Differences in Ca2+ signaling underlie age-specific effects of secretagogues on colonic Clminus transport

Jayashree Venkatasubramanian1, Nataraja Selvaraj1, Maria Carlos2, Stanley Skaluba1, Mark M. Rasenick1, and Mrinalini C. Rao1

Departments of 1 Physiology and Biophysics and 2 Pediatrics, University of Illinois at Chicago, Chicago, Illinois 60612-7342


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, Galpha 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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); [gamma -32P]ATP (DuPont-NEN, Wilmington, DE); polyclonal Gq antibody (David Manning, Philadelphia, PA); Pansorbin cells (Calbiochem, San Diego, CA); and IP3 radioimmunoassay and enhanced chemiluminescence (ECL) kits (Amersham, Buckinghamshire, UK). All other reagents were of analytical grade and were purchased from Sigma Chemical (St. Louis, MO).

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

Cl- transport was measured using the halide-sensitive fluorescent probe, MQAE, as described earlier (14, 58). Briefly, colonocytes were loaded with 5 mM MQAE for 5 min at room temperature and for 90 min on ice in buffer A that contained (in mM) 5 HEPES (pH 7.4), 110 NaCl, 1 MgCl2, 1 CaCl2, 5 dextrose, 50 mannitol, and 1 K2SO4. Cells were then Cl- depleted for 30 min in buffer B that contained (in mM) 5 HEPES (pH 7.4), 110 sodium isethionate, 1 MgSO4, 5 dextrose, 50 mannitol, 1 K2SO4, and 1 CaSO4. Fluorescence was measured at Exlambda 350 nm and Emlambda 460 nm, using a Photon Technology International Alphascan spectrofluorimeter (Princeton, NJ). In each test sample, ~104 cells were used. When cells were transferred from a Cl--free to a Cl--containing buffer, Cl- entered the cell via all available transporters, that is, the Na+-K+-2Cl- cotransporter and Cl- channels. MQAE fluorescence is quenched by Cl-, and the rate of quenching is a reflection of Cl- permeability via these pathways (58). Cl- influx was measured under basal conditions ± secretagogues ± DPC ± furosemide and/or ± DPC + furosemide. DPC (50 µM) and furosemide (10 µM) specifically inhibit Cl- channels and the Na+-K+-2Cl- cotransporter, respectively (58). Transport was calculated as described (58) and expressed as Cl- influx or inhibitor-sensitive Cl- influx in millimolar per second. In the present study and in earlier reports from this laboratory (58, 59), none of the secretagogues affect the inhibitor-insensitive component of Cl- transport, suggesting that they primarily alter DPC- and/or furosemide-sensitive transporters. In a variety of cell types, including airway epithelia, T84 cells, and primary human colonocytes, secretagogue-stimulated Cl- influx, as measured by MQAE [or 6-methoxy-N-(3-sulfopropyl)quinolinium] fluorescence, mimicked Cl- transport, as measured by radioisotope tracers.

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 (Exlambda 340/380 nm; Emlambda 505 nm).

Intracellular 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 [alpha -32P]GTP (AAGTP), a hydrolysis-resistant photoaffinity GTP analog. The cells were incubated in a cocktail that contained 50 µM GDP + 1 µM AAGTP + 1 mM MgCl2 ± neurotensin (0.5-20 µM) or ± TDC (25-200 µM) at room temperature for 5 min. They were then transferred to an aluminum block on ice and ultraviolet irradiated at a 3-cm distance for 4 min. The reaction was quenched with ice-cold Hanks' buffer (pH 7.4) that contained 1 mM MgCl2 and 4 mM DTT. After labeling, the samples were precipitated with 1 ml 10% TCA on ice (1 h), treated with acidified ether (1 ml) for 30 min, and the pellet dissolved in Laemmli buffer and subjected to SDS-PAGE (10% gel). The AAGTP-labeled proteins were then visualized and quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

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 Galpha q and Galpha 11 members of the Gq family. AAGTP-labeled cells were irradiated and frozen immediately in RIPA buffer that contained 50 mM Tris · HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate. The cells were thawed, homogenized, and centrifuged at 10,000 g for 5 min. The supernatant was incubated with antibody (1:20) overnight at 4°C. The immune complex was precipitated by incubation for 1 h at 4°C with RIPA buffer-prewashed Pansorbin cells and centrifuged. The pellet was washed twice in RIPA buffer, and the protein recovered by heating at 60°C for 10 min in 50 µl Laemmli buffer. Proteins were analyzed by immunoblotting.

