1 Department of Physiology and 2 Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong, China
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ABSTRACT |
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The effect of baicalein on mucosal ion
transport in the rat distal colon was investigated in Ussing chambers.
Mucosal addition of baicalein (1-100 µM) elicited a
concentration-dependent short-circuit current
(Isc) response. The increase in
Isc was mainly due to Cl
secretion. The presence of mucosal indomethacin (10 µM) significantly reduced both the basal and subsequent baicalein-evoked
Isc responses. The baicalein-induced
Isc were inhibited by mucosal application of
diphenylamine-2-carboxylic acid (100 µM) and glibenclamide (500 µM)
and basolateral application of chromanol 293B (30 µM), a blocker of
KvLQT1 channels and Ba2+ ions (5 mM). Treatment
of the colonic mucosa with baicalein elicited a threefold increase in
cAMP production. Pretreating the colonic mucosa with carbachol (100 µM, serosal) but not thapsigargin (1 µM, both sides) abolished the
baicalein-induced Isc. Addition of baicalein
subsequent to forskolin induced a further increase in
Isc. These results indicate that the baicalein
evoked Cl
secretion across rat colonic mucosa, possibly
via a cAMP-dependent pathway. However, the action of baicalein cannot
be solely explained by its cAMP-elevating effect. Baicalein may
stimulate Cl
secretion via a cAMP-independent pathway or
have a direct effect on cystic fibrosis transmembrane conductance regulator.
Ussing chambers; isolated crypts; intracellular cyclic adenosine 5'-monophosphate; colonic mucosa
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INTRODUCTION |
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INTESTINAL FLUID
SECRETION is a passive process driven by osmotic forces generated
by ion transport. The main determinant of a luminally directed osmotic
gradient is the mucosal transport of Cl into the lumen.
Intestinal Cl
secretion is an energy-dependent process
that generates an electrical potential difference across the mucosal
epithelium (i.e., electrogenic). Cations are drawn into the lumen by
the established electrochemical gradient, and water loss is an
obligatory consequence of the efflux of salt. If Cl
secretion exceeds the capacity for distal salt and water reabsorption, then diarrhea ensues (7, 8).
The mechanisms of colonic Cl secretion are well
understood. Increase in cAMP level activates Cl
secretion
via the luminal cystic fibrosis transmembrane conductance regulator
(CFTR) Cl
channels (12). In addition, cAMP
also increases basolateral K+ conductance, probably via
KvLQT1-type K+ channels, which hyperpolarize
the membrane and provide driving force for apical Cl
exit. This cAMP-dependent K+ conductance can be blocked by
293B (38). The involvement of Ca2+-activated
Cl
channel (CaCC) in Cl
secretion remains
controversial in intact enterocytes. However, increase in intracellular
free Ca2+ concentration ([Ca2+]i)
activates basolateral SK4-type K+ channels
(37), which provide additional driving force for luminal Cl
exit through CFTR.
In Japan and China, Scutellariae radix (dried root of
Scutellariae baicalensis) has been employed for centuries as
an important medicine, which is used as an anti-inflammatory and smooth
muscle relaxant. It contains a large amount of flavonoids such as
baicalein, baicalin, and wogonin. These flavonoids are known to have
multiple biological effects such as anti-inflammatory
(22), antitumor (16), and antiproliferative
(14) effects. Baicalein and baicalin also potentiate the
contractile response in rat mesenteric artery through inhibition of
nitric oxide formation and/or release in the endothelium
(35). S. radix is often used in combination with Coptidis rhizoma (dried rhizome of Coptis
chinensis Franch.) in traditional folk remedies. C. rhizoma has been employed for the treatment of gastroenteritis and
