Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M411553200 on January 6, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11859-11868, March 25, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11859    most recent
M411553200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saksena, S.
Right arrow Articles by Dudeja, P. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saksena, S.
Right arrow Articles by Dudeja, P. K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Involvement of c-Src and Protein Kinase C{delta} in the Inhibition of Cl-/OH- Exchange Activity in Caco-2 Cells by Serotonin*

Seema Saksena, Ravinder K. Gill, Sangeeta Tyagi, Waddah A. Alrefai, Zaheer Sarwar, Krishnamurthy Ramaswamy, and Pradeep K. Dudeja{ddagger}

From the Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612

Received for publication, October 12, 2004 , and in revised form, December 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter and intercellular messenger regulating various gastrointestinal functions, including electrolyte transport. To date, however, no information is available with respect to its effects on the human intestinal apical anion exchanger Cl-/OH- (). The present studies were therefore undertaken to examine the direct effects of serotonin on OH- gradient-driven 4,4'-diisothiocyanato-stilbene-2, 2'-disulfonic acid-sensitive 36Cl- uptake utilizing the post-confluent transformed human intestinal epithelial cell line Caco-2. Our results demonstrate that serotonin inhibits Cl-/OH- exchange activity in Caco-2 cells via both tyrosine kinase and Ca2+-independent protein kinase C{delta}-mediated pathways involving either 5-HT3 or 5-HT4 receptor subtype. The data consistent with our inference are as follows. (i) The short term treatment of cells with 5-HT (0.1 µM) for 15-60 min significantly decreased Cl-/OH- exchange (50-70%, p < 0.05). (ii) The specific agonists for 5-HT3, m-chlorophenylbiguanide, and 5-HT4, 3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, mimicked the effects of serotonin. (iii) Tropisetron dual inhibitor for both the 5-HT3/4 receptor subtypes significantly blocked the inhibition, whereas specific 5-HT3 (Y-25130) or 5-HT4 receptor (RS39604) antagonist failed to block the inhibitory effects of 5-HT. (iv) The Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl ester) had no effect on the serotonin-induced inhibition. (v) The specific protein kinase C (PKC) inhibitors chelerythrine chloride or calphostin C completely blocked the inhibition by 5-HT. (vi) The specific inhibitor for PKC{delta}, rottlerin, significantly blocked the inhibition by 5-HT. (vii) The specific tyrosine kinase inhibitor, herbimycin, or Src family kinase inhibitor, PP1, abolished the 5-HT-mediated inhibition of Cl-/OH- exchange activity. (viii) 5-HT stimulated tyrosine phosphorylation of c-Src kinase and PKC{delta}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The gastrointestinal tract is an important source of the endogenous amine, serotonin, mainly stored in the mucosal enterochromaffin cells and in the enteric nervous system (1). 5-HT1 is continuously released from the enterochromaffin cells in the intestinal mucosa into the portal circulation and the gut lumen in response to a variety of luminal stimuli such as change in pH or osmolarity and mechanical and chemical stimuli (2). 5-HT is an important neurotransmitter and intercellular messenger to modulate gastrointestinal functions (3). 5-HT has been shown to alter gastrointestinal motility (4), alter gastric acid secretion, and enhance intestinal secretions (1, 5). Most of the previous studies regarding the effects of serotonin on ion transport were mainly based on the electrophysiological data including short circuit current measurements and unidirectional fluxes. These studies showed that 5-HT stimulated electrogenic Cl- secretion and inhibited Na+ and Cl- absorption in the rat colon (6), jejunum (7), ileum (8), and guinea pig ileum (9). 5-HT has also been shown to inhibit the NaCl-absorptive process in the rabbit ileum and gall bladder (10) but has no effect in rabbit and guinea pig colon (10, 11). Moreover, Sundaram et al. (12) have demonstrated that 5-HT inhibits Cl-/HCO-3 exchange activity in villus cells and stimulates HCO-3 secretion in crypt cells of the rabbit ileum. In contrast, 5-HT appears to have no significant effect on coupled NaCl absorption in the human jejunum (13).

5-HT exerts its effects by binding to 5-HT receptors, which are distributed on the neuronal, muscular, and epithelial structures (14). Based on pharmacological studies and molecular cloning (15), the existence of at least seven 5-HT receptors (5-HT1-7) subdivided into 14 subtypes has been discovered. With the exception of 5-HT5 and 5-HT6, 5-HT1A, 5-HT1P, 5-HT2, 5-HT3, and 5-HT4 have been reported to be expressed in the gut (16). 5-HT1, 5-HT2, and 5-HT4 receptors are coupled via guanine nucleotide binding (G) proteins to their effectors, whereas 5-HT3 receptor is a ligand-gated ion channel permeable to anions and cations (17). Previous studies in different animal models have shown that 5-HT1 receptors mediate peristaltic reflex and do not seem to play a major role in intestinal fluid transport, whereas 5-HT2, 5-HT3, and 5-HT4 receptors produce a variety of responses in the GI tract such as alterations in intestinal fluid transport (18) and modulation of motility (19).

5-HT seems to induce Cl- secretion by stimulating neural 5-HT3 receptors in the distal colon of guinea pig (20) and rat small intestine (21) and non-neural 5-HT4 receptors in the rat colon (22), human jejunum (13), and ileum (23). Recent binding studies have shown the presence of 5-HT4 receptors on the brush border membranes of the rabbit jejunal enterocytes (24). Studies have also demonstrated the existence of 5-HT2 receptors on the basolateral membranes of epithelial cells from rabbit ileum (25), human sigmoid colon (26), and guinea pig small intestinal crypt cells (27).

Although 5-HT has been known to play a major role in regulating a number of physiological functions in the GI tract, elevated levels of serotonin have been implicated in the patho-physiology of disease states such as carcinoid syndrome (7), celiac disease (28), irritable colon (29), and dumping syndrome (30). Previous studies have shown that 5-HT administration in vivo induces Cl- secretion in the rabbit small intestine (31) and acts as a mediator of diarrhea in patients with carcinoid syndrome associated with a net secretion of water and electrolytes (32). Therefore, these studies suggest an important role of 5-HT in modifying electrolyte transport process that could involve either stimulation of Cl- secretion or a decrease in Na+ and Cl- absorption. However, to date, the effects of 5-HT on the human intestinal apical membrane Cl-/OH- exchange activity have not been investigated, and the signal transduction pathways involved in 5-HT-mediated alteration of NaCl absorption have not been delineated.

In this regard, most of the effects of 5-HT on the intestinal ion transport have been shown to be mediated through various intracellular mediators such as phosphoinositides, Ca2+, and prostaglandins (33). Activation of 5-HT4 receptors has been reported to change the levels of cAMP and cGMP (34). However, studies of Eklund et al. (35) and Donowitz et al. (31) have shown that cAMP plays no role in mediating the secretory effects of 5-HT in cat and rabbit intestine. Recent studies (36) point out the role of nitric oxide in mediating the effects of 5-HT-induced Cl- secretion in the rat colon.

The current studies were therefore undertaken to examine the following: (i) the possible regulation of Cl-/OH- exchange activity in Caco-2 cells (a well established model for the human intestinal transport studies) by serotonin; (ii) to characterize the 5-HT receptor subtype(s) involved; and (iii) the signal transduction pathways involved in this process. Our current studies demonstrate that serotonin inhibits apical Cl-/OH- exchange activity in Caco-2 cells via both tyrosine kinase and protein kinase C{delta}-mediated pathways involving either the 5-HT3 or 5-HT4 receptor subtype.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Radionuclide 36Cl as HCl was obtained from PerkinElmer Life Sciences. Caco-2 cells were obtained from the ATCC (Manassas, VA). 5-Hydroxytryptamine-creatinine sulfate (5-HT, serotonin), 4,4'-diisothiocyanato-stilbene-2, 2'-disulfonic acid (DIDS), and tropisetron were obtained from Sigma. Specific agonists for 5-HT3, m-chlorophenylbiguanide, and 5-HT4, 3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, specific antagonists for 5-HT3 (Y-25130) and 5-HT4 (RS39604), were obtained from TOCRIS (Ellisville, MO). BAPTA-AM was obtained from Molecular Probes (Eugene, Oregon). Chelerythrine chloride, calphostin C, herbimycin, and 4-amino-5-(4methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine were obtained from Biomol (Plymouth Meeting, PA). Ro318220, Go6796, and rottlerin were procured from Calbiochem. Affinity-purified rabbit polyclonal antibody against PKC{delta}, goat anti-rabbit antibody conjugated to horseradish peroxidase, and protein A-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphorylated p60 (p-Src, Tyr416) and polyclonal anti-Lck were purchased from Cell Signaling (Cell Signaling Technology, Beverly, MA). Anti-phosphotyrosine (4G10) HRP-conjugated antibody, monoclonal anti-Src, anti-Fyn, or polyclonal anti-Yes and anti-Lyn were obtained from Upstate Biotechnology Inc. (Upstate Biotechnology, Lake Placid, NY). All other chemicals were of at least reagent grade and were obtained from Sigma or Fisher.