Statistics

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TDC Action on Colonocytes in Suspension

We demonstrated earlier that TDC increases Cl- transport only in adult, but not in weanling or newborn, distal colonocytes (14). These studies were performed on colonocytes attached to collagen IV matrix. Since the attached cell preparation requires a larger number of cells, we first examined if TDC could activate Cl- transport in adult distal colonocytes in suspension. Table 1 shows that TDC (50 µM) increased Cl- transport in colonocytes in suspension. To determine the underlying transport processes, the effects of DPC and furosemide either added individually or in combination were examined. As shown in Table 1, DPC (50 µM) and furosemide (10 µM), when added separately, inhibited basal and TDC-stimulated Cl- transport. However, maximal inhibition was seen when both inhibitors were added together. Thus TDC affects both the Cl- channels and the Na+-K+-2Cl- cotransporter. We previously demonstrated that in rabbit colonocytes of all ages, Cl- transport stimulated by the cAMP-dependent agents PGE1, forskolin, and 8-Br-cAMP or by the PKC activator, PDB, is inhibited by DPC and furosemide added separately or in combination (14, 58). This was also seen with the actions of neurotensin or serotonin in adult colonocytes (59). In these studies as well, maximal inhibition was seen in the presence of DPC + furosemide. Therefore, in subsequent experiments, Cl- transport was assessed in the presence or absence of both inhibitors added together.

                              
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Table 1.   Effect of inhibitors, added individually or in combination, on TDC-stimulated Cl- transport in adult colonocytes

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.


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Fig. 1.   Effect of taurodeoxycholic acid (TDC) on Cl- transport in adult distal colonocytes in suspension. Distal adult colonocytes in suspension were incubated with different doses of TDC for 5 min, and Cl- influx in the presence and absence of inhibitors [50 µM diphenylamine-2-carboxylate (DPC) + 10 µM furosemide] was measured as described in MATERIALS AND METHODS. TDC caused a dose-dependent increase in inhibitor-sensitive Cl- influx in colonocytes in suspension (n = 4; *P < 0.025, different from basal). In all subsequent figures, values are represented as inhibitor-sensitive Cl- influx, which is the difference between influx values in the absence and presence of inhibitors (50 µM DPC + 10 µM furosemide).

Segment and Chemical Specificity of Bile Acid Action

To determine whether other bile acids, present in abundance in the colon, regulate Cl- transport, and whether they show segment-specific effects, we studied the effects of TCDC (50 µM) and TC (50 µM) on Cl- transport in adult proximal and distal colonocytes. Although the dihydroxy bile acids TCDC and TDC stimulated Cl- influx in the distal colon, neither reagent had any effect in the proximal colon (Table 2). Furthermore, the trihydroxy primary bile acid, TC, did not alter Cl- transport in either colonic segment (Table 2).

                              
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Table 2.   Effect of various bile acids on Cl- transport in adult colonocytes

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.


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Fig. 2.   Effect of indomethacin on TDC-stimulated Cl- transport (A) and effect of TDC on cAMP content (B) and PKC activity (C) in primary cultures of adult rabbit distal colonocytes. A: effect of indomethacin (1 µM) on basal and TDC (50 µM)-stimulated Cl- influx was measured in colonocytes. Results are expressed as inhibitor-sensitive Cl- transport in millimolar per second. *P < 0.05, significantly different from basal values (n = 4). B: cells were incubated with buffer, forskolin (1 µM), or TDC (50 µM) for 5 min. cAMP content of these cells was measured as described in MATERIALS AND METHODS and expressed as fmoles/106 cells. *P < 0.025, different from basal values (n = 5). C: cells were treated with TDC (50 µM) or phorbol dibutyrate (1µM), and cytosolic and membrane-associated PKC activity was measured as described in MATERIALS AND METHODS. Protein kinase C (PKC) activity was expressed as pmol · min-1 · µg-1 protein. *P < 0.01 and **P < 0.001, different from basal values (n = 5). Each n value represents cells pooled from 3 adult rabbits. [cAMP]i, intracellular cAMP concentration.

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).


