Departments of 1 Physiology and Biophysics and 2 Pediatrics, University of Illinois at Chicago, Chicago, Illinois 60612-7342
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Taurodeoxycholic acid (TDC) stimulates Cl transport in
adult (AD), but not weanling (WN) and newborn (NB), rabbit colonic epithelial cells (colonocytes). The present study demonstrates that
stimuli like neurotensin (NT) are also age specific and identifies the
age-dependent signaling step. Bile acid actions are segment and bile
acid specific. Thus although TDC and taurochenodeoxycholate stimulate
Cl
transport in AD distal but not proximal colon,
taurocholate has no effect in either segment. TDC increases
intracellular Ca2+ concentration
([Ca2+]i) in AD, but not in WN and NB,
colonocytes. In AD cells, TDC (5 min) action on Cl
transport needs intra- but not extracellular Ca2+. NT,
histamine, and bethanechol increase Cl
transport and
[Ca2+]i in AD, but not WN, distal
colonocytes. However, A-23187 increased [Ca2+]i and Cl
transport in all
age groups, suggesting that Ca2+-sensitive Cl
transport is present from birth. Study of the proximal steps in
Ca2+ signaling revealed that NT, but not TDC, activates a
GTP-binding protein, G
q, in AD and WN cells. In
addition, although WN and AD colonocytes had similar levels of
phosphatidylinositol 4,5-bisphosphate, NT and TDC increased
1,4,5-inositol trisphosphate content only in AD cells.
Nonresponsiveness of WN cells to Ca2+-dependent stimuli,
therefore, is due to the absence of measurable phospholipase C
activity. Thus delays in Ca2+ signaling afford a crucial
protective mechanism to meet the changing demands of the developing colon.
taurodeoxycholic acid; neurohumoral modulators; rabbit colon; inositol trisphosphates
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN ADDITION TO ITS
PROMINENT ROLES in the reabsorption of fluid and electrolytes and
in the production of short chain fatty acids and secondary bile acids,
the colon secretes salt and water. Salt secretion, which is necessary
for maintaining fluidity, is governed by cellular Cl
transport processes, and the balance between colonic salt secretion and
absorption is carefully controlled by the coordinated action of
numerous systemic and luminal modulators. Perturbation of the balance
can lead to net Cl
, fluid secretion, and diarrhea
(10). Hormones and neurotransmitters that utilize
Ca2+ as a second messenger are often involved in modulating
processes that require minute by minute regulation, including that of
epithelial fluid transport. These processes are especially useful in
the gastrointestinal tract, where the epithelium has to cope with continuous fluctuations in the luminal milieu. These luminal challenges are heightened in the young animal, especially at parturition and
weaning (24). Ontogenic changes in nutrient transporters have been well documented (24), but there are only a few
studies that examine regulation of ion transport during development in the small intestine and colon (14, 21, 38, 41,
47-49).
In a variety of adult secretory epithelia, Cl secretion
occurs by the concerted action of the
Na+-K+-2Cl
cotransporter,
Na+-K+-ATPase, and K+ channels on
the basolateral membrane and Cl
channels such as cystic
fibrosis transmembrane conductance regulator and/or
Ca2+-activated Cl
channels on the apical
membrane (1). Neurohumoral agents that increase
intracellular Ca2+ concentration
([Ca2+]i) or cAMP stimulate intestinal
Cl
secretion (10).
Ca2+-dependent agents activate receptor-coupled G proteins
linked to stimulation of phospholipase C (PLC) (55). PLC
activation results in hydrolysis of phosphatidylinositol
4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and
1,4,5-inositol trisphosphate (IP3) (4). DAG
activates protein kinase C (PKC), and IP3 mobilizes stored
Ca2+ (4).
Regulation of [Ca2+]i may be more critical in the developing animal when key neurohumoral changes occur. Some studies report age-related differences in Ca2+ uptake into endoplasmic reticulum and in specific signaling proteins involved in the Ca2+ cascade, such as IP3 receptors and phosphatidylinositol kinase (15, 22, 23, 37, 56, 62, 68), but only a few demonstrate a link between biochemical differences and function. In rat brain, while the metabolism of IP3 by IP3 3'-kinase and IP3 5'-phosphatase increases during development, its functional ramifications are unknown (23, 37, 68). In contrast, the reduced contractile response to cholinergic stimulation in kitten gastric smooth muscle compared with the cat has been linked to a decrease in IP3 receptor density (15).
An intriguing example of developmental regulation in terms of
Cl secretion occurs with respect to bile acids
(47). In a healthy adult mammal, >95% of the bile acid
pool is reabsorbed in the distal ileum by Na+-dependent
bile acid transporters and recycled to the liver via enterohepatic
circulation (65). However, in the case of ileal malabsorption of bile acids, excessive production of secondary bile
acids by colonic bacteria elicits net Cl
secretion and
diarrhea. In the neonatal rabbit, although the ileum does not have a
bile acid uptake mechanism and the bile acid levels are similar to the
adult, no bile acid diarrhea is observed (47). In colonic
preparations comprising the epithelial, subepithelial, and muscle
layers, bile acids stimulated electrogenic Cl
transport
only in adult, but not in neonatal, rabbit distal colon (41,
48).
The taurodeoxycholic acid (TDC) signaling mechanism in stimulating
colonic Cl transport is unclear because different
processes have been implicated. In the rat (50) and mouse
distal colon (20), roles for paracrine mediators, such as
prostaglandins (PG) and histamine, respectively, secreted by the
epithelial and subepithelial components, have been demonstrated. In the
human colon carcinoma cell line HT-29, Huang et al. (29)
showed that bile acids act via PKC and suggested that the bile acids
may substitute for phosphatidyl serine. In T84 cells, Devor et al.
(16) reported that bile acids act by increasing
IP3 production and [Ca2+]i but do
not require extracellular Ca2+
([Ca2+]o) for their initial effect. In
contrast, Dharmsathaphorn et al. (17) reported that
TDC-induced Cl
secretion was reduced in the absence of
[Ca2+]o. In adult rabbit, Freel
(18) suggested that bile acids act by altering
paracellular permeability. In the only study on bile acid mechanisms in
the developing colon, TDC was shown to increase intracellular cAMP in a
Ca2+-dependent manner in adult but not in neonatal rabbits
(49). Thus TDC appears to act via cAMP, Ca2+,
PKC, histamine, and/or a combination thereof, and any of them could be
developmentally regulated.
In a colonic developmental model, this laboratory has shown that
newborn, weanling, and adult rabbit colonocytes exhibit basal Cl transport and respond to PGE1, forskolin,
8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), or the PKC
activator phorbol dibutyrate (PDB) (14). Thus all the
major steps of the cAMP cascade and the steps leading from
[Ca2+]i increases and PKC activation to
Cl
transport are operative from an early age in rabbit.
However, TDC stimulated Cl
transport only in the adult
but not in weanling or neonatal rabbit colonocytes (14, 40,
48).
