of action for
InsP4. However,
as outlined in the introduction, the role of a
Ca2+-activated
Cl
channel in mediating
intestinal Cl
secretion in
an intact epithelium remains obscure and awaits direct
electrophysiological confirmation.
Inhibition of KCa by fatty acids.
Ca2+-dependent agonists are known
to increase AA levels in a wide range of tissues. This can occur in
several ways (1): 1) Ca2+ can directly activate
PLA2,
2) either DAG itself or PKC can
activate PLA2, or
3) DAG lipase can directly generate
AA from DAG. The generation of AA by
Ca2+-mediated agonists lags behind
the rise in intracellular Ca2+.
Thus an effect of AA on the transporters associated with
Cl secretion would be
temporally appropriate to explain the dissociation between the
Ca2+ and
Isc. Numerous
agonists, both Ca2+ dependent and
cAMP dependent, including the kinins (30), bile acids (11), adenosine
(6), vasoactive intestinal polypeptide (5), and
Ca2+ ionophores (23), have been
shown to increase the levels of AA in intestinal tissues. We previoulsy
demonstrated that both taurodeoxycholic acid and
Ca2+ ionophores activate
KCa in the colonic cell line T84
(14). In the present communication, we demonstrate that an additional second messenger known to be generated by these agonists, AA, potently
inhibits KCa. Thus AA may serve as
an important second messenger in the
Cl
secretory response to
these agonists.
Effect of cPLA2 inhibition.
Our results demonstrate that inhibition of
cPLA2 potentiates the subsequent
response to the Ca2+-dependent
agonist carbachol. This is consistent with the notion that the
generation of AA by carbachol attenuates the
Cl secretory response. As
we have demonstrated that AA is a potent negative modulator of
KCa, and
KCa is known to be activated by carbachol (14), the most parsimonious explanation of these results is
that the activation of cPLA2 by
carbachol results in the liberation of AA, which subsequently inhibits
KCa, resulting in an attenuation of the Cl
secretory
response. Others have previously reported that inhibition of AA
generation by blocking DAG lipase activity results in a potentiation of
both carbachol-induced (31) and histamine-induced (5)
Cl
secretory responses in
T84 cells. These results are consistent with the hypothesis that AA
acts as a negative modulator of
Ca2+-dependent
Cl
secretion. Also, the
kinins (30) and bile acids (11), both of which are
Ca2+-dependent agonists, have
previously been shown to increase intestinal AA levels. We previously
demonstrated that the bile acid taurodeoxycholic acid similarly
activates KCa in T84 cells (14).
However, direct measurements of AA generation and
cPLA2 activation during agonist stimulation in T84 cells are required to confirm this proposal.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the excellent technical assistance of Cheng Zhang Shi in both tissue culture and Ussing chamber experiments.
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FOOTNOTES |
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This work was supported by Cystic Fibrosis Foundation Grant DEVOR96PO (to D. C. Devor) and National Institute of Diabetes and Digestive and Kidney Diseases Research Grant DK-31091 (to R. A. Frizzell).
Address for reprint requests: D. C. Devor, Dept. of Cell Biology and Physiology, S312 BST, 3500 Terrace St., University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (E-mail: dd2+{at}pitt.edu).
Received 7 February 1997; accepted in final form 20 September 1997.
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REFERENCES |
---|
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---|
1.
Abdel-Latif, A. A.
Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers.
Pharmacol. Rev.
38:
227-272,
1986[Medline].
2.
Anderson, M. P.,
and
M. J. Welsh.
Fatty acids inhibit apical membrane chloride channels in airway epithelia.
Proc. Natl. Acad. Sci. USA
87:
7334-7338,
1990[Abstract].
3.
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].
4.
Baro, I.,
B. Roch,
A. Hongre,
and
D. Escande.
Concomitant activation of Cl and K+ currents by secretory stimulation in human epithelial cells.
J. Physiol. (Lond.)
478:
469-482,
1994[Abstract].
5.
Barrett, K. E.
Effect of the diglyceride lipase inhibitor, RG80267, on epithelial chloride secretion induced by various agents.
Cell. Signal.
7:
225-233,
1995[Medline].
6.
Barrett, K. E.,
and
T. D. Bigby.
Involvement of arachidonic acid in the chloride secretory response of intestinal epithelial cells.
Am. J. Physiol.
264 (Cell Physiol. 33):
C446-C452,
1993
7.
Bohme, M.,
M. Diener,
and
W. Rummel.
Calcium- and cyclic-AMP-mediated secretory responses in isolated colonic crypts.
Pflügers Arch.
419:
144-151,
1991[Medline].
8.
Clarke, L. L.,
B. R. Grubb,
J. R. Yankaskas,
C. U. Cotton,
A. McKenzie,
and
R. C. Boucher.
Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(/
) mice.
Proc. Natl. Acad. Sci. USA
91:
479-483,
1994[Abstract].
9.
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].
10.
Cohn, J. A.