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Fig. 3.   Effect of 50 µM TDC on intracellular Ca2+ in newborn, weanling, and adult distal colonocytes (A) and effects of chelating extracellular Ca2+ ([Ca2+]o) (B) or intracellular Ca2+ concentration ([Ca2+]i) (C) on TDC-induced Cl- transport in adult distal colonocytes. A: primary cultures of distal colonocytes of all ages were loaded with fura 2-AM, and intracellular calcium measured ± 50 µM TDC and was expressed as a fluorescence ratio (Exlambda 340/380 nm; Emlambda 505 nm). *P < 0.05, different from basal values (n = 5). B: adult distal colonocytes were loaded with 6-methoxy-quinolyl acetoethyl ester (MQAE) and transferred to either a Cl--free buffer or Cl--free buffer with low Ca2+ and 1 mM EGTA ([Ca2+]o ~10 pM). Cl- influx measured ± TDC (50 µM) or ± A-23187 (1 µM) and is represented as inhibitor-sensitive Cl- influx. *P < 0.025, different from basal values (n = 4). **P < 0.05, different from ionophore-stimulated Cl- influx in the presence of [Ca2+]o = 1 mM. C: cells were loaded with MQAE for 90 min at 4°C and transferred to a Cl--free buffer as described in MATERIALS AND METHODS. [Ca2+]i was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 25 µM) for 20 min, and Cl- influx was measured ± TDC (50 µM) or ± PGE1 (1 µM) in the presence of [Ca2+]o ~10 pM. *P < 0.01, different from basal values (n = 4).

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.

Together, the results presented in this section demonstrate that TDC stimulates Cl- transport in adult rabbit distal colonocytes by increasing [Ca2+]i.

Effect of Ca2+-Dependent Secretagogues on Cl- Transport and Intracellular Ca2+

We next determined whether other Ca2+-dependent neurohumoral modulators also exhibit age-specific differences in their secretagogue action. We previously demonstrated in adult rabbit distal colonocytes that at concentrations of 1 µM, neurotensin or histamine caused a significant increase in Cl- influx (59). However, at a dose of 1 µM, neither neurotensin, histamine, nor bethanechol altered Cl- transport in weanling colonocytes (data not shown). Therefore, we examined the effect of a 10-fold higher concentration. As shown in Fig. 4A, in marked contrast to adult colonocytes (59), even at 10 µM each, none of these agents altered Cl- transport in weanling colonocytes (n = 4). However, forskolin increased Cl- influx in both adult and weanling colonocytes (Fig. 4A), indicating that these cells are capable of exhibiting cAMP-sensitive Cl- transport.


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Fig. 4.   Effects of Ca2+-dependent secretagogues on Cl- influx in adult and weanling colonocytes (A) and on [Ca2+]i in weanling colonocytes (B). A: Cl- transport was measured as described in MATERIALS AND METHODS in overnight cultures of colonocytes. Transport, measured ± secretagogues and ± furosemide (10 µM) + DPC (50 µM), is depicted as inhibitor-sensitive Cl- influx, which is the difference in flux in the presence and absence of inhibitors. B: weanling colonocytes were loaded with fura 2-AM and the effects of sequential addition of secretagogues (a-d) on [Ca2+]i estimated. A representative trace, depicting fluorescence ratio (Exlambda 340/380 nm; Emlambda 505 nm) is shown and the quantitation provided. Values in A and B are means ± SE (n = 4, each in triplicate); *P < 0.025 different from basal.

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

The inability of Ca2+-dependent secretagogues to stimulate Cl- transport or [Ca2+]i in weanling colonocytes could be due to an overall "immature" Ca2+ response pathway. We therefore compared the effects of the Ca2+ ionophore A-23187 (1 µM) on Cl- transport and [Ca2+]i in the distal colonocytes of all age groups. Distal colonocyte preparations, regardless of age, responded to the ionophore with increases in [Ca2+]i (Fig. 5A) and Cl- transport (Fig. 5B). This suggests that the distal steps in Ca2+ signaling, leading from an increase in [Ca2+]i to Cl- transport, are extant in weanling colonocytes. The nonresponsiveness of the weanling cells to Ca2+-dependent secretagogues must, therefore, lie at a proximal signal transduction step. Since a number of Ca2+-dependent modulators act via a receptor right-arrow G protein right-arrow PLC cascade, likely candidates could be G protein or PLC activation.