In the present study, we demonstrate that the stimulation of
Cl transport in adult rabbit is bile acid and segment
specific. We also show that other Ca2+-dependent
neurohumoral agents exhibit age-specific responsiveness similar to that
of bile acids. We have explored the cellular basis of these differences
in adult and weanling rabbit colonocytes. We report that this
age-related difference is due to the inability of bile acids and other
Ca2+-dependent agents to increase
[Ca2+]i in the distal colonocytes of young
rabbits. The age-dependent regulation is not at the level of G protein
activation but lies at the level of PLC activity.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials
Materials were purchased from the following sources: 6-methoxy-quinolyl acetoethyl ester (MQAE), fura 2, fura 2-AM, and Pluronic F-127 (Molecular Probes, Eugene, OR); Ham's F-12 nutrient mixture, fetal calf serum (FCS), PKC and cAMP assay kits (Life Technologies, Grand Island, NY); sterile lactated Ringer (Baxter Health Care, Deerfield, IL); diphenylamine-2-carboxylate (DPC; Aldrich, Milwaukee, WI); [Tissue Procurement and Cell Isolation
New Zealand White adult (6 mo old), weanlings (25-28 days old), and newborn (7-9 days old) rabbits were procured from Lesser Rabbits (Delfield, WI) and housed at the institutional Biological Resources Laboratory (according to guidelines of the American Association for Accreditation of Laboratory Animal Care). Animal protocols were approved by the Institutional Animal Care Committee. Proximal colon, from the ileocecal valve to the hepatic flexure, and distal colon, from the anal verge to the splenic flexure, were excised and the mucosa separated from underlying muscle by blunt dissection and subjected to enzymatic digestion, followed by serial centrifugation as described earlier (14, 61). This yields a pellet fraction of colonocytes that is crypt enriched (3) and cultured overnight, in suspension, in a Ham's F-12 nutrient medium containing supplements as previously described (3, 54). The primary cultures of colonocytes are chiefly epithelial in origin as visualized by cytokeratin staining (54). The colonocyte preparation used in our studies is a mixture of crypts that contain clusters of "polarized" cells and clusters of single cells. Tissue from all newborn animals from one litter (~6 pups) was pooled to yield sufficient colonocytes. For the same reason, tissue from two weanling animals were pooled. For adults, tissue from a single rabbit yielded sufficient colonocytes for each experiment. All functional studies used colonocytes cultured overnight.Cl Transport
The secretagogue doses were selected based on earlier studies in rabbit
colonocytes, and concentrations used represented the near maximal dose
of each secretagogue (14). To study the effects of bile
acids, TDC (1-200 µM), taurochenodeoxycholic acid (TCDC; 50 µM), and taurocholic acid (TC; 50 µM) were used. Forskolin (1 µM)
or PGE1 (1 µM) was used to assess cAMP-stimulated
Cl transport. The Ca2+ ionophore A-23187 (1 µM) was used to study the Ca2+ signaling pathway.
Indomethacin (1 µM) was used to determine the involvement of PG in
bile acid-stimulated Cl
transport.
In studies where extracellular Ca2+ was depleted,
MQAE-loaded, Cl-depleted cells in buffer B
were centrifuged and transferred to buffer C that contained
(in mM) 5 HEPES (pH 7.4), 110 sodium isethionate, 1 MgSO4,
5 dextrose, 50 mannitol, 1 K2SO4, 0.01 CaSO4, and 1 EGTA for 10 min before fluorescence was
measured. To chelate [Ca2+]i, cells loaded
with MQAE were incubated in buffer that contained 25 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for 20 min at room temperature, centrifuged, and resuspended in
a Ca2+-free buffer. Cl
transport was measured
in the presence of secretagogues.
[Ca2+]i Measurements
The AM of fura 2 was used to measure [Ca2+]i using a modification of our earlier protocol (58). Briefly, colonocytes were resuspended in 1 ml of buffer D that contained (in mM) 110 NaCl, 5 KCl, 5 dextrose, 5 K2(S2O5), 10 Tris (pH 7.4), 30 mannitol, and 0.1 CaCl2. FCS (50 µl) + 20% Pluronic F-127 (1.25 µl) + fura 2-AM (2 µM) were added to 2 ml cell suspension and incubated at 37°C for 90 min. The cells were centrifuged to remove extracellular fura 2 and resuspended in buffer D + 1 mM CaCl2. [Ca2+]i in the isolated colonocyte population is reported as a fluorescence ratio (ExIntracellular cAMP Concentration Measurements
Adult distal colonocytes (24-h cultures) were treated with either TDC (50 µM) or forskolin (1 µM) for 5 min. cAMP content in cells was measured using the Life Technologies enzyme-linked immunoassay kit as per the manufacturer's protocol and expressed as fmol/106 cells.PKC Activity
Adult distal colonocytes were incubated with buffer alone, TDC (50 µM), or PDB (1 µM) for 5 min. Cells were frozen and rapidly homogenized in a buffer (HB) that contained (in mM) 10 HEPES-Tris (pH 7.4), 3 EGTA, 10 mannitol, 0.1 phenylmethylsulfonyl fluoride, 2 dithiothreitol (DTT), and 0.001 mg/ml of leupeptin, pepstatin, and aprotinin, followed by sonication (5-s pulse; 20 Hz). The homogenate was sequentially centrifuged at 2,000 g for 5 min and 100,000 g for 30 min. The resultant supernatant and pellet were the cytosol and membrane fractions, respectively. The membrane fraction was solubilized in HB that contained 0.5% Triton X-100. Cytosol and the solubilized membrane fraction were partially purified using a DEAE cellulose column. The DEAE column was scaled down by a factor of three due to low-PKC content (see RESULTS). PKC activity of the samples was measured with a commercially available kit (Life Technologies) specific for the Ca2+-dependent isoforms of PKC. A PKC consensus sequence-containing peptide was used as the substrate, and a pseudosubstrate was used to determine specific activity.IP3 Measurement
The kit allowed for measurement of IP3 and PIP2 levels. Colonocytes, from one adult or pooled from two weanling rabbits, were incubated with buffer alone, bethanechol (1 or 10 µM), neurotensin (1 or 10 µM), TDC (50 µM), or PGE1 (1 µM) for 5 min at room temperature. The incubation was terminated with an equal volume of ice-cold 15% (vol/vol) TCA. With the use of the manufacturer's protocol, IP3 was recovered in the soluble fraction and PIP2 from the TCA-insoluble pellet. PIP2 in these crude lipid extracts was converted to IP3 by alkaline hydrolysis. The recovery of IP3 from PIP2 was 81% and factored into the calculations for PIP2 amount. The IP3 released by alkaline hydrolysis was directly proportional to the total picomoles of PIP2 in the lipid pellet. IP3 in the various fractions was measured by a competitive binding assay according to the manufacturer's protocol.G Protein Activation