Cell Culture
Caco-2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter glucose, 2 mM glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, 10 mM Hepes, 1% essential and nonessential amino acids, and 20% fetal bovine serum, pH 7.4, in 5% CO2, 95% O2 at 37 °C. For the uptake experiments, cells from passages between 20 and 25 were plated in 24-well plates at a density of 2 x 104 cells/ml. Confluent monolayers were used for transport experiments at the10th to the 12th day post-plating (i.e. 5-7 days post confluence). To study the effect of serotonin (5-HT) on Cl-/OH- exchange activity, cells were acutely exposed to 100 nM 5-HT for 15, 30, or 60 min.

In a separate set of experiments, cells were pretreated with tropisetron (dual 5-HT3/5-HT4 antagonist, 1 µM) or 5-HT3 receptor antagonist Y25130 (300 nM), or 5-HT4 receptor antagonist RS39604 (300 nM), or Ca2+ chelator BAPTA-AM (20 µM), or the specific PKC inhibitors chelerythrine chloride (2 µM) and calphostin C (200 nM), or the specific PKC{alpha} inhibitor Go6976 (5 nM), the specific PKC{epsilon} inhibitor Ro318220 (100 nM), the specific PKC{delta} inhibitor rottlerin (10 µM), or the general tyrosine kinase inhibitor herbimycin (1 µM) or the Src family kinase inhibitor PP1 (10 µM) for 1 h prior to the addition of serotonin (100 nM). These inhibitors were also co-incubated along with serotonin for another 1 h. In another set of experiments, cells were treated with specific 5-HT3 (m-chlorophenylbiguanide, 1-100 µM) and 5-HT4 (3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, 0.1-10 µM) agonists for 1 h.

36Cl-
Uptake Chloride uptake experiments were performed essentially as described by Olsnes et al. (37) with some modifications (38, 39). Caco-2 cells were incubated with Dulbecco's modified Eagle's base media containing 20 mM Hepes/KOH, pH 8.5, for 30 min at room temperature. The media were removed, and the cells were rapidly washed with 1 ml of tracer-free uptake mannitol buffer containing 260 mM mannitol, 20 mM Tris/MES, pH 7.0. The cells were then incubated with the uptake buffer for a 5-min period. This period was chosen because it falls within the linear range of Cl- uptake in this system (38, 39). For 36Cl- uptake studies, the uptake buffer was the mannitol buffer containing 1.4 µCi of 36Cl- (2.9 mM) of hydrochloric acid (specific activity-17.12 mCi/g) ± 0.3 mM DIDS. The uptake was terminated by removing the buffer and washing the cells rapidly two times with 1 ml of ice-cold phosphate-buffered saline, pH 7.2 (PBS). Finally, the cells were solubilized by incubation with 0.5 N NaOH for 4 h. The protein concentration was measured by the method of Bradford (40), and the radioactivity was counted by a Packard liquid scintillation analyzer, TRI-CARB 1600-TR (Packard Instrument Co.). 0.3 mM DIDS-sensitive chloride uptake was considered as Cl-/OH- exchange, and the uptake values were expressed as nmol/mg protein/5 min.

Protein Tyrosine Phosphorylation
Caco-2 cells grown to confluence in 6-well plates at a density of 10 x 104 (Corning Glass) were treated with serotonin (100 nM) for the 15-, 30-, and 60-min period or with the specific Src kinase inhibitor PP1 (10 µM). Cells were washed with ice-cold PBS three times and lysed in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, and 1x complete protease inhibitor mixture. The cells were homogenized by passing 10 times through a 26-guage needle. The lysate was centrifuged at 5000 rpm for 5 min at 4 °C. Equal amounts (~75 µg/sample) of protein, as determined by the Bradford assay, were combined with the SDS sample buffer and boiled for 5 min. Proteins were separated by electrophoresis on 8% SDS-polyacrylamide gels and transblotted to nitrocellulose membranes. The protein-bound nitrocellulose membranes were first incubated for 20 min at room temperature in blocking buffer containing 1x PBS and 3% nonfat dry milk. Nitrocellulose membranes were then incubated with the anti-phosphotyrosine HRP-conjugated antibody (1:1000 dilution) in the blocking buffer overnight at 4 °C. The membranes were rinsed in water and washed with the wash buffer containing 1x PBS and 0.05% Tween 20 for 45 min with agitation, during which time the wash buffer was changed every 5 min. Tyrosine-phosphorylated bands were visualized with ECL detection reagents.

Immunoprecipitation and Immunoblot Analysis
After treatment of Caco-2 cells with serotonin (100 nM) for different times or specific Src kinase inhibitor PP1 (10 µM), specific 5-HT3 receptor antagonist Y25130 (300 nM), 5-HT4 receptor antagonist RS39604 (300 nM), or addition of both 5HT3 and 5HT4 receptor antagonists, cells were washed with phosphate-buffered saline, and lysates were prepared as described above. The protein content of the resulting supernatant was adjusted to contain 500 µg in 200 µl of lysis buffer and incubated with the polyclonal antibody against novel PKC{delta} (2 µg) overnight at 4 °C. After incubation, immune complexes were precipitated by using protein A-agarose beads. In a separate set of experiments, 5-HT-treated lysates at different time points were incubated with monoclonal anti-c-Src or anti-Fyn or polyclonal anti-Yes, anti-Lck, or anti-Lyn overnight at 4 °C. After incubation, the immune complexes were precipitated using protein A- or G-agarose beads. The immunoprecipitates were collected by centrifugation and washed four times with the lysis buffer. Proteins were separated by electrophoresis on 8 or 12% SDS-polyacrylamide gels and transblotted to nitrocellulose membranes.

To detect PKC{delta} phosphorylation, the protein-bound nitrocellulose membranes were incubated with anti-phosphotyrosine-HRP-conjugated antibody, and bands were visualized with the ECL method. To detect the phosphorylation of each of the Src family members, the protein-bound nitrocellulose membranes were first incubated for 60 min at room temperature in blocking buffer containing 1x TBS (Tris-buffered saline), 0.1% Tween 20 with 5% nonfat dry milk. Nitrocellulose membranes were then incubated with the anti-phospho-Src Tyr416 (1:1000 dilution) in the dilution buffer containing 1x TBS, 0.1% Tween 20 with 5% bovine serum albumin overnight at 4 °C. The membranes were washed with the wash buffer containing 1x TBS and 0.1% Tween 20 for 15 min with agitation, during which time the wash buffer was changed every 5 min. Finally, the membranes were probed with HRP-conjugated goat anti-rabbit IgG antibody (1:2000 dilution), and bands were visualized with ECL detection reagents.

Membrane Translocation of PKC{delta}
Subcellular Fractionation—Caco-2 cells grown to confluence in 6-well plates at a density of 10 x 104 (Corning Glass) were treated with serotonin (100 nM) for different times, washed with ice-cold PBS three times, and scraped into 400 µl of the cold homogenization buffer (HB) containing 20 mM Tris-HCl, pH 7.5, 250 mM sucrose, 4 mM EDTA, 2 mM EGTA, and 1x complete protease inhibitor mixture. The cells were homogenized on ice with 25 strokes of a glass tissue homogenizer. The resulting homogenate was ultracentrifuged at 59,000 rpm for 50 min at 4 °C (OptimaTM TLX Ultracentrifuge; Beckman Instruments). The supernatant was designated the cytosolic fraction. The pellet was resuspended in 150 µl of the HB containing 0.5% (v/v) Triton X-100 by brief sonication and incubated on ice for 30 min. At the end of the incubation period, the samples were centrifuged at 14,000 rpm for 20 min at 4 °C. The resulting supernatant was designated the membrane fraction.