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Fig. 5.   Effect of A-23187 (1 µM) on [Ca2+]i (A) and Cl- transport (B) in distal colonocytes from newborn, weanling, and adult rabbits. A: primary cultures of distal colonocytes were loaded with fura 2-AM and [Ca2+]i measured ± 1 µM A-23187 and expressed as a fluorescence ratio (Exlambda 340/380 nm; Emlambda 505 nm). *P < 0.05, different from basal (n = 4). B: distal colonocytes were loaded with MQAE as described in MATERIALS AND METHODS and Cl- influx measured ± 1 µM A-23187. Values are expressed as inhibitor-sensitive Cl- influx in millimolar per second. *P < 0.025, different from basal (n = 5).

Activation of G Proteins

In heterotrimeric G protein-coupled signaling systems, binding of ligand to receptor increases GTP binding to Galpha . Therefore, assessment of GTP binding would indicate both receptor binding and activation of G proteins. This was first examined by utilizing the photoaffinity azido analog, AAGTP (52), and neurotensin as the representative agent. Neurotensin was selected because it is known to activate the G protein right-arrow PLC cascade in other systems and it was the most potent stimulator of Ca2+-mediated Cl- transport in adult rabbit colonocytes (59). Adult and weanling colonocytes were permeabilized to increase accessibility of AAGTP and then exposed to buffer or neurotensin. The major protein labeled with AAGTP in adult colonocytes was a 42-kDa protein, and this labeling was set at 100% (Fig. 6A). Neurotensin caused a dose-dependent increase in the labeling of the 42-kDa protein with increases of 146 ± 22% at 0.5 µM, 165 ± 16% at 1 µM, 183 ± 23% at 10 µM, and 184 ± 14% at 20 µM (n = 8). At 20 µM neurotensin, but not in control samples, the labeling of a 40-kDa species was also observed. As shown in Fig. 6B, 1 µM neurotensin similarly stimulated the labeling of a 42-kDa protein in weanling colonocytes (control 100%; neurotensin 180 ± 8%; n = 8). These data suggest that adult and weanling colonocytes possess neurotensin receptors as well as neurotensin-activatable G protein. In addition, there is a basal level of activation of a 42-kDa protein in control cells.


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Fig. 6.   Effect of neurotensin on G protein activation in colonocytes (A and B) and identification of 42-kDa protein as Galpha q (C and D). Cells were labeled with azidoanilido [alpha -32P]GTP (AAGTP) ± neurotensin as described in MATERIALS AND METHODS. A: adult colonocytes, dose-dependent effects of neurotensin on G protein activation (AAGTP labeling), expressed as percent stimulation over basal (top). B: weanling colonocytes, neurotensin-stimulated G protein activation in weanling colonocytes, expressed as percent stimulation over basal (top). In A and B, values are means ± SE; n = 8, each in duplicate; *P < 0.01, different from control. A and B: bottom shows representative autoradiograms. C and D: identification of the 42-kDa protein. C: adult colonocytes labeled with AAGTP + neurotensin (1 µM) were subjected to immunoprecipitation (IP) and immunoblotting with anti-Gq antibody (see MATERIALS AND METHODS). Lane 1: non-IP AAGTP-labeled cells; lane 2: IP with normal rabbit serum (NRS); lane 3: IP with anti-Gq; and lane 4: non-IP lysates of Sf9 cells transfected with Galpha q. D: autoradiogram of the nitrocellulose membrane from C exposed to PhosphorImager screen. Similar data were obtained in duplicate experiments.

To identify the 42-kDa protein, immunoprecipitation of solubilized total protein from AAGTP-labeled colonocytes was performed using an antibody that recognizes both Galpha q and Galpha 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 Galpha 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 Galpha q or Galpha 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%).


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Fig. 7.   Effect of TDC on G protein activation in adult (A) and weanling (B) colonocytes. Cells were labeled with AAGTP ± TDC (25-200 µM) as described in MATERIALS AND METHODS. A: adult colonocytes, dose-dependent effects of TDC on G protein activation (AAGTP labeling), expressed as percent activation over basal. B: weanling colonocytes, G protein activation in response to TDC (50 and 100 µM), expressed as percent activation over basal. In A and B, values are means ± SE; n = 8, each in duplicate (*P < 0.01). Representative autoradiograms are shown (bottom).

Thus neurotensin, but not TDC, increases Galpha 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.