secretory diarrhea (32). Berberine is the major active
components of C. rhizoma. Recently, the pharmacological
effects of berberine on colonic secretion have been extensively
studied. Recent reports suggest that its antisecretory activity is due
to a direct effect on colonic epithelium via a blockage of
K+ channels that are responsible for K+
recycling during Cl secretion (33). This
provides a plausible mechanism to partially explain its therapeutic
benefit seen in vivo. In contrast, the effect of baicalein on
electrolyte transport processes across colonic mucosa has not been
reported. Therefore, the aim of this study was to examine the action of
baicalein in active ion transport across rat colonic mucosa. Results
demonstrate that baicalein stimulates Cl
secretion,
probably via a cAMP-dependent pathway. Therefore, in contrast to
berberine, it is a prosecretory compound. The presence of baicalein in
S. radix may limit the effectiveness of C. rhizoma in treating secretory diarrhea.
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MATERIALS AND METHODS |
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Ussing chamber experiments. Both male and female Sprague-Dawley rats of ~400 g body wt were used. They were killed by CO2 asphyxiation in accordance with a protocol approved by Animal Research Ethics Committee of our university. A segment of the distal colon (~5 cm) was removed and rinsed with ice-cold Krebs-Henseleit (KH) solution. Two pieces of distal colon were used from each animal. The muscle layers were separated from the mucosal sheet as follows. The colon was pinned with the mucosal side facing down on a dissection plate, and then the muscular layer was carefully separated from the mucosa by blunt dissection. Finally, the isolated mucosal sheet was cut into an appropriate size (area of exposed tissue 0.45 cm2) and mounted between two halves of modified Ussing chambers. The chambers were filled with 20 ml of identical KH solution on both sides of the epithelium. The solution was gassed with 95% O2 and 5% CO2 to maintain the pH at 7.4 and to provide adequate agitation. The bath temperature was kept at 37°C by water jackets. An equilibration period of 30 min was given before the experiments.
Electrodes for measuring transepithelial potential difference (PD) and passing current were connected to the chambers. The transepithelial PD was then clamped at 0 mV, and the short-circuit current (Isc) was recorded with MC6-VC4 voltage-current clamp amplifier (Physiologic Instruments, San Diego, CA) and displayed using a chart recorder (Kipp and Zonen, Delft, The Netherlands). A transepithelial PD of 1 mV was applied periodically, and the resultant change in current was used to calculate the transepithelial resistance (Rt) using Ohm's law. The methodology for tissue preparation and measurement of electrical parameters across the mucosal sheets has been described earlier (15). During the washout treatment, the bathing solution was carefully sucked out by two syringes and replaced with normal KH solution. These maneuvers were repeated seven times. The whole procedure took <15 min.Measurement of [Ca2+]i in isolated colonic crypts. For the isolation of colonic crypts, the distal colon was exposed to a Ca2+ chelating solution [composition (in mM): 96 NaCl, 1.5 KCl, 10 HEPES, 27 Na EDTA, 45 sorbitol, and 28 sucrose] for 30 min at room temperature. A pellet of isolated crypts was formed by centrifugation at 200 rpm for 1 min. The isolated crypts were resuspended in the normal KH solution.
The isolated crypts bathed in normal KH solution were attached to the glass coverslips (Fisher Scientific, Pittsburgh, PA) precoated with Cell-Tak (Becton-Dickinson). The crypts were then loaded with fura 2-AM, a Ca2+-sensitive fluorescent dye, by incubation (45 min, 37°C) in normal KH solution containing 3 µM dye. The coverslip was mounted in a temperature-controlled perfusion chamber (Warner Instrument) on the stage of an inverted microscope (Nikon TE300). Fura 2 fluorescence ratio (340/380 nm) was recorded by the PTI Ratio-Master fluorescence system (Photon Technology International, Lawrenceville, NJ). The cells were continuously superfused with normal KH solution. Changes in [Ca2+]i elicited corresponding changes in the fura 2 fluorescence ratio, and this allowed changes in [Ca2+]i to be monitored using a standard microspectrofluorimetric technique (18).Measurement of cAMP.
Cytosolic cAMP concentrations were measured by RIA. After a 30-min
period of equilibration in normal KH solution at 37°C, the isolated
mucosal sheet was treated with DMSO, IBMX, baicalein, forskolin with
IBMX, baicalein with forskolin and IBMX, and PGE2 with
IBMX. The mucosal sheets were further incubated for 2 min and then
rapidly frozen in liquid nitrogen and stored at 70°C until
homogenized in 0.5 ml of ice-cold 6% trichloracetic acid using a glass
homogenizer. The homogenate was centrifuged at 2,000 g for
10 min at 4°C. The supernatant was extracted three times with three
volumes of diethyl ether before lyophylization. The amount of cAMP was
determined by RIA with a 125I-labeled cAMP RIA kit
(Amersham Pharmacia Biotech, Little Chalfont, England). The tissue
residue was dissolved in 2 M NaOH, and protein content was determined
using a protein assay kit (Sigma, St. Louis, MO) with bovine serum
albumin as the standard. The concentration of cAMP was presented as
picomoles per milligram of protein.