Gel Electrophoresis and Western Blotting—Equal amounts of protein (~75 µg/sample), as determined by the Bradford assay, were combined with Laemmli's Sample Buffer containing 5% (v/v) {beta}-mercaptoethanol and then boiled for 5 min. Proteins were separated by electrophoresis on 8% SDS-polyacrylamide gels and trans-blotted to nitrocellulose membranes. The protein-bound nitrocellulose membranes were first incubated for 1 h at room temperature in blocking buffer containing 1x PBS, 0.1% Tween 20, and 5% nonfat dry milk. Nitrocellulose membranes were then incubated with the polyclonal antibody specific to PKC{delta} (1:800 dilution) in the blocking buffer containing 1x PBS, 0.1% Tween 20, and 1% nonfat dry milk for 1 h at room temperature and rinsed for 30 min with a wash buffer containing 1x PBS and 0.1% Tween 20. Finally, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:2000 dilution) for 1 h at room temperature and washed for 45 min with agitation, during which the wash buffer was changed every 5 min. PKC bands were visualized with ECL detection reagent.

Statistical Analysis
Results were expressed as mean ± S.E. Each independent set represents mean ± S.E. of data from at least 9 wells used on three separate occasions. Student's t test was used for statistical analysis. p < 0.05 or less was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Acute Effects of 5-HT
To examine the effects of 5-HT on the Cl-/OH- exchange activity, Caco-2 cells were incubated with 5-HT at a concentration of 100 nM for different time points, and the DIDS-sensitive Cl-/OH- exchange activity was assessed. As shown in Fig. 1, a significant decrease (~60%) in Cl-/OH- exchange activity was observed as early as 15 min, and the inhibition remained about the same at 30 and 60 min.



View larger version (8K):
[in this window]
[in a new window]
  		FIG. 1.
Effect of 5-HT on apical Cl-/OH- exchange activity in Caco-2 cells. Caco-2 cells were incubated with 0.1 µM concentration of 5-HT in the cell culture medium for 15, 30, or 60 min. Cells were then washed with 1x PBS and were base-loaded with Hepes/KOH medium adjusted to pH 8.5 for 30 min. Cl-/OH- exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min. Results are expressed as % of control and represent mean ± S.E. of 6-9 separate experiments performed in triplicate. *, p < 0.05 compared with control. Control value: 1.41 ± 0.29 nmol/mg/5 min.

 
Role of 5-HT Receptor Subtypes in the 5-HT-mediated Inhibition of Cl-/OH- Exchange Activity in Caco-2 Cells
Effect of Tropisetron (Dual 5-HT3/4 Receptor Antagonist)—The receptor subtype mediating the 5-HT-induced inhibition of Cl-/OH- exchange activity was characterized with the use of the 5-HT receptor antagonist tropisetron. When employed at a final concentration of 0.1 µM (concentration at which it is a selective 5-HT3 receptor antagonist), it had no effect on the 5-HT-mediated inhibition of Cl-/OH- exchange activity (data not shown). However, when used at a concentration of 1 µM (1 h), at which it is known to antagonize the 5-HT4 as well as the 5-HT3 receptors, tropisetron caused a significant reversal in the 5-HT-mediated inhibition of Cl-/OH- exchange activity (Fig. 2A). Because tropisetron is characterized as a weak competitive 5-HT4 antagonist (having higher affinity for the 5-HT3 receptor), we therefore used more specific and selective individual receptor antagonists.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2.
Role of 5-HT antagonists in 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. Caco-2 cells were preincubated with dual 5-HT3/4 receptor antagonist tropisetron (1 µM) (A); Y25130 (300 nM), 5-HT3 receptor antagonist (B); RS39604 (RS) (300 nM, 5-HT4 receptor antagonist (C); or both Y2513 and RS39604 (Y + RS) (300 nM) (D) for 60 min in the cell culture medium and then co-incubated with 0.1 µM of 5-HT for another 1 h. Cl-/OH- exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min (as mentioned in the legend to Fig. 1). Results are expressed as % of control or actual value and represent mean ± S.E. of 6-9 separate experiments performed in triplicate. *, p < 0.05 compared with control. Control values: 1.50 ± 0.22 (A), 1.14 ± 0.12 (B), 0.74 ± 0.08 (C), and 1.33 ± 0.12 (D) at nmol/mg/5 min.

 
Effect of 5-HT Receptor Antagonists—The specific 5-HT3 receptor antagonist Y25130 (300 nM) failed to block the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells (Fig. 2B), indicating that if 5-HT3 receptor is blocked, 5-HT can still mediate its effects through the 5-HT4 receptor. Similar results were observed with the selective 5-HT4 receptor antagonist RS39604 (300 nM, 1000-fold selectivity over 5-HT3 receptor) (41) (Fig. 2C).

Studies were also performed in the presence of the 5-HT3 (Y25130) and 5-HT4 (RS39604) receptor antagonists together on the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. The inhibitory effects of 5-HT were significantly abolished in the presence of both the 5-HT3 and 5-HT4 antagonists added together (Fig. 2D). Hence, the observed findings with the dual and individual receptor antagonists suggest that either of the 5-HT3 or 5-HT4 receptor subtypes could be sufficient for 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. When 5-HT3 receptor is blocked, 5-HT could act through the 5-HT4 receptor, and when the 5-HT4 receptor is blocked, 5-HT could act via 5-HT3 receptor.

Effect of 5-HT Receptor Agonists
To confirm further that both 5-HT3 and 5-HT4 receptor subtypes are involved in this process, specific 5-HT3, m-chlorophenylbiguanide (1-100 µM, 1 h), and 5-HT4, 3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile (0.1-10 µM, 1 h), agonists were used. Both 5-HT3 (Fig. 3A) and 5-HT4 (Fig. 3B) agonists significantly decreased the Cl-/OH- exchange activity in Caco-2 cells. These results indicate that 5-HT mediates its effects via activation of either 5-HT3 or 5-HT4 receptor as both 5-HT3 and 5-HT4 agonists could mimic the effects of 5-HT. Moreover, no additive effects were observed when both the 5HT3 and 5HT4 agonists were added together (75 ± 2% as compared with control).



View larger version (9K):
[in this window]
[in a new window]
  		FIG. 3.
Effect of 5-HT agonists in 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. Caco-2 cells were incubated with different concentrations of 5-HT3 receptor agonist m-chlorophenylbiguanide (1-100 µM) (a) or 5-HT4 receptor agonist 2-[1-(4-pieronyl) piperazinyl]benzothiazole (0.1-10 µM) (b) in the cell culture medium for 60 min. Cl-/OH- exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min (as mentioned in the legend to Fig. 1). Results are expressed as % of control value and represent mean ± S.E. of 6-9 separate experiments performed in triplicate. *, p < 0.05 compared with control. Control values: 0.94 ± 0.12 (a) and 1.58 ± 0.12 (b) at nmol/mg/5 min.

 
Effect of Ca2+ Chelator BAPTA-AM on 5-HT-mediated Inhibition of Cl-/OH- Exchange Activity in Caco-2 Cells
Previous studies have suggested that the effects of 5-HT on NaCl absorption (10) and Cl- secretion (42) are mediated by an increase in the intracellular calcium levels. We thus examined the role of calcium in mediating the effects of 5-HT by using the Ca2+ chelator BAPTA-AM (20 µM, 1 h). As shown in Table I, in the presence of BAPTA-AM there was essentially no effect on the 5-HT-mediated inhibition of Cl-/OH- exchange activity. However, previous findings from our laboratory demonstrated that exactly under the same experimental conditions, BAPTA-AM (20 µM, 1 h) significantly blocked the Ca2+-dependent effects of serotonin on Na+/H+ exchange activity (43) and the H2O2 effects on Cl-/OH- exchange activity (44) in Caco-2 cells. Collectively, these observations suggest that the inhibitory effects of 5-HT on the Cl-/OH- exchange activity in Caco-2 cells are not related to the intracellular calcium levels and further confirm that BAPTA-AM is an optimal chelating agent of calcium in Caco-2 cells.