Protein kinase C mediates cholinergically regulated protein phosphorylation in a Cl secreting epithelium.
Am. J. Physiol.
258 (Cell Physiol. 27):
C227-C233,
1990
11.
DeRubertis, F. R.,
P. A. Craven,
and
R. Saito.
Bile salt stimulation of colonic epithelial proliferation: evidence for involvement of lipoxygenase products.
J. Clin. Invest.
74:
1614-1624,
1984[Medline].
12.
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
13.
Devor, D. C.,
J. N. Forrest, Jr.,
W. K. Suggs,
and
R. A. Frizzell.
cAMP-activated Cl channels in primary cultures of spiny dogfish (Squalus acanthias) rectal gland.
Am. J. Physiol.
268 (Cell Physiol. 37):
C70-C79,
1995
14.
Devor, D. C.,
and
R. A. Frizzell.
Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C1271-C1280,
1993
15.
Devor, D. C.,
M. C. Sekar,
R. A. Frizzell,
and
M. E. Duffey.
Taurodeoxycholate activates K+ and Cl conductances via an IP3-mediated release of Ca2+ from intracellular stores.
J. Clin. Invest.
92:
2173-2181,
1993[Medline].
16.
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
17.
Devor, D. C.,
A. K. Singh,
R. J. Bridges,
and
R. A. Frizzell.
Modulation of Cl secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK by NS004, NS1619, and 1-EBIO.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L785-L795,
1996
18.
Devor, D. C.,
A. K. Singh,
R. J. Bridges,
and
R. A. Frizzell.
Psoralens: novel modulators of Cl secretion.
Am. J. Physiol.
272 (Cell Physiol. 41):
C976-C988,
1997
19.
Devor, D. C.,
A. K. Singh,
R. A. Frizzell,
and
R. J. Bridges.
Modulation of Cl secretion by benzimidazolones. I. Direct activation of a Ca2+-dependent K+ channel by 1-ethyl-2-benzimidazolinone, 1-EBIO.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L775-L784,
1996
20.
Devor, D. C.,
A. K. Singh,
R. A. Frizzell,
and
R. J. Bridges.
Inhibition of intestinal Cl secretion by clotrimazole: direct effect on basolateral membrane K+ channels.
Am. J. Physiol.
273 (Cell Physiol. 42):
C531-C540,
1997
21.
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
22.
Dharmsathaphorn, K.,
and
S. J. Pandol.
Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.
J. Clin. Invest.
77:
348-354,
1986[Medline].
23.
Dickinson, K. E. J.,
R. A. Frizzell,
and
M. C. Sekar.
Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 receptor.
Eur. J. Pharmacol.
225:
291-298,
1992[Medline].
24.
Hwang, T.-C.,
S. E. Guggino,
and
W. B. Guggino.
Direct modulation of secretory chloride channels by arachidonic and other cis unsaturated fatty acids.
Proc. Natl. Acad. Sci. USA
87:
5706-5709,
1990[Abstract].
25.
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
26.
Kachintorn, U.,
M. Vajanaphanich,
K. E. Barrett,
and
A. E. Traynor-Kaplan.
Elevation of inositol tetrakisphosphate parallels inhibition of Ca2+-dependent Cl secretion in T84 cells.
Am. J. Physiol.
264 (Cell Physiol. 33):
C671-C676,
1993
27.
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].
28.
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 epithelial cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C15-C22,
1992
29.
Kubo, M.,
and
Y. Okada.
Volume-regulatory Cl channel currents in cultured human epithelial cells.
J. Physiol. (Lond.)
456:
351-371,
1992[Abstract].
30.
Lawson, L. D.,
and
D. W. Powell.
Bradykinin-stimulated eicosanoid synthesis and secretion by rabbit ileal components.
Am. J. Physiol.
252 (Gastrointest. Liver Physiol. 15):
G783-G790,
1987
31.
Lindeman, R. P.,
and
H. S. Chase, Jr.
Protein kinase C does not participate in carbachol's secretory action in T84 cells.
Am. J. Physiol.
263 (Cell Physiol. 32):
C140-C146,
1992
32.
Mandel, K. G.,
K. Dharmsathaphorn,
and
J. A. McRoberts.
Characterization of a cyclic AMP-activated Cl transport pathway in the apical membrane of a human colonic epithelial cell line.
J. Biol. Chem.
261:
704-712,
1986
33.
Meeves, H.
Modulation of ion channels by arachidonic acid.
Prog. Neurobiol.
43:
175-186,
1994[Medline].
34.
Morris, A. P.,
S. A. Cunningham,
D. J. Benos,
and
R. A. Frizzell.
Cellular differentiation is required for cAMP but not Ca2+-dependent Cl secretion in colonic epithelial cells expressing high levels of cystic fibrosis transmembrane conductance regulator.
J. Biol. Chem.
267:
5575-5583,
1992
35.