Solutions.
The normal KH solution has the following compositions (in mM): 117 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 24.8 NaHCO3, 2.56 CaCl2, and 11.1 D-glucose. The solution was
continuously bubbled with 95% O2 and 5% CO2
to maintain the pH at 7.4. Cl-free solution was prepared
by isosmotically replacing NaCl and KCl with Na gluconate and K
gluconate, respectively. CaCl2 was replaced with 11 mM Ca
gluconate to counteract the chelation effect of gluconate anion.
NaHCO3 was also replaced by HEPES in the Cl
-
and HCO
Chemicals.
The following drugs were used: baicalein, forskolin, atropine, TTX,
carbachol (CCh), IBMX, indomethacin, acetazolamide, -conotoxin GVIA
(
-CTX), PGE2, glibenclamide, bumetanide (Sigma),
diphenylamine-2-carboxylic acid (DPC), DIDS, thapsigargin (Calbiochem,
La Jolla, CA), and Cell-Tak. Chromanol 293B was a generous gift from
Dr. H. J. Lang (Aventis Pharma Deutschland, Frankfurt, Germany).
Stock solutions of all the chemicals were dissolved in DMSO. Final DMSO
concentrations never exceeded 0.1% (vol/vol). Preliminary experiments
indicated that the vehicle did not alter any baseline
electrophysiological parameters (data not shown).
Data analysis.
For Isc measurements, positive currents are
defined as those that would be carried by anions moving from the
serosal to mucosal compartments and are shown as upward deflections of
the traces. Changes in Isc
(Isc) were quantified by subtracting the
current flowing at the peak of a response from its respective baseline values, which is the current flowing immediately before drug administration.
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RESULTS |
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Effect of baicalein on baseline Isc.
The basal Isc and Rt in
these tissues were 43.1 ± 3.4 µA/cm2 and 106 ± 6 · cm2 (n = 80),
respectively. Subsequent addition of baicalein to the mucosal
side induced a triphasic Isc response. As shown
in Fig. 1A, 30 µM baicalein
stimulated a transient increase in Isc up to
8.5 ± 1.7 µA/cm2 above baseline within one-half
minute, but subsequently the Isc returned below
prestimulated level with an average of 2.4 ± 1.8 µA/cm2 (n = 8). Afterwards, baicalein
evoked a second, more sustained increase in Isc
(15.9 ± 2.4 µA/cm2). Rt was
slightly reduced from 126.3 ± 11.0 (before baicalein) to
115.2 ± 11.0
· cm2 (at the second peak),
but the changes were statistically significant (P < 0.05).
Addition of DMSO (up to 1% vol/vol) alone to the mucosal and serosal
sides did not change the basal Isc
(n = 8). In some of the preparations, however,
baicalein induced a monophasic Isc increase (34 of 149; Fig. 1D) or biphasic Isc
response (72 of 149; Fig. 1C), which only consisted of an
initial peak followed by a more sustained Isc.
In these cases, the maximal baicalein-induced Isc increases were used to compare between
control and treated groups. The cumulative concentration-response
curves of baicalein-induced Isc are shown in
Fig. 1B. The apparent EC50 of baicalein is
16.7 ± 3.0 µM (Fig. 1B).
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Effect of TTX and atropine on baicalein-evoked increase in
Isc.