Colonocytes (~3 × 106) from adult and weanling rabbits were permeabilized using 0.005% saponin at room temperature for 30 min. G protein activation was carried out as described previously (46, 52) using azidoanilido [Immunoblotting
Proteins of cell homogenate, membrane, or cytosolic fractions (see PKC Activity for isolation procedure) were resolved by SDS-PAGE (10% gels) and electrotransferred to Hybond nitrocellulose membrane (7). The membrane was washed in Tris-buffered saline [TBS; 50 mM Tris · HCl (pH 7.4) and 150 mM NaCl] that contained 0.05% (vol/vol) Tween 20 (TBS-T) for 20 min, followed by 4× 5-min washes in TBS-T. The blot was then blocked in Blotto (5% nonfat dry milk in TBS-T) for either 1 h at room temperature or overnight at 4°C. These blots were then incubated with anti-Gq antibody (1:1,000) in Blotto overnight at 4°C. The blots were washed in TBS-T (5 min × 2, 10 min × 2), followed by incubation with a peroxidase-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature. The blots were washed (5 min × 3, 10 min × 2 in TBS-T) and the reaction products visualized by ECL.Immunoprecipitation
The identity of the labeled G protein was determined by immunoprecipitation with specific polyclonal Gq antibodies that recognize both GStatistics
Statistical significance of differences between means ± SE, within an experiment, was determined by paired Student's t-test. Analysis of variance (ANOVA) test was used to determine statistical significance when more than two means were compared. For adults, each n value depicts a separate animal. For weanlings, each n value represents a pool of colonocytes obtained from two weanlings. For newborns, colonocytes were pooled from all the pups (~6) of a litter. Within an experiment, samples were examined at least in duplicate, but often in triplicate, and their values were averaged to provide n = 1. Values are means ± SE where n = number of animals or number of pools as described above. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TDC Action on Colonocytes in Suspension
We demonstrated earlier that TDC increases Cl
|
As shown in Fig. 1, the TDC-stimulated
increase in Cl influx is dose dependent, reaching a
maximum at 50 µM. There was no TDC-stimulatable Cl
transport in the presence of furosemide (10 µM) + DPC (50 µM) (Fig. 1) at any of the doses tested. As in our earlier studies (14, 58, 59), none of the agents tested in this study
altered the inhibitor-insensitive Cl
flux values, i.e.,
inhibitors ± reagent were similar. Thus in the rest of the
figures, Cl
influx is depicted as inhibitor-sensitive
values (shown as open circles in Fig. 1). Our earlier study on attached
colonocytes demonstrated that newborn and weanling cells were not
responsive to TDC at any dose (14). This lack of
responsiveness was confirmed in nonattached cells. Newborn and weanling
colonocytes in suspension showed no response to 50 µM TDC
[inhibitor-sensitive Cl
transport in newborn cells
(basal 0.98 ± 0.02; + TDC 1.1 ± 0.08; n = 5) and weanling colonocytes (basal 0.9 ± 0.06; + TDC 0.85 ± 0.05; n = 5)]. Since TDC evoked similar responses in
colonocytes in suspension (Table 1 and Fig. 1) and in those attached to
a matrix (14), all subsequent experiments were performed
on cells in suspension.
|
Segment and Chemical Specificity of Bile Acid Action
To determine whether other bile acids, present in abundance in the colon, regulate Cl
|
Intracellular Mechanism of Action of TDC
Role of paracrine modulators.
We first examined whether TDC acted via histamine and/or PG. We
previously demonstrated in rabbit distal colonocytes that the action of
histamine is indomethacin sensitive, implying a mediatory role of PG
(59). Therefore, we postulated that if indomethacin
altered TDC action, it would imply involvement of PG and perhaps
histamine. As shown in Fig.
2A, indomethacin (1 µM)
affected neither basal (basal 1 ± 0.15 and + indo 0.93 ± 0.14 mM/s) nor TDC-stimulated Cl transport (TDC
2.1 ± 0.12 and TDC + indo 1.9 ± 0.10 mM/s) in adult
rabbit distal colonocytes.
|
Effect on [cAMP]i. To determine whether bile acid increases [cAMP]i in adult distal colonocytes, we measured the effect of TDC on cAMP levels by an enzyme-linked immunoassay. As shown in Fig. 2B, whereas 1 µM forskolin stimulated cAMP significantly (~3-fold), TDC had no effect.
Effect on PKC activity.
We previously demonstrated that PDB (1 µM) stimulated
Cl secretion in colonocytes of all three age groups
(14), suggesting that PKC and its distal pathways were
present from birth. To determine whether TDC utilizes this pathway in
adult distal colon, we compared the effects of TDC and PDB on PKC
activity. It must be noted that rabbit colon shows very low PKC
activity, and cells from three adult rabbits had to be pooled to obtain
each n value. In addition, the DEAE anion exchange
chromatography step, used to purify partially cytosolic and solubilized
membrane fractions, had to be scaled down by a factor of three. As
shown in Fig. 2C, PDB (1 µM) caused a fourfold increase in
cytosolic (0.36 ± 0.15 to 1.54 ± 0.16 pmol · min
1 · µg
1
protein; n = 5, P < 0.01) and a
fivefold increase in membrane-associated PKC activity (0.6 ± 0.3 to 3.2 ± 1.1 pmol · min
1 · µg
1
protein; n = 5, P < 0.001). However,
50 µM TDC did not significantly alter either the cytosolic or
membrane-associated PKC activity.
Effect on [Ca2+]i.
We next investigated the involvement of
[Ca2+]i in TDC action in adult distal
colonocytes. TDC (50 µM) significantly increased [Ca2+]i in adult colonocytes [basal vs. TDC
(fluorescence ratio 340/380) 1.9 ± 0.2 vs. 2.8 ± 0.3;
n = 5, P < 0.05; Fig.
3A]. However, TDC failed to
increase [Ca2+]i levels in weanling and
newborn distal colonocytes [basal vs. 50 µM TDC (fluorescence ratio
340/380) (n = 4); weanling 2.05 ± 0.2 vs.
1.98 ± 0.3; newborn 1.68 ± 0.2 vs. 1.71 ± 0.2]. This age-related pattern was similar to that of TDC action on
Cl transport (Fig. 1 and Ref. 14).
|
Effect of [Ca2+]o on
TDC-induced Cl transport.
To determine whether [Ca2+]o is important in
the action of TDC, the effect of lowering
[Ca2+]o on TDC-stimulated Cl
transport was examined (Fig. 3B). Chelating 10 µM
[Ca2+]o with 1 mM EGTA was estimated to yield
a [Ca2+]o ~10 pM by the method of Tsien and
Pozzan (66). As shown in Fig. 3B, this
level of [Ca2+]o did not affect TDC-induced
Cl
influx {basal vs. TDC (mM/s);
[Ca2+]o = 1 mM: 1.0 ± 0.2 vs.
1.6 ± 0.2; [Ca2+]o ~10 pM: 0.89 ± 0.1 vs. 1.5 ± 0.2}. However, response to A-23187 (1 µM) is
diminished when [Ca2+]o is ~10 pM {basal
vs. A-23187 (mM/s); [Ca2+]o = 1 mM:
1.0 ± 0.2 vs. 1.8 ± 0.2; [Ca2+]o
~10 pM: 0.89 ± 0.1 vs. 1.4 ± 0.15, P < 0.05, different from [Ca2+]o = 1 mM}.
These results suggest that whereas short-term TDC action does not
require [Ca2+]o, A-23187 allows entry of
Ca2+ from extracellular and intracellular stores.
Effect of BAPTA on TDC-induced Cl transport.