View this table:
[in this window]
[in a new window]
  		TABLE I
5-HT-mediated inhibition of Cl–/OH– exchange activity in Caco-2 cells is Ca2+-independent

Caco-2 cells were preincubated with BAPTA-AM (20 µM), an intracellular chelator of Ca2+, for 60 min in the cell culture medium and then co-incubated with 0.1 µM of 5-HT for another 1 h. Cl–/OH– exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min. Values represent mean ± S.E. of 6-9 separate experiments performed in triplicate.

 
Possible Role of PKA
Previous studies have suggested the involvement of 5-HT4 receptors in the stimulation of cAMP (36). Therefore, it was of interest to examine the role of PKA in 5-HT-mediated effects on Cl-/OH- exchange activity in Caco-2 cells. The PKA inhibitor (Rp)-cAMP (25 µM), however, showed no significant effects on the inhibition of Cl-/OH- exchange activity by 5-HT (Table II).


View this table:
[in this window]
[in a new window]
  		TABLE II
Effect of PKA inhibitor on 5-HT-mediated inhibition of Cl–/OH– exchange activity in Caco-2 cells

Caco-2 cells were preincubated for 60 min in the presence of (Rp)-cAMP in the cell culture medium and then co-incubated with 5-HT (0.1 µM) for a period of 60 min. Cl–/OH– exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min. Values represent mean ± S.E. of 6-9 separate experiments performed in triplicate.

 
Role of PKC
Effect of PKC Inhibitors on the Ability of 5-HT to Inhibit DIDS-sensitive Cl- Uptake in Caco-2 Cells—Earlier studies have shown that serotonin can exert its effects on motility in rat stomach (45) and L-leucine transport in rabbit jejunum (25) through the activation of PKC. Hence, we next investigated the role of PKC in the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. For these studies, cells were pretreated with specific PKC inhibitors for 1 h prior to the addition of 5-HT (100 nM), followed by co-incubation for another 1 h. Chelerythrine chloride (2 µM) or calphostin C (200 nM) (Table III) completely abolished the 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. These results suggest that the observed decrease in Cl-/OH- exchange activity involved PKC activation.


View this table:
[in this window]
[in a new window]
  		TABLE III
Effect of PKC inhibitors on 5-HT-induced inhibition of Cl–/OH– exchange activity in Caco-2 cells

Caco-2 cells were preincubated for 60 min in the presence of chelerythrine chloride or calphostin C in the cell culture medium and then co-incubated with 5-HT (0.1 µM) for a period of 60 min. Cl–/OH– exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min. Values represent mean ± S.E. of 6-9 separate experiments performed in triplicate.

 
Effect of Isoform-specific Inhibitors of PKC on the 5-HT-induced Inhibition of Cl-/OH- Exchange Activity in Caco-2 Cells—To test which particular PKC isoform is involved in this effect of 5-HT, we used the specific PKC inhibitors (e.g. Go6976) to inhibit conventional (diacylglycerol- and Ca2+-dependent) PKC (e.g. PKC{alpha} and Ro318820), and rottlerin to inhibit novel (diacylglycerol-dependent and Ca2+-independent) PKCs (e.g. PKC{epsilon} and {delta}). Both Go6976 (5 nM) and Ro318220 (100 nM), added 1 h prior to 5-HT addition followed by co-incubation for another 1 h, failed to block the inhibitory effect of 5-HT on Cl-/OH- exchange activity (Table IV). However, rottlerin (10 µM, 1 h prior to 5-HT addition, followed by co-incubation for another 1 h) significantly attenuated the inhibitory effect of 5-HT on Cl-/OH- exchange activity in Caco-2 cells (Fig. 4). The data suggest that the PKC{delta} isoform sensitive to rottlerin might be the key PKC isoform responsible for mediating the inhibitory effects of 5-HT on chloride uptake, whereas PKC{alpha} or other isoforms sensitive to Go6976 and PKC{epsilon} do not appear to have any role in this process.


View this table:
[in this window]
[in a new window]
  		TABLE IV
No role for PKC{alpha} and PKC{epsilon} in 5-HT-induced inhibition of Cl–/OH– exchange activity in Caco-2 cells

Caco-2 cells were preincubated for 60 min in the presence of PKC{alpha} inhibitor, Go6976, or PKC{epsilon} inhibitor, Ro318220, in the cell culture medium and then co-incubated with 5-HT (0.1 µM) for a period of 60 min. Cl–/OH– exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min. Values represent mean ± S.E. of 6-9 separate experiments performed in triplicate.

 



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 4.
Effect of specific inhibitor of PKC{delta} on 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. Caco-2 cells were preincubated for 60 min in the presence of the PKC{delta} inhibitor rottlerin (10 µM) in the cell culture medium and then co-incubated with 5-HT (0.1 µM) for a period of 60 min. Cl-/OH- exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min (as mentioned in the legend to Fig. 1). Results represent mean ± S.E. of 6-9 separate experiments performed in triplicate. *, p < 0.05 compared with control. Control value: 0.86 ± 0.15 nmol/mg/5 min.

 
Translocation of PKC{delta} in Caco-2 Cells by Serotonin—Because PKC{delta} was found to be the possible isoform responsible for the regulation of the Cl-/OH- exchanger in Caco-2 cells, we characterized the time course for PKC{delta} membrane translocation upon serotonin treatment. Translocation of PKC{delta} from the cytosol to the membrane fractions was apparent as early as 15 min after 5-HT (100 nM) addition, and continued for at least 1 h (Fig. 5).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5.
Time course of membrane translocation of PKC{delta} in Caco-2 cells in response to 5-HT. Caco-2 cells were treated with 0.1 µM of 5-HT for the 15-, 30-, or 60-min period. The cytosolic (C) and membrane (M) fractions were subjected to SDS-PAGE and probed with specific PKC{delta} antibody to examine subcellular distribution (A). 5-HT induced translocation of PKC{delta} from cytosol to the membrane fraction as early as 15 min and continued for at least 60 min. The data were quantified by densitometric analysis and expressed as relative arbitrary units (membrane PKC{delta} compared with actin) and represent the mean ± S.E. of three determinations (B). *, p < 0.05 compared with untreated control.

 
Role of Tyrosine Kinases
Effect of Herbimycin A and PP1 on 5-HT-mediated Inhibition of Cl-/OH- Exchange Activity in Caco-2 Cells—Previous studies have suggested that in addition to the serine/threonine phosphorylation of PKC{delta} (46), several extracellular signals like growth factors (47), hormones (48), phorbol esters (49) and reactive oxygen species (50) in a number of cell types also induce tyrosine phosphorylation of PKC{delta}. Therefore, it was considered of interest to determine whether serotonin induces tyrosine phosphorylation of proteins in Caco-2 cells, and if so, are tyrosine kinases mediating the inhibitory effects of 5-HT on the Cl-/OH- exchange process in Caco-2 cells by phosphorylating (activating) PKC{delta}? Western blot analysis of the total lysates showed that serotonin (15-60 min) stimulated tyrosine phosphorylation of proteins in the range of ~66 and 220 kDa (Fig. 6). Because PKC{delta} is an 87-kDa protein, it might be one of the downstream target molecules of tyrosine kinases. Furthermore, to determine the role of tyrosine kinases in the 5-HT-induced inhibition of Cl-/OH- exchange activity, the effect of different tyrosine kinase inhibitors, herbimycin A (a nonspecific inhibitor, 1 µM, 1 h), tyrphostin A25 (a nonspecific inhibitor, 25 µM, 1 h), and PP1 (a specific Src family kinase inhibitor, 10 µM, 1 h) on the 5-HT-mediated inhibition of Cl-/OH- exchange activity was examined. Both herbimycin A and PP1 inhibited the 5-HT-mediated decrease in Cl-/OH- exchange activity in Caco-2 cells (Table V), thereby suggesting the involvement of tyrosine kinases in the 5-HT-mediated inhibition of Cl-/OH- exchange activity. Similar results were also seen with tyrphostin A25 (data not shown).