To test the effect of the neuronal blocker TTX on baicalein-evoked
Isc, the colon was first stimulated with 30 µM
mucosal baicalein. Afterwards, the Isc was
reversed back to basal level on washout of baicalein. The colon was
then stimulated again with baicalein in the absence or presence of 1 µM serosal TTX (Fig. 2, A
and C). Serosal addition of TTX did not significantly alter basal Isc (Fig. 2C). In control
experiments (Fig. 2B), the baicalein-induced increase in
Isc did not differ significantly with the second
response after washout (first peak, 5.4 ± 1.7 µA/cm2; second peak, 22.5 ± 3.5 µA/cm2; after washout: first peak, 2.9 ± 1.0 µA/cm2; second peak, 33.0 ± 6.2 µA/cm2; n = 10, P > 0.05). In the presence of TTX (Fig. 2D), baicalein induced
an increase of Isc (first peak, 2.8 ± 1.3 µA/cm2; second peak, 43.1 ± 13.9 µA/cm2; n = 4), which was not different
from the first baicalein-induced Isc responses
(first peak, 7.4 ± 1.5 µA/cm2; second peak,
31.0 ± 8.6 µA/cm2; P > 0.05). The
TTX-dependent changes of Isc were compared with the second control responses (after mock washout), and there was no
statistical significance (P > 0.05) between the two
groups of data. In addition, the neuronal Ca2+-channel
blocker -CTX was used.
-CTX (0.5 µM) reduced the basal Isc by 7.2 ± 2.2 µA/cm2 but
did not affect the subsequent baicalein-evoked
Isc increase (control: 22.7 ± 2.8 µA/cm2, n = 5; after
-CTX: 20.0 ± 2.4 µA/cm2, n = 4; P > 0.05).
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Ionic basis of baicalein-evoked Isc.
The increase in Isc could be attributed to
either enhanced movement of positive charges from the luminal to the
serosal side (i.e., Na+) or movement of negative charges
into the lumen (i.e., Cl and/or HCO
in the bathing solutions was
replaced by gluconate, mucosal application of baicalein (100 µM) only
evoked a maximal increase in Isc up to 6.8 ± 2.2 µA/cm
2 (n = 7). The response
that persisted under these conditions was concentration dependent and
was not abolished when both Cl
and HCO
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Involvement of prostanoid synthesis.
PGs are potent stimulators of intestinal secretion (24).
Therefore, the effect of PG synthesis inhibitor indomethacin on baicalein-induced Isc increase was determined.
Figure 5A shows that mucosal
addition of indomethacin (10 µM) reduced the basal Isc from 26.1 ± 3.3 to 8.9 ± 3.0 µA/cm2 (n = 4). The baicalein-evoked
first Isc response was absent after indomethacin
treatment. Indomethacin also significantly reduced the baicalein-evoked
second peak increase in Isc to 3.1 ± 0.7 µA/cm2 from the control value of 31.7 ± 6.6 µA/cm2 (P < 0.05, Fig. 5B).
Indomethacin increased the Rt from 87.5 ± 10.2 to 99.0 ± 12.6 · cm2
(n = 9). The increase in Rt
caused by indomethacin was statistically significant (P > 0.05). However, the inhibitory effect of indomethacin could be due
to the fact that baicalein depends on a basal activity of CFTR, which
is maintained by PGs. In Fig. 5C, after the first baicalein
response was washed out, intracellular cAMP level was enhanced by the
stimulator of the adenylate cyclase forskolin (0.3, 1, and 3 µM,
mucosal) in the presence of indomethacin (10 µM, mucosal). After the
cAMP-dependent pathway was activated, the Isc
was restored to the level before the first addition of baicalein. Under
such conditions, 30 µM baicalein evoked an Isc of 23.9 ± 4.3 µA/cm2, which is not statistically
significant from the first baicalein-evoked Isc
responses (
Isc = 16.4 ± 4.9 µA/cm2, n = 4, P > 0.05). These data indicate that the inhibitory effect of indomethacin
can be overcome by elevating intracellular cAMP. Figure
6A shows that, in the presence
of 10 µM indomethacin, exogenous application of PGE2 (100 µM, serosal) stimulated an increase in Isc of
80.0 ± 7.7 µA/cm2 (n = 9), which is
not statistically different from the control responses without
indomethacin (64.0 ± 15.7 µA/cm2, n = 7, P > 0.05). After a maximal concentration of
PGE2 (
Isc = 45.3 ± 7.0 µA/cm2), however, the Isc response
to baicalein was reduced significantly from 27.2 ± 4.2 to
13.1 ± 4.8 µA/cm2 (Fig. 6B,
n = 5, P < 0.05). Therefore, we cannot
completely rule out the possibility that PG release is involved in
mediating the baicalein-induced secretion.