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Fig. 8.   Effect of Ca2+-dependent secretagogues on 1,4,5-inositol trisphosphate (IP3) levels (A) and effect of neurotensin on total phosphatidylinositol 4,5-bisphosphate (PIP2) level (B) in adult and weanling colonocytes. IP3 levels and PIP2 levels in the primary cultures of colonocytes were measured as described in MATERIALS AND METHODS. A: IP3 levels in response to neurotensin (1 µM), bethanechol (Bch; 10 µM), TDC (50 µM), and PGE1. Values represent means ± SE, n = 5; in the adult, neurotensin-, bethanechol-, and TDC-treated values are P < 0.01, different from control values. B: PIP2 levels ± neurotensin (1 µM). Values represent means ± SE, n = 3, aP < 0.025, different from control values. In IP3 and PIP2 measurements, each n value was the average of triplicate determinations.

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Cl- transport is restricted to the dihydroxy bile acids, TCDC and TDC, with the trihydroxy primary bile acid TC having no effect. Furthermore, the effect of these bile acids is segment specific, affecting only the distal and not the proximal colonocytes. It is interesting that taurocholate does not have an effect in the proximal colon despite the apparent presence of a taurocholate-transporting organic anion transporter (OATP). With the use of a rat liver cDNA probe, OATP mRNA was detected in the proximal but not in the distal rabbit colon (32). Details on the effects of bile acids on ion transport in the intact rabbit proximal colonic tissues are sparse. Luminal deoxycholate stimulates migratory action potentials in rabbit proximal colonic smooth muscle (63). However, in mouse, the proximal colon is much less responsive to bile acids than the distal colon (20), indicating that segmental differences in bile acid action may be a common feature. This segment specificity of bile acid action may have physiological relevance. The ~5% of primary bile acids produced by the liver that enters the colon daily are dehydroxylated into secondary bile acids by colonic bacteria. Whereas lithocholic acid is largely excreted, deoxycholic acid reenters the portal circulation by passive diffusion along the length of the colonic lumen. Any residual primary bile acids could also be transported by mechanisms such as OATP. Thus the proximal colon's insensitivity to the secretagogue action of bile acids prevents unnecessary fluid loss in a milieu where the bile acid levels are fluctuating and higher than in the more distal colonic segments.

Mechanism 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 Cl- transport at the level of the colonocyte per se, in this species. Thus in Fig. 1, colonocytes in suspension are shown to stimulate Cl- transport in a dose-dependent manner, corroborating our earlier report on adult colonocytes attached to collagen IV matrix (14) and those of others on intact epithelial sheets (36, 49). Recently, Mauricio et al. (36) reported that either luminal or basolateral application of 100 µM deoxycholic acid stimulates electrogenic Cl- secretion in rabbit distal colonic mucosa. In contrast, only apical and not basolateral application of deoxycholate altered K+ secretion. Thus with respect to Cl- secretion, bile acids are equally effective from either surface, further validating our colonocyte cell model for studying Cl- transport.

In 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 PKCbeta 1 (45). In isolated rat hepatocytes, tauroursodeoxycholic acid, but not TC, activates PKCalpha (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

As shown in Fig. 4, the age-dependent refractoriness is not restricted to bile acids but extends to a variety of Ca2+-dependent secretagogues such as bethanechol, histamine, and neurotensin. These agents are potent secretagogues in adults but fail to stimulate Cl- transport in weanlings. As in the action of TDC, this is due to an inability of these agents to raise [Ca2+]i in the weanling animal. Activators of the cAMP cascade do not show this age-dependent effect (Fig. 4, Ref. 14).

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-gamma , 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-gamma 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 Galpha q in other cell types (39). However, the present study shows that TDC did not activate Gq or any other G protein detectable by AAGTP labeling in either weanling or adult rabbits (Fig. 7). Yet, TDC activated PLC to increase IP3 (Fig. 8) and [Ca2+]i (Fig. 3) in adult distal colonocytes. Therefore, although neurotensin and TDC both stimulate PLC, they may be respectively activating PLC via a G protein-dependent and a G protein-independent mechanism. It remains to be determined whether TDC and neurotensin activate the same or different PLC isoforms/subtypes. Although conventional dogma (8, 35, 43) would indicate that neurotensin may be activating Gq and PLC-beta , whereas TDC would be activating a PLC-gamma isoform with the increasing evidence of cross talk and compartmentalization, it is conceivable that the two pathways converge on a common PLC target.

Physiological/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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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