View larger version (121K):
[in this window]
[in a new window]
  		FIG. 6.
5-HT induces tyrosine phosphorylation of total proteins in Caco-2 cells. Caco-2 cells were incubated with 5-HT (0.1 µM) in the cell culture medium for 15 or 60 min. After washing the cells with 1x PBS, the extracted proteins (75 µg) were subjected to Western blot analysis on 8% SDS-polyacrylamide gel utilizing horseradish peroxidase-conjugated anti-phosphotyrosine antibody. 5-HT treatment to cells induced phosphorylation of total proteins in the size range of 66-220 kDa. The blot shown is representative of three experiments.

 


View this table:
[in this window]
[in a new window]
  		TABLE V
Role of tyrosine kinases (TK) in 5-HT induced inhibition of Cl–/OH– exchange activity in Caco-2 cells

Caco-2 cells were preincubated with herbimycin A, the general tyrosine kinase inhibitor or PP1, the Src family kinase inhibitor for 60 min in the cell culture medium and then co-incubated with 0.1 µM of 5-HT for another 1 h. Cl–/OH– exchange activity was measured as DIDS-sensitive (300 µM) 36Cl uptake at 5 min. Values represent mean ± S.E. of 6-9 separate experiments performed in triplicate.

 
5-HT-induced Tyrosine Phosphorylation of PKC{delta} in Caco-2 Cells—We next examined the tyrosine phosphorylation profile of PKC{delta} in response to 5-HT. As shown in Fig. 7, 5-HT stimulated tyrosine phosphorylation of PKC{delta} (PKC{delta} activation) in Caco-2 cells as early as 15 min, which continued for at least 1 h. To confirm further that tyrosine kinases, especially the Src family kinases, are upstream mediators of PKC{delta} and that PKC{delta} lies downstream to the tyrosine kinases in the 5-HT-induced signal transduction cascade, the effect of the specific Src family kinase inhibitor PP1 (10 µM, 1 h) on the 5-HT-mediated increase in PKC{delta} tyrosine phosphorylation was investigated. PKC{delta} activation at 60 min was significantly decreased in the presence of the Src family kinase inhibitor PP1. These findings further confirm that the Src family kinases are the upstream effectors of PKC{delta} in the 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. To delineate further the involvement of either the 5HT3 or 5HT4 receptor subtype in the 5HT-stimulated tyrosine phosphorylation of PKC{delta}, we studied PKC{delta} phosphorylation in the presence of the specific 5HT3, Y25130 (300 nM, 1 h), or 5HT4, RS39604 (300 nM, 1 h) receptor antagonist, or the addition of both the 5HT3 and 5HT4 receptor antagonists. 5HT3 or 5HT4 receptor antagonist alone failed to block the 5-HT-stimulated PKC{delta} tyrosine phosphorylation at 60 min; however, addition of 5HT3 and 5HT4 receptor antagonists together significantly ablated the 5-HT-induced increase in PKC{delta} tyrosine phosphorylation, indicating that 5-HT effects on Cl-/OH- exchange activity via PKC{delta} phosphorylation could be mediated through either the 5-HT3 or 5-HT4 receptor.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 7.
PKC{delta} tyrosine phosphorylation in Caco-2 cells in response to 5-HT. Caco-2 cells were incubated with 5-HT (0.1 µM) in the cell culture medium for different time intervals ranging from 15 to 60 min. Cells were also pretreated with the Src kinase inhibitor PP1 (10 µM) for 60 min and then co-incubated with 5-HT (0.1 µM) for another 60 min, or cells were pretreated with either specific 5HT3, Y25130 (Y) (300 nM, 1 h), or 5HT4, RS39604 (RS) (300 nM, 1 h) receptor antagonist, or addition of both the 5HT3 and 5HT4 receptor antagonists for 60 min and then co-incubated with 5-HT (0.1 µM) for another 60 min. Cells were washed with 1x PBS, lysed, and immunoprecipitated (IP) with anti-PKC{delta} antibody. Immunoprecipitates were analyzed by 8% SDS-PAGE, followed by transfer of proteins to nitrocellulose, and probed with anti-phosphotyrosine antibody (p-PKC{delta}). 5-HT-induced phosphorylation of PKC{delta} at 60 min was blocked by the Src family kinase inhibitor PP1. Increased PKC{delta} phosphorylation by 5-HT at 60 min was unaffected in the presence of either specific 5-HT3 or 5-HT4 antagonist and completely blocked when both 5HT3 and 5HT4 antagonists were added together. The blots were stripped and re-probed with the anti-PKC{delta} antibody (PKC{delta}) to indicate equal loading of protein in each lane (upper panel). The data were quantified by densitometric analysis and expressed as arbitrary units and represent mean ± S.E. of three determinations (lower panel). *, p < 0.05 compared with untreated control.

 
PKC{delta} Phosphorylation in Response to Serotonin Is Dependent on c-Src—The ability of PP1 to inhibit PKC{delta} phosphorylation by serotonin suggested an important role for the Src family kinases. Although PP1 is a specific inhibitor for the Src family kinases, it does not define the precise Src kinase involved. Therefore, we next investigated the role of the specific individual Src family kinase member. The serotonin-induced activation/phosphorylation of each of the Src family members, c-Src, Fyn, Yes, Lck, and Lyn, was evaluated. Src kinase activation requires carboxyl-terminal Tyr530 dephosphorylation and subsequent kinase domain Tyr416 autophosphorylation (51). 5-HT-stimulated c-Src phosphorylation in Caco-2 cells was observed as early as 15 min and persisted at a longer time point (60 min). There was no effect of 5HT on Fyn phosphorylation (Fig. 8). Most interestingly, the other Src family members, Yes, Lck, and Lyn, could not be detected in Caco-2 cells. These findings indicate that the c-Src kinase is the upstream effector of PKC{delta} in the 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 8.
5-HT induces c-Src phosphorylation in Caco-2 cells. Caco-2 cells were incubated with 5-HT (0.1 µM) in the cell culture medium for 15, 30, and 60 min. Cells were washed with 1x PBS, lysed, and immunoprecipitated (IP) with anti-c-Src or anti-Fyn antibody. Immunoprecipitates were analyzed by 12% SDS-PAGE, followed by transfer of proteins to nitrocellulose and probed with phospho-Src family (Tyr416) antibody (p c-Src or pFyn). 5-HT-induced tyrosine phosphorylation of c-Src was observed as early as 15 min and remained about the same at 60 min. 5-HT showed no effect on Fyn phosphorylation. The blots were stripped and re-probed with the anti-c-Src (c-Src) or anti-Fyn (Fyn) antibody to indicate equal loading of protein in each lane (upper panel). The data were quantified by densitometric analysis and expressed as arbitrary units and represent mean ± S.E. of three determinations (lower panel). *, p < 0.05 compared with untreated control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
5-HT is an important signaling molecule in the initiation of peristaltic reflex and in the stimulation of secretory processes in the gut (14). There are pronounced differences observed in the release mechanism of 5-HT and the effects of 5-HT on GI motility and electrolyte transport relating to species differences and regional heterogeneity (14, 52, 53). In the present study, 5-HT (0.1 µM) decreased Cl-/OH- exchange activity by ~60% in Caco-2 cells. These data are in accordance with the previous in vitro electrophysiological studies showing that 5-HT inhibits coupled NaCl absorption in the small intestine and gall bladder of rabbit (10) and rat colon (6). 5-HT has also been shown to inhibit the Cl-/HCO-3 exchange activity in villus cells and stimulate HCO-3 secretion in crypt cells of the rabbit ileum (12). Earlier studies have also demonstrated 5-HT to increase electrogenic chloride secretion in the guinea pig ileum (9), rat ileum (8), jejunum (7), distal colon (6), and human jejunum (13). In vivo studies in the rabbit ileum (31) have also shown 5-HT to induce Cl- secretion. However, our results are in contrast to the findings of Kellum et al. (13), showing that 5-HT has no significant effect on coupled NaCl absorption in the human jejunum. This discrepancy might indicate that the response to 5-HT varies among different regions of the human intestine.