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Effect of K+-channel blockers.
The basolateral membrane K+ channels are important in
maintaining the driving force for cAMP-dependent Cl
secretion. Forskolin (1 µM, mucosal) evoked a sustained increase in
Isc of 52.2 ± 10.7 µA/cm2
(n = 7, Fig.
7A). Serosal addition of
chromanol 293B (30 µM), a blocker of KvLQT1 channels,
inhibited the Isc by 70%, indicating that
forskolin activated a 293B-sensitive K+ channel. Similarly,
baicalein-induced Isc responses were inhibited by serosal addition of chromanol 293B (Fig. 7B,
n = 6) to a similar degree. Figure 7C shows
that the baicalein-induced Isc was completely inhibited by the serosal application of Ba2+ (5 mM,
n = 9). When added before baicalein, 293B reduced the basal current by 6.4 ± 1.3 µA/cm2. Subsequent
application of baicalein only evoked an increase in
Isc of 12.6 ± 3.0 µA/cm2
(n = 6; control, 24.4 ± 2.1 µA/cm2,
n = 5, P < 0.05). In the presence of
293B, baicalein did not evoke any triphasic current response. Two of
six responses were biphasic, and the rest were monophasic. On the other
hand, the Ca2+-activated K+-channel blocker
charybdotoxin (100 µM, serosal) was without effect (n = 6, data not shown). Adding Ba2+ (5 mM) to the serosal
bath also reduced the baicalein-evoked Isc from
21.4 ± 2.1 to 5.7 ± 1.0 µA/cm2
(n = 6). Therefore, activation of Ba2+- or
293B-sensitive K+ channels is important to maintain
Cl
secretion stimulated by baicalein.
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Interaction of baicalein with CCh, thapsigargin, and forskolin.
As shown in Fig. 8A, serosal
application of the calcium secretagogue CCh (100 µM) to the rat colon
evoked a transient fall in Isc
(Isc = 82.2 ± 7.2 µA/cm2) followed by an increase in positive
Isc (
Isc = 145.2 ± 22.7 µA/cm2). Subsequent application of
mucosal baicalein (30 µM) only evoked a small increase in
Isc of 9.6 ± 5.2 µA/cm2,
whereas application of forskolin (1 µM, mucosal) further increased the Isc by 35.9 ± 9.4 µA/cm2. In contrast, a maximal dose of forskolin (30 µM) elicited an Isc response of 65.8 ± 14.8 µA/cm2 (Fig. 8B) and addition of
baicalein (30 µM) subsequent to forskolin induced a further
Isc of 35.0 ± 8.5 µA/cm2
(n = 4). The increase in Isc is
not statistically different from the control responses
(
Isc = 28.6 ± 4.0 µA/cm2, n = 4, P > 0.05). In the presence of forskolin, baicalein did not produce any
triphasic current response and all the responses were monophasic.
Although these results strongly suggest that baicalein and CCh share a
common intracellular pathway (i.e., a rise in
[Ca2+]i), Fig. 8C shows that
thapsigargin, a microsomal Ca2+-ATPase inhibitor, elicited
a sustained increase in Isc
(
Isc = 39.1 ± 5.1 µA/cm2, n = 6). When the current had
reached a plateau, mucosal baicalein produced an
Isc response (
Isc = 38.3 ± 3.6 µA/cm) that was not significantly different from
that produced by the same concentration of baicalein
(
Isc = 34.1 ± 3.7 µA/cm2, n = 6, P > 0.05)
without prior treatment of thapsigargin. As shown in Fig.
8D, thapsigargin stimulated a sustained increase in
Isc (
Isc = 57.9 ± 11.0 µA/cm2) and inhibited the subsequent
CCh-evoked positive Isc by 80% (
Isc = 25.4 ± 8.0 µA/cm2). Afterwards, the Isc
decayed toward its basal level, and the subsequent baicalein-evoked
Isc (
Isc = 4.1 ± 1.3 µA/cm2) was inhibited (n = 7, P < 0.05 compared with baicalein-evoked Isc shown in Fig. 8C). It therefore
appears that the baicalein-induced Isc was
inhibited only after prior treatment of the colonic mucosa with CCh,
but this is not due to its Ca2+-mobilizing effect.