To confirm the role of Ca2+ in TDC signaling in adult
distal colonocytes, the effect of chelating
[Ca2+]i with BAPTA on TDC-induced
Cl
transport was examined. Whereas the 340/380 emission
ratio in control cells loaded with fura 2-AM was 1.82 ± 0.06, it
was 1.0 in BAPTA-treated cells, indicating complete chelation. As shown in Fig. 3C, 25 µM BAPTA abolished TDC-stimulated
Cl
influx [basal vs. TDC (mM/s) (n = 4);
control cells 1.05 ± 0.4 vs. 2.4 ± 0.2; P < 0.01; cells + BAPTA 1.0 ± 0.1 vs. 1.04 ± 0.2]. In
contrast, BAPTA pretreatment had no effect on the ability of 1 µM
PGE1 to stimulate Cl
transport (Fig.
3C). The transport studies were carried out in a minimally
Ca2+-free (~10 pM) buffer.
Effect of Ca2+-Dependent
Secretagogues on Cl Transport and Intracellular
Ca2+
|
When [Ca2+]i was estimated in fura 2-loaded adult colonocytes, a significant increase over basal was seen in the presence of bethanechol, neurotensin, and histamine (10 µM each; fluorescence ratio 340/380, basal 0.60; bethanechol 1.12; neurotensin 1.20; histamine 1.02; n = 2, in triplicate). These results confirmed earlier findings from this laboratory that neurotensin (59) and bethanechol (57) increased [Ca2+]i. Again, in marked contrast to the adult, none of these agents, even at 10 µM, altered [Ca2+]i in weanling colonocytes (Fig. 4B, n = 4). This age dependence is similar to the effect of TDC (Fig. 3A).
Effect of Ca2+ Ionophore on
Cl Transport in Distal Colonocytes
|
Activation of G Proteins
In heterotrimeric G protein-coupled signaling systems, binding of ligand to receptor increases GTP binding to G
|
To identify the 42-kDa protein, immunoprecipitation of solubilized
total protein from AAGTP-labeled colonocytes was performed using an
antibody that recognizes both Gq and G
11.
The samples were subjected to SDS-PAGE and sequentially followed
by Western blotting with the same anti-Gq antibodies (Fig.
6C) and PhosphorImager analyses (Fig. 6D). The
antibody immunoprecipitated a 42-kDa protein from cell lysates
(lane 3) that had an identical mobility to a G
q standard (lane 4; Ref. 46).
Anti-Gq antibodies also identified a 42-kDa protein in
membranes not subjected to immunoprecipitation (lane 1).
Equally important, the immunoprecipitated protein was labeled with
AAGTP (Fig. 6D, lane 3). Normal rabbit serum
failed to immunoprecipitate a 42-kDa labeled protein (lane
2). However, the normal rabbit serum identified
lower-molecular-weight species, which we consider nonspecific, as there
was no band in the corresponding autoradiograms (Fig. 6D;
lane 2). This result demonstrated that the 42-kDa
neurotensin-activated protein is most likely G
q or G
11 and that there are no age-related differences in
neurotensin receptor-G protein coupling.
In contrast to neurotensin and bethanechol, the mechanism by which TDC
increases [Ca2+]i in any cell type, including
adult colonocytes, is not clear. We therefore determined whether TDC
acts via a G protein-dependent mechanism. The data are shown in Fig.
7A, with representative autoradiograms (bottom) and the corresponding quantitation
of multiple autoradiograms (top). In adult distal
colonocytes, TDC at doses >50 µM caused a decrease in AAGTP labeling
of the 42-kDa protein [Fig. 7A; 50 µM TDC 92.6 ± 20% (not significant); 100 µM TDC 80.1 ± 13%,
n = 8, P < 0.05; 200 µM TDC 65 ± 19%, n = 8, P < 0.01]. In
weanling colonocytes, 50 µM TDC did not alter labeling, whereas 100 µM TDC again caused a decrease, although statistically insignificant,
in labeling of the 42-kDa protein (Fig. 7B; 50 µM TDC
108 ± 18%; 100 µM TDC 79 ± 19%).
|
Thus neurotensin, but not TDC, increases G protein labeling in adult
and weanling colonocytes. Furthermore, the predominant G protein in
rabbit distal colonocytes appears to be the 42-kDa Gq. The
major findings of these experiments are that there are no age-specific
differences in G protein activation, which could account for the
developmental differences in regulation of Cl
transport
by Ca2+-dependent secretagogues.
Effect of Ca2+-Dependent Secretagogues on PLC Activity
The difference in Ca2+ signaling between young and adult rabbits could be due to differences at the level of PLC, either in its activity and/or substrate availability. This was addressed in an assay system wherein both product (IP3) and substrate (PIP2) content could be measured in response to Ca2+-dependent secretagogues. As shown in Fig. 8A, neurotensin (5 µM) caused an 80-fold increase in IP3 levels over basal (1.0 ± 0.2 pmol/106 cells) in adult cells. This dose of neurotensin is maximal, because 10 µM had similar effects (75.5 pmol IP3; n = 2, each in duplicate). Whereas TDC (50 µM) and bethanechol increased IP3 50-fold, PGE1 (1 µM) had no effect. The IP3 levels in unstimulated weanling and adult colonocytes were similar (Fig. 8A). However, neurotensin at 1 µM (Fig. 8A) or at 10 µM (1.23 pmol; n = 2, each in duplicate) and TDC (50 µM; Fig. 8A) failed to increase IP3 levels in weanling colonocytes. This suggests that the acquisition of responsiveness to neurotensin and to TDC from weanling to adult lies at the step of generating IP3 from PIP2, a step catalyzed by PLC.
|
To determine whether the inability to increase IP3 in weanlings is due to insufficient substrate for PLC, PIP2 levels were assessed in weanling and adult colonocytes. As shown in Fig. 8B, nonstimulated weanling and adult colonocytes had similar PIP2 levels. Neurotensin (1 µM) caused a significant reduction in PIP2 levels in adult cells but had no effect on PIP2 levels in weanling colonocytes (Fig. 8B). The neurotensin-stimulated increase in IP3 in adult colonocytes of ~80 pmol corresponds with the ~75 pmol decrease it caused in PIP2 levels (Fig. 8, A and B). Clearly, IP3 can be generated in weanling colonocytes, because under basal conditions, similar amounts of IP3 are present in weanling and adult cells. Furthermore, these are well within the detection limits of the assay. Therefore, the nonresponsiveness of weanling distal colon to Ca2+-dependent secretagogues is not due to a lack of substrate but an inability to activate PLC.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study reports the novel finding that
Ca2+-dependent signaling of colonic epithelial
Cl transport is ontogenically regulated and demonstrates
that the site of this regulation lies at the level of PLC activity. The impact of these observations is underscored by their physiological relevance. Epithelia in general, and those lining the gastrointestinal tract in particular, are faced with frequent osmotic and secretory challenges from their luminal surface while having to maintain a
transepithelial fluid balance (10). This demands
minute-by-minute regulation of vectorial ion transport, including
Cl
secretory pathways. Ca2+-dependent
modulators are often considered the fine-tuners of epithelial fluid
homeostasis, causing rapid and transient effects on Cl
secretion (51). The biochemical basis for the transience
is not fully elucidated, but duration of the stimulus, receptor
desensitization, activation of negative modulators, and crosstalk of
cascades have been implicated (1, 31, 67). Many of these
studies have been conducted in cell lines that preclude examination of
age-related changes. The current study examines changes during
development in colonocytes isolated from newborn, weanling, and adult rabbits.