The changes in electrolyte transport induced by 5-HT in the gut involve mainly the 5-HT2, 5-HT3, and 5-HT4 receptors (14). The receptor mechanisms in 5-HT-induced intestinal secretory responses have been reported to vary between regions and across species. For example, 5-HT2 receptors mediate 5-HT-induced secretion in the dog jejunum (54) and rabbit ileum (55) and are at least partly responsible together with the 5-HT3 receptors in the rat jejunum in vivo (21). Binding studies show the presence of 5-HT2 receptors on the basolateral epithelial membranes of the rabbit intestine (55), human sigmoid colon (26), and guinea pig small intestinal crypt cells (27). 5-HT3 receptors along with 5HT2 and 4 have been shown to be implicated in the 5-HT-induced inhibition of L-leucine transport in rabbit jejunum (25). 5-HT4 receptors mediate the 5-HT-induced secretory response in rat jejunum, ileum (21), and distal colon (22). 5-HT4 receptors have also been demonstrated in vitro to have a role in 5-HT-induced changes in short circuit current in the human jejunum and ileum (13, 23). Recent binding studies have shown the presence of 5-HT4 receptors on the brush border membranes of rabbit jejunal enterocytes (24). Therefore, to determine which receptor subtype is involved in the 5-HT-mediated inhibition of Cl-/OH- exchange activity, we studied the effect of the dual 5-HT3/4 antagonist tropisetron. Tropisetron (1 µM) significantly blocked the 5-HT-mediated inhibition of Cl-/OH- exchange activity, thereby suggesting the involvement of either the 5-HT3 or 5-HT4 receptor or involvement of both the receptors.

Tropisetron is a registered anti-emetic drug and exerts its effects by acting on both peripheral and central 5-HT3 receptors, when administered at low doses (in nM range, in vitro), and appears to be a weak competitive 5-HT4 antagonist, when administered in high doses (in µM range, in vitro) (56). The anti-secretory effects of tropisetron have been well defined in rodents, pig, and man (14). Thus, the dose (1 µM) selected to examine the effects of tropisetron on the Cl-/OH- exchange activity in Caco-2 cells is in the same range as used by previous investigators (13, 14). However, its lack of selectivity for the 5-HT4 receptor (pA2 6-6.5) and having a higher affinity for the 5-HT3 receptor (pA2 8-10) limits its usefulness (22). Hence, it is difficult to ascertain whether the inhibitory effect of tropisetron was due to 5-HT3 or 5-HT4 antagonism. To rule out this uncertainty, we used more potent, specific, and selective individual receptor antagonists. The 5-HT3, Y25130 (300 nM), or 5-HT4, RS 39604 (300 nM), antagonist when used alone failed to block the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. However, when 5HT3 and 5-HT4 antagonists were added together, the inhibitory effects of 5-HT were completely abolished, indicating that the 5-HT-mediated inhibition of Cl-/OH- exchange activity might occur through either the 5-HT3 or 5-HT4 receptor subtype. It should also be noted that both 5-HT3 (m-chlorophenylbiguanide, 1-100 µM, 1 h) and 5-HT4 (3-(4-allylpiperazin-1-yl)-2-quinoxaline chloronitrile, 0.1-10 µM, 1 h) agonists decreased Cl-/OH- exchange activity in Caco-2 cells. These data further confirm the involvement of 5-HT3 and 5-HT4 receptors in mediating the effects of 5-HT on Cl-/OH- exchange activity as both the agonists mimicked the effects of 5-HT. It should be noted that for the above-mentioned studies, Caco-2 cells grown on plastic supports (10-12 day post-plating) were utilized. Upon confluency, Caco-2 cells become well polarized with well defined tight junctions, exhibit several functional properties, such as carrier-mediated transport systems, receptors, and apical membrane enzyme activities, and closely resemble the native epithelium (59). Our results showing the effects of 5HT, specific 5HT3 or 5HT4 agonists/antagonists from the apical side in polarized Caco-2 cells, further suggest that 5HT3 and 5HT4 receptors are present on the apical membrane.

Previous studies in rodents and dog have shown that the effects of serotonin on electrolyte transport are associated with an increase in the intracellular calcium levels (6, 10, 33). However, in our studies, in the presence of the known Ca2+ chelator BAPTA-AM (20 µM), there was no change in the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells. Our results indicate that the effects of 5-HT on the Cl-/OH- exchange activity are independent of intracellular calcium and are consistent with the previous study of Hansen (14) showing that 5-HT evoked secretion in the hen colon independent of Ca2+. 5-HT4 receptors have also been linked to stimulation of cAMP (36). Conversely, although 5-HT4 receptors have been implicated to mediate secretory effects, they have not been shown to significantly increase levels of cAMP in the intestinal mucosa (35). Our results showing that the well known PKA inhibitor (Rp)-cAMP (25 µM) had no effect on the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells (Table II) are also in accordance with the previous findings.

To test whether PKC is involved in the 5-HT-mediated regulation of Cl-/OH- exchange activity, we studied the effect of the PKC inhibitors, chelerythrine chloride (2 µM) and calphostin C (200 nM). Both the inhibitors abolished the inhibitory effects of 5-HT on Cl-/OH- exchange activity in Caco-2 cells. Our results suggest that the PKC pathway is involved in the 5-HT-induced decrease of Cl-/OH- exchange activity. To further characterize which PKC isoform might be involved in the 5-HT-induced regulation of Cl-/OH- exchange activity in Caco-2 cells, we examined the effect of specific PKC isoform inhibitors. The PKC family is composed of at least 10 isoforms that are divided into the following three groups, conventional PKCs, novel PKCs, and atypical PKCs based on their structure and cofactor regulation.

Specific PKC{alpha} (Go6976, 5 nM) and PKC{epsilon} (Ro318220, 100 nM) inhibitors failed to block the 5-HT-mediated decrease in Cl-/OH- exchange activity; however, the specific PKC{delta} inhibitor rottlerin (10 µM) significantly abolished the 5-HT-induced decrease in Cl-/OH- exchange activity in Caco-2 cells. These results implicate the role of the novel PKC{delta} isoform and not PKC{alpha} or PKC{epsilon} in the 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. Furthermore, translocation of PKC{delta} from the cytosol to the membrane fractions at 15-60 min in response to 5-HT (0.1 µM) confirmed the role of PKC{delta} in 5-HT-mediated effects. It should also be noted that the translocation of PKC{delta} within 1 h of 5-HT treatment is consistent with our functional data showing inhibition of Cl-/OH- exchange activity in Caco-2 cells.

Previous studies have shown PKC{delta} to be phosphorylated on the tyrosine residues by the Src family kinases (nonreceptor tyrosine kinases) including Fyn, Src, and Lyn (57). Therefore, the role of tyrosine kinase inhibitors tyrphostin A25 (a nonspecific inhibitor, 25 µM, 1 h), herbimycin A (a nonspecific inhibitor, 1 µM, 1 h), and PP1 (a specific Src family kinase inhibitor, 10 µM, 1 h) in the 5-HT-induced decrease in Cl-/OH- exchange activity in Caco-2 cells was studied. These inhibitors significantly blocked the 5-HT-induced inhibition of Cl-/OH- exchange activity in Caco-2 cells, thereby suggesting the involvement of tyrosine kinases in the 5-HT-mediated inhibition. Additionally, the observed increase in PKC{delta} tyrosine phosphorylation at 60 min by 5-HT was significantly blocked by the Src family kinase inhibitor PP1. These findings further implicate the Src family kinases to be the proximal tyrosine kinases acting on PKC{delta} in the 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. Most interestingly, the 5-HT-stimulated PKC{delta} tyrosine phosphorylation at 60 min remained unaffected in the presence of either 5HT3 (Y25130) or 5HT4 (RS39604) receptor antagonist but was significantly abolished upon addition of both 5HT3 and 5HT4 receptor antagonists, thereby confirming the involvement of either the 5HT3 or 5HT4 receptor subtype in the 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells via the activation of PKC{delta}.