Needleman, P.,
J. Turk,
B. A. Jakschik,
A. R. Morrison,
and
J. B. Lefkowith.
Arachidonic acid metabolism.
Annu. Rev. Biochem.
55:
69-102,
1986[Medline].
36.
Ordway, R. W.,
J. J. Singer,
and
J. V. Walsh, Jr.
Direct regulation of ion channels by fatty acids.
Trends Neurosci.
14:
96-100,
1991[Medline].
37.
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
38.
Roch, B.,
I. Baro,
A. Hongre,
and
D. Escande.
ATP-sensitive K+ channels regulated by intracellular Ca2+ and phosphorylation in normal (T84) and cystic fibrosis (CFPAC-1) epithelial cells.
Pflügers Arch.
426:
355-363,
1995.
39.
Schultz, B. D.,
A. D. G. DeRoos,
C. J. Venglarik,
A. K. Singh,
R. A. Frizzell,
and
R. J. Bridges.
Glibenclamide blockade of CFTR chloride channels.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L192-L200,
1996
40.
Shen, B.-Q.,
R. A. Barthelson,
W. Skach,
D. C. Gruenert,
E. Sigal,
R. J. Mrsny,
and
J. H. Widdicombe.
Mechanism of inhibition of cAMP-dependent epithelial chloride secretion by phorbol esters.
J. Biol. Chem.
268:
19070-19075,
1993
41.
Sheppard, D. N.,
and
M. J. Welsh.
Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
J. Gen. Physiol.
100:
573-591,
1992[Abstract].
42.
Shinomura, T.,
Y. Asaoka,
M. Oka,
K. Yoshida,
and
Y. Nishizuka.
Synergistic action of diacylglycerol and unsaturated fatty acids for protein kinase C activation: its possible implications.
Proc. Natl. Acad. Sci. USA
88:
5149-5153,
1991[Abstract].
43.
Tabcharani, J. A.,
A. Boucher,
J. W. L. Eng,
and
J. W. Hanrahan.
Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides, and kinases.
J. Membr. Biol.
142:
255-266,
1994[Medline].
44.
Tabcharani, J. A.,
X. Chang,
J. R. Riordan,
and
J. W. Hanrahan.
Phosphorylation-regulated Cl channel in CHO cells stably expressing the cystic fibrosis gene.
Nature
352:
628-631,
1991[Medline].
45.
Traynor-Kaplan, A. E.,
T. Buranawuti,
M. Vajanaphanich,
and
K. E. Barrett.
Protein kinase C activity does not mediate the inhibitory effect of carbachol on chloride secretion by T84 cells.
Am. J. Physiol.
267 (Cell Physiol. 36):
C1224-C1230,
1994
46.
Vaandrager, A. B.,
N. Van Den Berghe,
A. G. M. Bot,
and
H. R. De Jonge.
Phorbol esters stimulate and inhibit Cl secretion by different mechanisms in a colonic cell line.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G249-G256,
1992
47.
Vajanaphanich, M.,
C. Schultz,
M. T. Rudolf,
M. Wasserman,
P. Enyedl,
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].
48.
Valverde, M. A.,
G. M. Mintenig,
and
F. V. Sepulveda.
Cl currents of unstimulated T84 intestinal epithelial cells studied by intracellular recording.
J. Membr. Biol.
137:
237-247,
1994[Medline].
49.
Veeze, H. J.,
D. J. J. Halley,
J. Bijman,
J. C. de Jongste,
H. R. de Jonge,
and
M. Sinaasappel.
Determinants of mild clinical symptoms in cystic fibrosis patients: residual chloride secretion measured in rectal biopsies in relation to the genotype.
J. Clin. Invest.
93:
461-466,
1994[Medline].
50.
Walters, R. J.,
and
F. V. Sepulveda.
A basolateral K+ conductance modulated by carbachol dominates the membrane potential of small intestinal crypts.
Pflügers Arch.
419:
537-539,
1991[Medline].
51.
Warhurst, G.,
N. B. Higgs,
A. Tonge,
and
L. A. Turnberg.
Stimulatory and inhibitory actions of carbachol on chloride secretory responses in human colonic cell line T84.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G220-G228,
1991
52.
Wong, S. M. E.,
R. P. Lindeman,
S. Parangi,
and
H. S. Chase, Jr.
Role of calcium in mediating action of carbachol in T84 cells.
Am. J. Physiol.
257 (Cell Physiol. 26):
C976-C985,
1989
53.
Wong, S. M. E.,
A. Tesfaye,
M. C. DeBell,
and
H. S. Chase, Jr.
Carbachol increases basolateral K+ conductance in T84 cells: simultaneous measurements of cell [Ca] and gK explore calcium's role.
J. Gen. Physiol.
96:
1271-1285,
1990[Abstract].
54.
Zhou, L.,
C. R. Dey,
S. E. Wert,
M. D. DuVall,
R. A. Frizzell,
and
J. A. Whitsett.
Correction of the lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR.
Science
266:
1705-1708,
1994[Medline].