The effects of the muscarinic agonist bethanechol, the neurohormone neurotensin, the immunomodulator histamine, and the bile acid TDC were examined. Whereas the first three agents have been clearly shown to act via increasing intracellular Ca2+, the cellular mechanisms by which bile acids act in the colon appear to vary with the model system studied. These range from PG (50) to intracellular cAMP (49), Ca2+ (16, 17), and PKC (29, 45). Therefore, the present study first undertook a stepwise characterization of these putative modes of TDC action in a single model to help identify the basis for age-specific differences in effects of TDC.
Specificity of TDC Action
Results in Table 2 demonstrate that the ability of bile acids to stimulate ClMechanism of TDC Action
Although it is conceivable that TDC could also act by altering paracellular permeability in adult rabbit (18), our data clearly indicate that TDC can stimulate ClIn other models, TDC has been postulated to act via modulators
associated with the immune pathway such as histamine and PG. Studies in
rat colon (13) and in human sigmoid colonic biopsies (2) demonstrated that 5-10 µM bile acids increase
mucosal PG release. High concentrations (0.3 mM) of lipophilic
dihydroxy bile acids activate histamine release from mouse colon
(20). Furthermore, the secretory effect of 1 mM
chenodeoxycholic acid was inhibited by H1 histamine receptor
antagonists and partially by the cyclooxygenase inhibitor indomethacin
(19). We previously demonstrated in rabbit distal
colonocytes that the action of histamine is indomethacin sensitive
(59). However, the data in Fig. 2A show that
indomethacin neither affected basal nor TDC-stimulated Cl
transport, ruling out either histamine or PG as mediators of TDC action
in rabbit colon.
There are various reports on the involvement of cAMP in TDC signaling.
Thus in human colonic mucosa, 10 nM deoxycholic acid or
chenodeoxycholic acid induced a dose-dependent inhibition of adenylate
cyclase activity (64), but TDC did not increase cAMP in
HT-29 cells (30). In intact rabbit colon, Potter et al.
(49) reported that TDC increases tissue cAMP content in
adult but not in neonate. In contrast, as shown in Fig. 2B,
TDC failed to increase intracellular cAMP in adult rabbit distal
colonocytes, whereas forskolin caused a significant increase.
Similarly, Mauricio et al. (36) recently reported that 100 µM deoxycholic acid did not increase cAMP production in rabbit distal
colonic mucosa. We postulate that the increases in cAMP content seen in
intact rabbit distal colon (49) could be due to the effect
of TDC on subepithelial elements.
In platelets and HT-29 cell homogenates, 50 µM-1 mM deoxycholate
activates Ca2+-dependent PKC isoforms 10-fold
(29). These authors suggested that amphipathic bile acids
act in a phosphatidylserine-like manner to activate PKC. In human
colonic mucosa, secondary bile acids best activate PKC
1
(45). In isolated rat hepatocytes, tauroursodeoxycholic acid, but not TC, activates PKC
(5, 6). All these data suggest the involvement of conventional, i.e.,
Ca2+-dependent PKC isoforms, in bile acid action. In
contrast, data in Fig. 2C showed that 1 µM PDB, but not 50 µM TDC, altered Ca2+-dependent PKC activity, indicating
that the action of TDC in adult rabbit distal colonocytes does not
involve conventional PKC isoforms.
The results in Fig. 3 clearly demonstrate that the major, if not only,
mechanism of TDC action in adult rabbit colonocytes is via
[Ca2+]i. Thus in Fig. 3A, TDC is
shown to increase [Ca2+]i in adult distal
colonocytes, and in Fig. 3C, this increase in
[Ca2+]i is shown to be essential, since
chelating [Ca2+]i with BAPTA-AM abolished
TDC-induced Cl transport. This effect was specific
because BAPTA-AM did not diminish the effects of PGE1.
Furthermore, Fig. 3B shows that the action of TDC over 5 min
does not require [Ca2+]o. Equally important,
Fig. 3A demonstrates that TDC did not increase [Ca2+]i in colonocytes of weanling or newborn
animals, two age groups in which it also fails to increase
Cl
transport (Fig. 1 and Ref. 14). This
age-specific effect rests at the step of or before increasing
[Ca2+]i, since the ionophore A-23187
increased [Ca2+]i and Cl
transport in all age groups (Fig. 5). In contrast to TDC, the effects
of A-23187 appear to utilize both extra- and intracellular Ca2+ (Fig. 3B).
The role of extracellular Ca2+ in the action of a
secretagogue is primarily dependent on length of exposure. In many cell
types, after initial depletion, the [Ca2+]i
stores are replenished from the extracellular medium via activation of
capacitative Ca2+ entry mechanisms (4).
However, the dependence of the initial response on
[Ca2+]o varies with the cell type and
secretagogue. In our studies, over 5 min, pharmacological ionophores
clearly utilize extra- and intracellular stores (Fig. 3B),
whereas TDC utilizes only intracellular stores (Fig. 3, B
and C). Our findings are consistent with previous data that
bile acid-induced secretion in rabbit distal colon does not require
[Ca2+]o (19) and that
deoxycholate mobilizes Ca2+ from internal stores in HT-29
cells (30). In contrast, in T84 cells, initial effects of
TDC on Cl transport were reported to be independent
(16) or partly dependent (17) on
[Ca2+]o. It is conceivable that although
[Ca2+]o is not required to initiate the
response to TDC in rabbit colonocytes, it may play a role in the
long-term maintenance of intracellular stores.
Thus our results demonstrate that in rabbit colonocytes, TDC stimulates
Cl transport over 5 min by releasing Ca2+
from intracellular stores, a mechanism that is absent in young animals.
The lack of TDC effects in weanling (25 days old) colon are intriguing
since, unlike in neonate at this age, the ileal bile acid transporter
is fully functional in rabbits (60).
Age-Specific Stimulation of Cl Transport by Other
Secretagogues
Mechanism(s) Underlying Age-Specific Effects
A major mechanism by which Ca2+-dependent secretagogues raise [Ca2+]i is by activation of PLC. This has been well documented for neurotensin and bethanechol (10, 39), and bile acids have been shown to increase IP3 levels in T84 cells (16) and in rat (11) and human colon (40). Data in Fig. 8 demonstrate that the absence of an increase in [Ca2+]i in weanling is due to the incapability of bethanechol, neurotensin, and TDC to activate PLC and increase IP3. In marked contrast, these agents cause a 50- to 80-fold increase in IP3 content in adults (Fig. 8A). The effects are specific to Ca2+-dependent secretagogues since PGE1 did not increase IP3 at any age (Fig. 8A). Figure 8B shows that absence of IP3 production is not due to the lack of PIP2 substrate in weanling animals and, therefore, rests at the ability of extracellular signals to activate PLC.Studies on the ontogeny of epithelial Cl transport
processes and/or their regulation by Ca2+ signaling are
sparse (14, 40, 48). Interestingly, the rotaviral polypeptide NSP4 stimulates [Ca2+]i in adult
and neonatal mice but stimulates Ca2+-activated
Cl
channel transport only in neonates (38).