We also examined the role of specific individual Src family kinase members involved in the 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. Tyrosine phosphorylation/activation profile of each of the Src family members induced by serotonin was studied. c-Src phosphorylation in Caco-2 cells induced by 5-HT was observed as early as 15 min and continued until 60 min. Fyn phosphorylation was not affected by 5-HT. However, Yes, Lck, and Lyn could not be detected in Caco-2 cells. These findings further suggest the role of c-Src kinase in the 5-HT-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells. It should be noted that the effects of 5-HT on Cl-/OH- exchange activity appear to be specific, involving distinct signaling pathways, and were not secondary to changes in 5-HT-mediated decrease in Na+/H+ exchanger activity. Parallel studies in our laboratory performed under similar conditions showed that serotonin also decreased both NHE2 and NHE3 activities via a mechanism involving increase in intracellular calcium levels (43). These disparate mechanisms of serotonin action on NHE3 and Cl-/OH- exchange activities (which are known to be functionally coupled to mediate NaCl absorption) are intriguing. However, the evidence in the literature does support the involvement of distinct mechanisms for their regulation in various systems. For example, our previous studies showed distinct regulatory mechanisms for NHE3 and Cl-/OH- exchange activities in response to nitric oxide in Caco-2 cells. Nitric oxide inhibited NHE3 activity via the activation of c-GMP-dependent protein kinase (58), whereas inhibition of Cl-/OH- exchange activity occurred through both c-GMP-dependent protein kinase- and PKC-mediated pathways (39). Similar mechanistic differences in the regulation of NHE3 and Cl-/OH- exchange activities by H2O2 in Caco-2 cells were also observed. H2O2 inhibited Cl-/OH- exchange activity in Caco-2 cells via a complex signaling pathway involving Fyn kinase, phospholipase C{gamma}1, and PKC{alpha} (44) but had no significant effects on NHE activity.2 Therefore, although NHE3 and Cl-/OH- exchange processes are functionally coupled, the mechanisms of regulation can be distinct. The importance of the involvement of distinct pathways in the regulation of such functionally coupled pathways is not clear at present and may require more extensive studies to further address this question.

Based on the above results, we have proposed a model for the effect of serotonin on Cl-/OH- exchange activity in Caco-2 cells (Fig. 9). 5-HT decreases apical Cl-/OH- exchange activity either through 5-HT3 or 5-HT4 receptor subtypes via a signal transduction pathway, which involves the c-Src kinase and the Ca2+-independent PKC{delta} isoform. The c-Src kinase appears to be the upstream mediator of PKC{delta}. PKA is not involved.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 9.
Proposed model of mechanism of inhibition of Cl-/OH- exchange activity in Caco-2 cells by 5-HT.

 
In conclusion, our studies for the first time demonstrate inhibition of the human intestinal apical membrane Cl-/OH- exchange activity by serotonin (utilizing Caco-2 cells as an experimental model) via either 5-HT3 or 5-HT4 receptor and indicate that the mechanism of 5-HT-mediated decrease in apical Cl-/OH- exchange activity involves both tyrosine kinase and novel PKC{delta}-mediated pathways. Understanding the role of 5-HT and involvement of specific receptors on GI functions together with additional new information on signal transduction pathways may aid in the development of better therapeutic approaches for diarrheal related disorders such as irritable bowel syndrome, celiac disease, and carcinoid syndrome.


   FOOTNOTES
 
* This work was supported by the Department of Veterans Affairs and by NIDDK Grants DK 68324 and DK 54016 (to P. K. D.), DK 67990 and DK 33349 (to K. R.), and DK 62221 (to W. A. A.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence and reprint requests should be addressed: University of Illinois, Medical Research Service (600/151), Jesse Brown Veterans Affairs Medical Center, 820 South Damen Ave., Chicago, IL 60612. Tel.: 312-569-7434; Fax: 312-569-6487; E-mail: pkdudeja{at}uic.edu.

1 The abbreviations used are: 5-HT, 5-hydroxytryptamine (serotonin); BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl ester); PKC, protein kinase C; PKA, cAMP-dependent protein kinase; GI, gastrointestinal; DIDS, 4,4'-diisothiocyanato-stilbene-2, 2'-disulfonic acid; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; pA2, the negative logarithm of the dissociation constant of an antagonist, considered the indirect measure of the antagonist's affinity for its receptors; TBS, Tris-buffered saline; HRP, horseradish peroxidase. Back