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Involvement of cAMP and
[Ca2+]i.
RIAs were performed to study the effects of baicalein on intracellular
cAMP levels in the colonic mucosa, and the results are shown in Fig.
9A. The intracellular cAMP
content under basal conditions with and without IBMX (100 µM, both
sides) was 1047.2 ± 102.2 (n = 7) and 1676.3 ± 357.4 pmol/mg protein (n = 7), respectively. After
incubation with mucosal baicalein (30 µM) in the presence of IBMX,
there was approximately a threefold increase in cAMP level (6273.0 ± 1872.6 pmol/mg protein, n = 6). The difference was
statistically significant (P < 0.05). Without IBMX,
baicalein alone did not evoke a significant increase in cAMP content.
In the presence of IBMX, forskolin (1 µM, mucosal) and
PGE2 (10 µM, serosal), serving as positive controls,
stimulated a rise in cAMP level to 11152.6 ± 949.9 (n = 5) and 11828.3 ± 1304.0 pmol/mg protein
(n = 6), respectively. Baicalein cannot produce further increases in cAMP content above forskolin and IBMX. For the increases in Isc, the effect of baicalein was potentiated
by more than fivefold in the presence of IBMX
(Isc = 114.6 ± 14.5 µA/cm2, n = 8 vs. control = 21.8 ± 2.5 µA/cm2, n = 5;
P < 0.05). Together, the data suggest that degradation of intracellular cAMP by phosphodiesterase(s) is one of the limiting factors controlling baicalein-evoked current responses.
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DISCUSSION |
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Baicalein is the major constituent and flavonoid derived from S. radix. It has a wide range of pharmacological activities such as anti-inflammatory effects (22), inhibition of platelet lipoxygenase (28), and antitumor effects on human cancer cell lines (16). Recently, it has been shown that baicalein reduces arachidonic acid liberation and PGE2 release through inhibition of the mitogen-activated protein kinase and cytosolic phospholipase A2 pathway in rat C6 glioma cells (26). Because S. radix- and C. rhizoma- derived compounds are frequently used together to treat diarrhea diseases in the East and PGE2 production in the digestive tract is closely associated with the onset of diarrhea (2), it would be expected that baicalein would be an antisecretory agent.
However, the findings of this study indicate that baicalein induced a
Cl-dependent secretory response in rat colon in vitro.
This is supported by the effect of Cl
ion replacement in
the bathing solutions, which greatly attenuated the
Isc. Removal of both Cl
and
HCO
secretion.
The increase in Isc was not due to electrogenic
Na+ absorption, because it was not inhibited by the ENaC
blocker, amiloride. In this study, the baicalein-evoked
Cl secretion was completely inhibited by DPC but not
DIDS, suggesting that CFTR is the likely candidate for apical
Cl
exit. DPC is known to have an effect on the
cAMP-dependent Cl
channel in various epithelia
(9). It has been shown that up to 500 µM DIDS has no
effect on the activity and conductance of CFTR (11).
Therefore, the insensitivity of the baicalein responses to 500 µM
DIDS (Fig. 4, B and C) is characteristic of
CFTR-mediated Cl
secretion, and so our data confirm that
the involvement of Ca2+-activated Cl
channels
in Cl
secretion in native colonic epithelium is unlikely
(12, 30). Application of CFTR blocker glibenclamide
inhibited the baicalein-evoked Isc by 61%,
further suggesting that Cl
exit through luminal CFTR.
The activation of basolateral K+ channels is essential in
driving apical Cl exit. The baicalein-evoked
Isc responses were inhibited by a nonspecific
K+-channel blocker, Ba2+, and a specific
blocker of the cAMP-activated KvLQT1 K+
channel, chromanol 293B (23). Chromanol 293B was also
effective in blocking forskolin-evoked Isc
responses. Similar findings have been reported in both human
(24) and rabbit colon (23). In T84 cells
(6) and rat colon (25), 293B selectively
blocks basolateral cAMP-dependent K+ conductance, leading
to inhibition of forskolin-dependent Cl
secretion.
Recently, the cAMP-activated KvLQT1 K+ channel
in rat colonic epithelial cells has been cloned. The K+
current activated by forskolin is blocked by 293B (20).