A few other studies, chiefly in adults, have examined the distribution
and expression of PLC in mammalian intestine (9, 33, 44).
In the adult rabbit (33) and rat (44) small
intestine, the major isoform reported is PLC-
, and this has been
linked to tyrosine kinase cascades. In the only study on developing
mammals in which homogenates of rat small intestine (muscle, lamina
propria, and epithelium) were used, PLC-
activity and expression was
highest in the weanling animal (44). Thus the data
presented here provide a new focus of regulation and allow one to
hypothesize that the developing gut protects itself from the dramatic
changes in its milieu by delaying the appearance of fully functional PLC.
There is considerable conjectural evidence to support this argument. First, the appearance of colonic enteroendocrine cells, the main source of neurotensin, increases with age (34). Second, age-related differences in phosphoinositide signaling have been reported in other tissues, albeit at different steps. For example, in rat brain, the capacity to metabolize, but not to generate IP3, increases with age (10, 37, 68), whereas in rabbit airway smooth muscles, IP3 production and metabolism increase with age (56). In cat gastric smooth muscle, IP3 receptor density increases with age (15). Third, in rat jejunum, Ca2+ sequestration into the endoplasmic reticulum has been shown to be age dependent (22, 62). This seems to be strain specific, with uptake decreasing with age in Sprague-Dawley rats (22) and increasing with age in Wistar-Kyoto rats (62).
Receptor-PLC Signaling
Figure 6 shows that the inability of neurotensin to activate PLC in weanlings is not due to a dearth of receptor-G protein coupling. Because AAGTP binds only to activated receptors, these data demonstrate that an active receptor and G protein are present in adults and weanlings and identify the protein as Gq. Although bile acids have been shown to stimulate PLC (11, 16, 40), none of those studies examined whether TDC utilizes a receptor-G protein-coupled pathway to activate PLC. The only possible linkage of G proteins to bile acids has been implicated with respect to a unique action of the monohydroxy secondary bile acid lithocholate. In guinea pig gastric chief cells, only lithocholic acid, but no other bile acids including TDC, can act on M3 cholinergic receptors to stimulate pepsinogen secretion (53). The M3 cholinergic signaling is linked to GPhysiological/Pathophysiological Implications
The present study shows a new facet of inositol phosphate signal regulation. By lacking PLC activity in the colon, the weanling animal has essentially shielded itself from inappropriate secretion in response to the ever-fluctuating levels of a major class of secretagogues. At the same time, a patent cAMP-sensitive mechanism from the neonatal age allows the animal to ensure basal secretion needed to maintain fluidity of colonic contents. The refractoriness to TDC in rabbits is a case in point. Bile acids can be potent secretagogues in mammalian colon. In a normal healthy adult mammal, this is not a problem, because the ~5% of bile acid output that enters the colon is dehydroxylated and passively reabsorbed. In cases of ileal bile acid malabsorption in adults, diarrhea ensues. The infant ileum, however, does not actively transport bile acids (27), and the infant produces bile acids that reach less than or equal to adult levels that enter the colon (28). In intact neonatal rabbit colon, no bile acid diarrhea was observed (47), and our data suggest that this is due to lack of PLC activity. In humans, although a number of studies imply that bile acid malabsorption is a major "cause" of refractory infantile diarrhea in children (12, 25, 26, 42), the cellular basis for this is not known. High levels of bile acid in the colon can contribute to diarrhea in one of two ways: 1) by directly stimulating secretion of salt and water, resulting in bile acid diarrhea, or 2) by causing inadequate solubilization of fatty acids and monoglycerides, resulting in high concentrations of hydroxy fatty acids in the colon that are potent secretagogues. It is noteworthy that the fecal fatty acid content was also high in infant patients with bile acid diarrhea (26, 28). It is conceivable that in humans, as in rabbits, there is an absence of bile acid-stimulated diarrhea, but that hydroxy fatty acid-stimulated secretion predominates. Thus the diarrhea associated with bile acid malabsorption in infants may be a tempered response and solely due to the latter agents. The cellular basis of developmental regulation of Ca2+-mediated colonic ion transport in human and in animal models clearly needs to be explored further, and this paper provides the framework for examining proximal steps in the signal transduction cascade. ![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institutes of Health Grants R01 DK-46910 and DK-38510 (to M. C. Rao), MH-39595 and AG-15482 (to M. M. Rasenick) and by the College Research Board, University of Illinois at Chicago (to M. C. Rao).
![]() |
FOOTNOTES |
---|
Present address of M. Carlos: Illinois Masonic Hospital and Rush Medical School, 836 W. Wellington Ave., Chicago, IL 60657.
Address for reprint requests and other correspondence: M. C. Rao, Dept. of Physiology and Biophysics (M/C 901), Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-7342 (E-mail: meenarao{at}uic.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 May 2000; accepted in final form 18 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barrett, K.
Integrated regulation of intestinal epithelial transport intercellular and intracellular pathways.
Am J Physiol Cell Physiol
272:
C1069-C1076,
1997
2.
Bartram, HP,
Scheppach W,
Englert S,
Dusel G,
Richter A,
Richter F,
and
Kasper H.
Effects of deoxycholic acid and butyrate on mucosal prostaglandin E2 release and cell proliferation in the human sigmoid colon.
JPEN J Parenter Enteral Nutr
19:
182-186,
1995[Abstract].
3.
Benya, R,
Schmidt L,
Sahi J,
Layden T,
and
Rao M.
Isolation characterization and attachment of rabbit distal colon epithelial cells.
Gastroenterology
101:
692-702,
1991[ISI][Medline].
4.
Berridge, MJ.
Elementary and global aspects of calcium signaling.
J Physiol (Lond)
499:
291-306,
1997[ISI][Medline].
5.
Beuers, U,
Probst I,
Soroka C,
Boyer JL,
Kullak-Ublick GA,
and
Paumgartner G.
Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes.
Hepatology
29:
477-482,
1999[ISI][Medline].
6.
Beuers, U,
Throckmorton DC,
Anderson MS,
Isales CM,
Thasler W,
Kullak-Ublick GA,
Sauter G,
Koebe HG,
Paumgartner G,
and
Boyer JL.
Tauroursodeoxycholic acid activates protein kinase C in isolated rat hepatocytes.
Gastroenterology
110:
1553-1563,
1996[ISI][Medline].
7.
Bhartur, SG,
Bookstein C,
Musch MW,
Boxer R,
Chang EB,
and
Rao MC.
An avian sodium-hydrogen exchanger.
Comp Biochem Physiol A Physiol
118:
883-889,
1997[ISI][Medline].
8.
Blank, JL,
Ross AH,
and
Exton JH.
Purification and characterization of two G-proteins that activate the 1 isozyme of phosphoinositide-specific phospholipase C. Identification as members of the Gq class.
J Biol Chem
266:
18206-18216,
1991
9.
Bolt, MJ,
Bissonnette BM,
Wali RK,
Hartmann SC,
Brasitus TA,
and
Sitrin MD.
Characterization of phosphoinositide-specific phospholipase C in rat colonocyte membranes.
Biochem J
292:
271-276,
1993[ISI][Medline].
10.
Chang, E,
and
Rao M.
Intestinal water and electrolyte transport: mechanisms of physiological and adaptive responses.
In: Physiology of the Gastrointestinal Tract, edited by Johnson L.. New York: Raven, 1994, p. 2027-2081.
11.
Craven, PA,
Pfanstiel J,
and
DeRubertis FR.
Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation and reactive oxygen formation by bile acids.
J Clin Invest
79:
532-541,
1987[ISI][Medline].
12.
De Belle, RC,
Vaupshas V,
Vitullo BB,
Haber LR,
Shaffer E,
Mackie GG,
Owen H,
Little JM,
and
Lester R.
Intestinal absorption of bile salts: immature development in the neonate.
J Pediatr
94:
472-476,
1979[ISI][Medline].
13.
DeRubertis, FR,
Craven PA,
and
Saito R.
Bile salt stimulation of colonic epithelial proliferation. Evidence for involvement of lipoxygenase products.
J Clin Invest
74:
1614-1624,
1984[ISI][Medline].
14.
Desai, G,
Sahi J,
Reddy P,
Vidyasagar D,
and
Rao M.
Chloride transport in primary cultures of mammalian colonocytes at different developmental stages.
Gastroenterology
111:
1541-1550,
1996[ISI][Medline].
15.
Deutsch, DE,
Bitar KN,
and
Hillemeier AC.
Access to intracellular calcium during development in the feline gastric antrum.
Pediatr Res
43:
369-373,
1998[Abstract].
16.
Devor, DC,
Sekar MC,
Frizzell RA,
and
Duffey ME.
Taurodeoxycholate activates potassium and chloride conductances via an IP3-mediated release of calcium from intracellular stores in a colonic cell line (T84).
J Clin Invest
92:
2173-2181,
1993[ISI][Medline].
17.
Dharmsathaphorn, K,
Huott PA,
Vongkovit P,
Beuerlein G,
Pandol SJ,
and
Ammon HV.
Cl secretion induced by bile salts. A study of the mechanism of action based on a cultured colonic epithelial cell line.
J Clin Invest
84:
945-953,
1989[ISI][Medline].
18.
Freel, RW.
Dihydroxy bile salt-induced secretion of rubidium ion across the rabbit distal colon.
Am J Physiol Gastrointest Liver Physiol
252:
G554-G561,
1987
19.
Freel, RW,
Hatch M,
Earnest DL,
and
Goldner AM.
Dihydroxy bile salt-induced alterations in NaCl transport across the rabbit colon.
Am J Physiol Gastrointest Liver Physiol
245:
G808-G815,
1983
20.
Gelbmann, CM,
Schteingart CD,
Thompson SM,
Hofmann AF,
and
Barrett KE.
Mast cells and histamine contribute to bile acid-stimulated secretion in the mouse colon.
J Clin Invest
95:
2831-2839,
1995[ISI][Medline].
21.
Ghishan, F.
Electrolyte fluxes in the small intestine during development. New York: Raven, 1989.
22.
Ghishan, FK,
and
Arab N.
Active calcium transport by intestinal endoplasmic reticulum during maturation.
Am J Physiol Gastrointest Liver Physiol
254:
G74-G80,
1988
23.
Heacock, AM,
Seguin EB,
and
Agranoff BW.
Developmental and regional studies of the metabolism of inositol 1,4,5-trisphosphate in rat brain.
J Neurochem
54:
1405-1411,
1990[ISI][Medline].
24.
Henning, S,
Rubin D,
and
Shulman R.
Ontogeny of the intestinal mucosa.
In: Physiology of the Gastrointestinal Tract, edited by Johnson L.. New York: Raven, 1994, p. 571-610.
25.
Heubi, JE,
Balistreri WF,
Fondacaro JD,
Partin JC,
and
Schubert WK.
Primary bile acid malabsorption: defective in vitro ileal active bile acid transport.
Gastroenterology
83:
804-811,
1982[ISI][Medline].
26.
Heubi, JE,
Balistreri WF,
Partin JC,
Schubert WK,
and
McGraw CA.
Refractory infantile diarrhea due to primary bile acid malabsorption.
J Pediatr
94:
546-551,
1979[ISI][Medline].
27.
Heubi, JE,
and
Fellows JL.
Postnatal development of intestinal bile salt transport. Relationship to membrane physico-chemical changes.
J Lipid Res
26:
797-805,
1985[Abstract].
28.
Heubi, JE,
Soloway RD,
and
Balistreri WF.
Biliary lipid composition in healthy and diseased infants, children, and young adults.
Gastroenterology
82:
1295-1299,
1982[ISI][Medline].
29.
Huang, XP,
Fan XT,
Desjeux JF,
and
Castagna M.
Bile acids, non-phorbol-ester-type tumor promoters, stimulate the phosphorylation of protein kinase C substrates in human platelets and colon cell line HT29.
Int J Cancer
52:
444-450,
1992[ISI][Medline].
30.
Huang, XP,
Heyman M,
Nath SK,
Castagne M,
and
Desjeux JF.
Distinct signaling mediates chloride secretion induced by tumor promoter bile acids and phorbol esters in human colonic cells.
Int J Oncology
6:
1159-1163,
1995[ISI].
31.
Ismailov, I,
Fuller C,
Berdiev B,
Shlyonsky V,
Benos D,
and
Barrett K.
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
32.
Jacquemin, E,
Hagenbuch B,
Stieger B,
Wolkoff AW,
and
Meier PJ.
Expression cloning of a rat liver Na+-independent organic anion transporter.
Proc Natl Acad Sci USA
91:
133-137,
1994[Abstract].
33.
Khurana, S,
Kreydiyyeh S,
Aronzon A,
Hoogerwerf WA,
Rhee SG,
Donowitz M,
and
Cohen ME.
Asymmetric signal transduction in polarized ileal Na+-absorbing cells: carbachol activates brush-border but not basolateral-membrane PIP2-PLC and translocates PLC-1 only to the brush border.
Biochem J
313:
509-518,
1996[ISI][Medline].
34.
Krause, WJ,
Yamada J,
and
Cutts JH.
Enteroendocrine cells in the developing opossum small intestine and colon.
J Anat
162:
83-96,
1989[ISI][Medline].
35.
Lee, CH,
Park D,
Wu D,
Rhee SG,
and
Simon MI.
Members of the Gq-subunit gene family activate phospholipase C beta isozymes.
J Biol Chem
267:
16044-16047,
1992
36.
Mauricio, AC,
Slawik M,
Heitzmann D,
von Hahn T,
Warth R,
Bleich M,
and
Greger R.
Deoxycholic acid (DOC) affects the transport properties of distal colon.
Pflügers Arch
439:
532-540,
2000[ISI][Medline].
37.
Moon, KH,
Lee SY,
and
Rhee SG.
Developmental changes in the activities of phospholipase C, 3-kinase, and 5-phosphatase in rat brain.
Biochem Biophys Res Commun
164:
370-374,
1989[ISI][Medline].
38.
Morris, AP,
Scott JK,
Ball JM,
Zeng QC,
O'Neal WK,
and
Estes MK.
NSP4 elicits age-dependent diarrhea and Ca2+-mediated I influx into intestinal crypts of cystic fibrosis mice.
Am J Physiol Gastrointest Liver Physiol
277:
G431-G444,
1999
39.