Although 293B inhibited a significant portion (70%) of the forskolin-
and baicalein-evoked Isc, a substantial
293B-insensitive Isc still remained (Fig. 7,
A and B). This may be due to basal
Cl
secretion driven by other basolateral K+
conductances similar to that reported in human bronchial epithelia (5). By using the Isc technique, it
is difficult to simply use different K+-channel blockers to
delineate which class of K+ channels is activated in each
phase of the baicalein responses. Further experiments using patch-clamp
recordings on isolated colonic crypts could be used to answer this
question. Moreover, the biphasic response to CCh is consistent with the
activation of apical K+ conductance. It is likely that CCh
stimulated a rise in [Ca2+]i and activated
the luminal Ca2+-sensitive K+ channels. The
transient increase in K+ secretion was then overridden by
the apical Cl
conductance. Similar results have been
shown in human colon, in which the Isc, due to
activation of apical K+ conductance, is masked by the
parallel activation of luminal Cl
channels, resulting in
lumen-negative Isc (24).
In the present study, the baicalein-induced Isc
increase was not inhibited by the specific neuronal blocker TTX,
neuronal Ca2+-channel blocker -CTX, or the muscarinic
acetylcholine-receptor antagonist atropine, suggesting that in rat,
colonic acetylcholine-containing submucosal neurons are not involved in
the mediation of baicalein-induced secretion. This is in contrast with
angiotensin II- and substance P-evoked Cl
secretory
response, which involves submucosal cholinergic neurons (13,
21). Therefore, baicalein may act directly on epithelial or
subepithelial cells in the mucosa to evoke Cl
secretion,
which involves PG synthesis. The present finding that the
baicalein-evoked response was attenuated by indomethacin, a
cyclooxygenase inhibitor, suggests the importance of PGs in baicalein-evoked secretory response. Indomethacin inhibits
Cl
secretion in stripped preparations of rat colon
(30). In this study, indomethacin reduced the basal
Isc by 64% and the baicalein-evoked Isc by 90% compared with the control. The
result suggests that there is an ongoing basal PG tone in the tissue,
and the baicalein-evoked Cl
secretion requires the PG
synthesis pathway in rat colon. After addition of indomethacin, there
was a fall in Rt, further indicating a role of
basal PG release on Cl
secretory activity of the colon.
Pretreating the mucosal layers with a maximal concentration of
PGE2 reduced the Isc response to
baicalein (Fig. 6B). Therefore, the possibility that the
baicalein-induced secretion is mediated, at least in part, by PG
release cannot be excluded.
Although it is likely that the Isc response of
the colonic mucosa to baicalein was mediated by an increase in cellular
cAMP level (Fig. 9A), which then activated the apical CFTR
channels and basolateral K+ conductances, not all of the
data were consistent with this hypothesis. Experiments on the
additivity of forskolin and baicalein on the cAMP pathway revealed that
even after a maximal stimulation of cAMP-dependent secretion by
forskolin, baicalein was still able to further increase the
Isc (Fig. 8B). Therefore, the
involvement of other second messengers cannot be excluded. Moreover,
the inhibitory effect of indomethacin on baicalein-evoked
Isc could be overcome by increasing the cellular
cAMP level by forskolin (Fig. 5C). The effect of baicalein
is therefore similar to that of the Ca2-dependent
secretagogue CCh. It has been previously reported that the effect of
CCh depends on PG synthesis, because the basal activation of CFTR by
cAMP is a prerequisite of Cl secretion (3,
24). Moreover, in the RIA study, baicalein increased cAMP
content only in the presence of IBMX. In the basal condition (control)
and after a maximal stimulation of adenylate cyclase activity (IBMX and
forskolin), addition of baicalein indeed reduced the cAMP levels. At
the moment, we do not have an explanation for this observation. The
possibility exists that multiple signaling pathways might be involved
in mediating the baicalein response and thus permitting the inhibition
of adenylate cyclase activity. However, this awaits further
investigation. Experiments should be conducted to access whether
baicalein exerts control over adenylate cyclase activity and/or
phosphodiesterase(s) activity under different conditions. Nonetheless,
the data further support that the Isc increase
induced by baicalein after forskolin (Fig. 8B) was not due
to a rise in cAMP levels (i.e., cAMP-independent effect). However, bear
in mind that, in this study, RIA of extracted cAMP at one time point
(2-min incubation) was performed. It is difficult to correlate exactly
the rise in cAMP content (static accumulated levels) to the increase in
Isc, which reflects dynamic and continuous changes due to the activation of cAMP-dependent cascades. In addition, baicalein evoked a more sustained increase in
Isc (tracings not shown) in the presence of
IBMX, suggesting that degradation of cAMP by phosphodiesterase(s) is
one of the limiting factors controlling baicalein-evoked current responses.