Nahorski, SR,
Tobin AB,
and
Willars GB.
Muscarinic M3 receptor coupling and regulation.
Life Sci
60:
1039-1045,
1997[ISI][Medline].
40.
Nomoto, K,
Morotomi M,
Miyake M,
Xu DB,
LoGerfo PP,
and
Weinstein IB.
The effects of bile acids on phospholipase C activity in extracts of normal human colon mucosa and primary colon tumors.
Mol Carcinog
9:
87-94,
1994[ISI][Medline].
41.
O'Loughlin, EV,
Hunt DM,
and
Kreutzmann D.
Postnatal development of colonic electrolyte transport in rabbits.
Am J Physiol Gastrointest Liver Physiol
258:
G447-G453,
1990
42.
Oelkers, P,
Kirby LC,
Heubi JE,
and
Dawson PA.
Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2).
J Clin Invest
99:
1880-1887,
1997
43.
Park, D,
Jhon DY,
Lee CW,
Lee KH,
and
Rhee SG.
Activation of phospholipase C isozymes by G protein -subunits.
J Biol Chem
268:
4573-4576,
1993
44.
Polk, DB.
Ontogenic regulation of phospholipase C-1 activity and expression in the rat small intestine.
Gastroenterology
107:
109-116,
1994[ISI][Medline].
45.
Pongracz, J,
Clark P,
Neoptolemos JP,
and
Lord JM.
Expression of protein kinase C isoenzymes in colorectal cancer tissue and their differential activation by different bile acids.
Int J Cancer
61:
35-39,
1995[ISI][Medline].
46.
Popova, JS,
Garrison JC,
Rhee SG,
and
Rasenick MM.
Tubulin, Gq, and phosphatidylinositol 4,5-bisphosphate interact to regulate phospholipase C 1 signaling.
J Biol Chem
272:
6760-6765,
1997
47.
Potter, G.
Development of colonic function.
In: Human Gastrointestinal Development, edited by Lebenthal E.. New York: Raven, 1989, p. 545-558.
48.
Potter, G,
Lester R,
Burlingame S,
Mitchell P,
and
Schmidt K.
Taurodeoxycholate and the developing rabbit distal colon: absence of secretory effect.
Am J Physiol Gastrointest Liver Physiol
253:
G483-G488,
1987
49.
Potter, G,
Sellin J,
and
Burlingame S.
Bile acid stimulation of cyclic AMP and ion transport in developing rabbit colon.
J Pediatr Gastroenterol Nutr
13:
335-341,
1991[ISI][Medline].
50.
Rampton, DS,
Breuer NF,
Vaja SG,
Sladen GE,
and
Dowling RH.
Role of prostaglandins in bile salt-induced changes in rat colonic structure and function.
Clin Sci (Lond)
61:
641-648,
1981[ISI][Medline].
51.
Rao, M,
and
de Jonge H.
Ca2+-dependent protein kinase: role in ion transport.
In: Secretory Diarrhea, edited by Lebenthal E,
and Duffey ME.. New York: Raven, 1990, p. 191-207.
52.
Rasenick, MM,
Talluri M,
and
Dunn WJ, III.
Photoaffinity guanosine 5'-triphosphate analogs as a tool for the study of GTP-binding proteins.
Methods Enzymol
237:
100-110,
1994[ISI][Medline].
53.
Raufman, JP,
Zimniak P,
and
Bartoszko-Malik A.
Lithocholyltaurine interacts with cholinergic receptors on dispersed chief cells from guinea pig stomach.
Am J Physiol Gastrointest Liver Physiol
274:
G997-G1004,
1998
54.
Reddy, P,
Sahi J,
Desai G,
Vidyasagar D,
and
Rao M.
Altered growth and attachment of rabbit colonocytes isolated from different developmental stages.
Pediatr Res
39:
287-294,
1996[Abstract].
55.
Rhee, SG,
and
Bae YS.
Regulation of phosphoinositide-specific phospholipase C isozymes.
J Biol Chem
272:
15045-15048,
1997
56.
Rosenberg, SM,
Berry GT,
Yandrasitz JR,
and
Grunstein MM.
Maturational regulation of inositol 1,4,5-trisphosphate metabolism in rabbit airway smooth muscle.
J Clin Invest
88:
2032-2038,
1991[ISI][Medline].
57.
Sahi, J,
Bissonnette G,
Goldstein J,
Layden T,
and
Rao M.
Effect of Ca2+-dependendent regulators in Cl transport and protein phosphorylation in human colon (Abstract).
FASEB J
10:
A544,
1996.
58.
Sahi, J,
Goldstein JL,
Layden TJ,
and
Rao MC.
Cyclic AMP- and phorbol ester-regulated Cl permeabilities in primary cultures of human and rabbit colonocytes.
Am J Physiol Gastrointest Liver Physiol
266:
G846-G855,
1994
59.
Sahi, J,
Wiggins MP,
Gibori GB,
Layden TJ,
and
Rao MC.
Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes.
J Cell Physiol
168:
276-283,
1996[ISI][Medline].
60.
Schwartz, SM,
Jing S,
Hosteller B,
and
Walkins JB.
Taurodeoxycholate transport in the developing ileum: structural/functional relationships (Abstract).
Gastroenterology
82:
1174A,
1982.
61.
Selvaraj, N,
Venkatasubramanian J,
Vidyasagar D,
and
Rao MC.
Ontogeny of cGMP stimulated chloride transport in rabbit colon (Abstract).
Gastroenterology
114:
G1636,
1998.
62.
Shibata, HJ,
and
Ghishan FK.
Intestinal calcium transport in spontaneously hypertensive rats and their genetically matched Wistar-Kyoto rats. I. Endoplasmic reticulum.
J Hypertens
8:
473-477,
1990[ISI][Medline].
63.
Shiff, SJ,
Soloway RD,
and
Snape WJ, Jr.
Mechanism of deoxycholic acid stimulation of the rabbit colon.
J Clin Invest
69:
985-992,
1982[ISI][Medline].
64.
Simon, B,
Cyzgan P,
Stiehl A,
and
Kather H.
Human colonic adenylate cyclase: effects of bile acids.
Eur J Clin Invest
8:
321-323,
1978[ISI][Medline].
65.
Suchy, F.
Bile formation: mechanisms and development.
In: Liver Diseases in Children, edited by Suchy MF.. St. Louis, MO: Mosby, 1994, p. 57-80.
66.
Tsien, R,
and
Pozzan T.
Measurement of cytosolic free Ca2+ with quin2.
Methods Enzymol
172:
230-262,
1989[ISI][Medline].
67.
Xie, W,
Kaetzel MA,
Bruzik KS,
Dedman JR,
Shears SB,
and
Nelson DJ.
Inositol 3,4,5,6-tetrakisphosphate inhibits the calmodulin-dependent protein kinase II-activated Cl conductance in T84 colonic epithelial cells.
J Biol Chem
271:
14092-14097,
1996
68.
Yamada, M,
Mizuguchi M,
Rhee SG,
and
Kim SU.
Developmental changes of three phosphoinositide-specific phospholipase C isozymes in the rat nervous system.
Brain Res Dev Brain Res
59:
7-16,
1991[ISI][Medline].