To elucidate the role of Ca2+ as a second messenger, the
[Ca2+]i of the isolated colonic crypts was
measured. In contrast to the effect of CCh (Fig. 9B),
baicalein did not evoke any discernible increase in
[Ca2+]i. Charybdotoxin, a
Ca2+-dependent K+-channel blocker, also
produced no noticeable effect on baicalein-induced Cl
secretion. In addition, the inhibitory effect of CCh on
baicalein-induced Isc appears to be independent
of [Ca2+]i. Thapsigargin, a
microsomal Ca2+-ATPase inhibitor that leads to the
depletion of Ca2+ store (31), did not affect
the baicalein-induced Isc. Thapsigargin, however, disrupted the mechanism permitting receptor-mediated control
over [Ca2+]i and inhibited the subsequent
effect of CCh. The results support the notion that intracellular
Ca2+ plays a negligible role in the inhibitory effect of
CCh. Several laboratories have investigated inhibitory influences of
CCh in human colonic epithelial cells (T84). For example, it has been shown that the inhibitory effect of CCh is attributable to a sustained elevation in inositol tetrakisphosphate, which exerts a direct inhibitory effect on the open probability of CaCC (17,
36). However, our data do not support the existence of CaCC in
the rat colon. Therefore, it is unlikely that CCh inhibited
thapsigargin-evoked Isc increase through this
mechanism. Another possibility is that CCh and baicalein may share a
common and yet unidentified intracellular second-messenger pathway.
Together, there is no direct evidence to suggest that the effect of
baicalein is mediated through an increase in
[Ca2+]i. The mechanism underlying the
inhibitory effect of CCh on baicalein-evoked Isc
remains to be elucidated and awaits further investigation. Another
possibility of a cAMP-independent action of baicalein is its direct
effect on CFTR. It has been suggested that certain flavonoids such as
quercetin may directly activate CFTR in rat colon independent of
intracellular cAMP level (4).
In traditional remedies, C. rhizoma and S. radix
are commonly used together to treat gastrointestinal diseases such as
diarrhea. Recently, it has been shown that the antisecretory mechanism
is due to the blocking effect of berberine, which is the major
constituent of C. rhizoma, on basolateral K+
conductance in colonic epithelia (33). It is interesting
that baicalein, in contrast to berberine, is a prosecretory compound. Although the presence of baicalein may counteract and limit the effectiveness of berberine in the treatment of diarrhea, baicalein may
have modulatory effects on the antisecretory action of
berberine-containing herbs. This may be important to maintain a basal
Cl secretion for lubrication of the mucosal surface layer
and for the flushing of intestinal contents during host defense against microbial invasions or artificial irritants.
In summary, the present study has demonstrated that Cl
secretion across the rat colonic mucosa could be stimulated by mucosal baicalein. Mechanisms involve luminal cAMP-dependent Cl
channels and serosal 293B-sensitive K+ channels. However,
the action of baicalein cannot be solely explained by its
cAMP-elevating effect. Baicalein may stimulate Cl
secretion via a cAMP-independent pathway or have a direct effect on CFTR.
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ACKNOWLEDGEMENTS |
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This work was supported by direct grant for research from the Chinese Univ. of Hong Kong (Ref. no. 2001.1.088) awarded to W. H. Ko.
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. H. Ko, Dept. of Physiology, The Chinese Univ. of Hong Kong, Shatin, N.T., Hong Kong, China (E-mail: whko{at}cuhk.edu.hk).
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.
10.1152/ajpgi.00291.2001
Received 3 June 2001; accepted in final form 16 November 2001.
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