2 S. Saksena, R. K. Gill, S. Tyagi, W. A. Alrefai, Z. Sarwar, K. Ramaswamy, and P. K. Dudeja, unpublished observations. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gershon, M. D., and Erde, S. M. (1981) Gastroenterology 80, 1571[Medline] [Order article via Infotrieve]
  2. Racke, K., and Schworer, H. (1991) Pharmacol. Res. 23, 13[CrossRef][Medline] [Order article via Infotrieve]
  3. Gershon, M. D., Takaki, M., Tamir, H., and Branchek, T. (1985) Experientia (Basel) 41, 863[CrossRef]
  4. Cooke, H. J. (1986) Gastroenterology 90, 1057[Medline] [Order article via Infotrieve]
  5. Zinner, M. J., McFadden, D., Sherlock, D., and Jaffe, B. M. (1986) Gastroenterology 90, 515[Medline] [Order article via Infotrieve]
  6. Zimmerman, T. W., and Binder, H. J. (1984) Gastroenterology 86, 310[Medline] [Order article via Infotrieve]
  7. Hardcastle, J., Hardcastle, P. T., and Redfern, J. S. (1981) J. Physiol. (Lond.) 320, 41[Abstract/Free Full Text]
  8. Moriarty, K. J., Higgs, N. B., Woodford, M., Warhurst, G., and Turnberg, L. A. (1987) Gut 28, 844[Abstract/Free Full Text]
  9. Leysen, J. (1984) Neuropharmacology 23, 247[CrossRef][Medline] [Order article via Infotrieve]
  10. Donowitz, M., Tai, Y. H., and Asarkof, N. (1980) Am. J. Physiol. 239, G463[Medline] [Order article via Infotrieve]
  11. Cooke, H. J., and Carey, H. V. (1985) Eur. J. Pharmacol. 111, 329[CrossRef][Medline] [Order article via Infotrieve]
  12. Sundaram, U., Knickelbein, R. G., and Dobbins, J. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6249[Abstract/Free Full Text]
  13. Kellum, J. M., Budhoo, M. R., Siriwardena, A. K., Smith, E. P., and Jebraili, S. A. (1994) Am. J. Physiol. 267, G357[Medline] [Order article via Infotrieve]
  14. Hansen, M. B. (1995) Pharmacol. Toxicol. 77, Suppl. 1, 3[Medline] [Order article via Infotrieve]
  15. Hoyer, D., Clarke, D. E., Fozard, J. R., Hartig, P. R., Martin, G. R., Mylecharane, E. J., Saxena, P. R., and Humphrey, P. P. (1994) Pharmacol. Rev. 46, 157[Abstract]
  16. Gaginella, T. S., Rimele, T. J., and Wietecha, M. (1983) J. Physiol. (Lond.) 335, 101[Abstract/Free Full Text]
  17. North, R. A. (1989) Br. J. Pharmacol. 98, 13[Medline] [Order article via Infotrieve]
  18. Burleigh, D. E., and Borman, R. A. (1993) Eur. J. Pharmacol. 241, 125[CrossRef][Medline] [Order article via Infotrieve]
  19. Bockaert, J., Fozard, J. R., Dumuis, A., and Clarke, D. E. (1992) Trends Pharmacol. Sci. 13, 141[CrossRef][Medline] [Order article via Infotrieve]
  20. Cooke, H. J., Wang, Y. Z., Frieling, T., and Wood, J. D. (1991) Am. J. Physiol. 261, G833[Medline] [Order article via Infotrieve]
  21. Beubler, E., and Horina, G. (1990) Gastroenterology 99, 83[Medline] [Order article via Infotrieve]
  22. Budhoo, M. R., and Kellum, J. M. (1994) J. Surg. Res. 57, 44[CrossRef][Medline] [Order article via Infotrieve]
  23. Borman, R. A., and Burleigh, D. E. (1993) Br. J. Pharmacol. 110, 927[Medline] [Order article via Infotrieve]
  24. Alcalde, A. I., Sorribas, V., Rodriguez-Yoldi, M. J., and Lahuerta, A. (2000) Eur. J. Pharmacol. 403, 9[CrossRef][Medline] [Order article via Infotrieve]
  25. Salvador, M. T., Rodriguez-Yoldi, M. C., Alcalde, A. I., and Rodriguez-Yoldi, M. J. (1997) Life Sci. 61, 309[CrossRef][Medline] [Order article via Infotrieve]
  26. Borman, R. A., and Burleigh, D. E. (1997) Ann. N. Y. Acad. Sci. 812, 224[Medline] [Order article via Infotrieve]
  27. Siriwardena, A. K., Budhoo, M. R., Smith, E. P., and Kellum, J. M. (1993) J. Surg. Res. 55, 55[CrossRef][Medline] [Order article via Infotrieve]
  28. Challacombe, D. N., Dawkins, P. D., and Baker, P. (1977) Gut 18, 882[Abstract/Free Full Text]
  29. Kyosola, K., Penttila, O., and Salaspuro, M. (1977) Scand. J. Gastroenterol. 12, 363[Medline] [Order article via Infotrieve]
  30. Thompson, J. H. (1971) Res. Commun. Chem. Pathol. Pharmacol. 2, 687[Medline] [Order article via Infotrieve]
  31. Donowitz, M., Charney, A. N., and Heffernan, J. M. (1977) Am. J. Physiol. 232, E85[Medline] [Order article via Infotrieve]
  32. Donowitz, M., and Binder, H. J. (1975) Am. J. Dig. Dis. 20, 1115[CrossRef][Medline] [Order article via Infotrieve]
  33. Hansen, M. B., and Skadhauge, E. (1997) Comp. Biochem. Physiol A 118, 283[Medline] [Order article via Infotrieve]
  34. Hoyer, D., and Schoeffter, P. (1991) J. Recept. Res. 11, 197[Medline] [Order article via Infotrieve]
  35. Eklund, S., Brunsson, I., Jodal, M., and Lundgren, O. (1987) Acta Physiol. Scand. 129, 115[Medline] [Order article via Infotrieve]
  36. Stoner, M. C., Scherr, A. M., Lee, J. A., Wolfe, L. G., and Kellum, J. M. (2000) Surgery 128, 240[CrossRef][Medline] [Order article via Infotrieve]
  37. Olsnes, S., Tonnessen, T. I., Ludt, J., and Sandvig, K. (1987) Biochemistry 26, 2778[CrossRef][Medline] [Order article via Infotrieve]
  38. Saksena, S., Gill, R. K., Syed, I. A., Tyagi, S., Alrefai, W. A., Ramaswamy, K., and Dudeja, P. K. (2002) Am. J. Physiol. 283, C1492
  39. Saksena, S., Gill, R. K., Syed, I. A., Tyagi, S., Alrefai, W. A., Ramaswamy, K., and Dudeja, P. K. (2002) Am. J. Physiol. 283, G626
  40. Bradford, M. M. (1976) Anal. Biochem. 72, 248[CrossRef][Medline] [Order article via Infotrieve]
  41. Hegde, S. S., Bonhaus, D. W., Johnson, L. G., Leung, E., Clark, R. D., and Eglen, R. M. (1995) Br. J. Pharmacol. 115, 1087[Medline] [Order article via Infotrieve]
  42. Bolton, J. E., and Field, M. (1977) J. Membr. Biol. 35, 159[CrossRef][Medline] [Order article via Infotrieve]
  43. Gill, R. K., Tyagi, S., Alrefai, W. A., Malakooti, J., Ramaswamy, K., and Dudeja, P. K. (2005) Gastroenterology, in press
  44. Saksena, S., Tyagi, S., Syed, I. A., Alrefai, W. A., Ramaswamy, K., and Dudeja, P. K. (2003) Gastroenterology 124, A143
  45. Cox, D. A., and Cohen, M. L. (1995) J. Pharmacol. Exp. Ther. 272, 143[Abstract/Free Full Text]
  46. Ohno, S., Mizuno, K., Adachi, Y., Hata, A., Akita, Y., Akimoto, K., Osada, S., Hirai, S., and Suzuki, K. (1994) J. Biol. Chem. 269, 17495[Abstract/Free Full Text]
  47. Denning, M. F., Dlugosz, A. A., Threadgill, D. W., Magnuson, T., and Yuspa, S. H. (1996) J. Biol. Chem. 271, 5325[Abstract/Free Full Text]
  48. Olivier, A. R., and Parker, P. J. (1994) J. Biol. Chem. 269, 2758[Abstract/Free Full Text]
  49. Soltoff, S. P., and Toker, A. (1995) J. Biol. Chem. 270, 13490[Abstract/Free Full Text]
  50. Konishi, H., Tanaka, M., Takemura, Y., Matsuzaki, H., Ono, Y., Kikkawa, U., and Nishizuka, Y. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11233[Abstract/Free Full Text]
  51. Bjorge, J. D., Jakymiw, A., and Fujita, D. J. (2000) Oncogene 19, 5620[CrossRef][Medline] [Order article via Infotrieve]
  52. Bonhaus, D. W., Wong, E. H., Stefanich, E., Kunysz, E. A., and Eglen, R. M. (1993) J. Neurochem. 61, 1927[Medline] [Order article via Infotrieve]
  53. Buchheit, K. H. (1989) Naunyn-Schmiedeberg's Arch. Pharmacol. 339, 704[Medline] [Order article via Infotrieve]
  54. Jaffe, B. M., Ferrara, A., and Sherlock, D. J. (1986) J. Pharmacol. Exp. Ther. 238, 536[Abstract/Free Full Text]
  55. Schuitz, B. D., Homaidan, F., Donowitz, M., and Sharp, G. W. G. (1988) Faseb. J. 2, A1722
  56. de Bruijn, K. M. (1992) Drugs 43, Suppl. 3, 11
  57. Konishi, H., Yamauchi, E., Taniguchi, H., Yamamoto, T., Matsuzaki, H., Takemura, Y., Ohmae, K., Kikkawa, U., and Nishizuka, Y. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6587[Abstract/Free Full Text]
  58. Gill, R. K., Saksena, S., Syed, I. A., Tyagi, S., Alrefai, W. A., Malakooti, J., Ramaswamy, K., and Dudeja, P. K. (2002) Am. J. Physiol. 283, G747
  59. Scaglione-Sewell, B., Abraham, C., Bissonnette, M., Skarosi, S. F., Hart, J., Davidson, N. O., Wali, R. K., Davis, B. H., Sitrin, M., and Brasitus, T. A. (1998) Cancer Res. 58, 1074[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
R. K. Gill, L. Shen, J. R. Turner, S. Saksena, W. A. Alrefai, N. Pant, A. Esmaili, A. Dwivedi, K. Ramaswamy, and P. K. Dudeja
Serotonin modifies cytoskeleton and brush-border membrane architecture in human intestinal epithelial cells
Am J Physiol Gastrointest Liver Physiol, October 1, 2008; 295(4): G700 - G708.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
A. Borthakur, R. K. Gill, S. Tyagi, A. Koutsouris, W. A. Alrefai, G. A. Hecht, K. Ramaswamy, and P. K. Dudeja
The Probiotic Lactobacillus acidophilus Stimulates Chloride/Hydroxyl Exchange Activity in Human Intestinal Epithelial Cells
J. Nutr., July 1, 2008; 138(7): 1355 - 1359.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Wu, C. S. Bohanan, J. C. Neumann, and J. B. Lingrel
KLF2 Transcription Factor Modulates Blood Vessel Maturation through Smooth Muscle Cell Migration
J. Biol. Chem., February 15, 2008; 283(7): 3942 - 3950.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
R. K. Gill, N. Pant, S. Saksena, A. Singla, T. M. Nazir, L. Vohwinkel, J. R. Turner, J. Goldstein, W. A. Alrefai, and P. K. Dudeja
Function, expression, and characterization of the serotonin transporter in the native human intestine
Am J Physiol Gastrointest Liver Physiol, January 1, 2008; 294(1): G254 - G262.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Kanchanapoo, M. Ao, R. Prasad, C. Moore, C. Kay, P. Piyachaturawat, and M. C. Rao
Role of protein kinase C-{delta} in the age-dependent secretagogue action of bile acids in mammalian colon
Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1851 - C1861.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
M. Donowitz and X. Li
Regulatory Binding Partners and Complexes of NHE3
Physiol Rev, July 1, 2007; 87(3): 825 - 872.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
G. Barthet, B. Framery, F. Gaven, L. Pellissier, E. Reiter, S. Claeysen, J. Bockaert, and A. Dumuis
5-Hydroxytryptamine4 Receptor Activation of the Extracellular Signal-regulated Kinase Pathway Depends on Src Activation but Not on G Protein or beta-Arrestin Signaling
Mol. Biol. Cell, June 1, 2007; 18(6): 1979 - 1991.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. Ogden, J. M. Thompson, Z. Hickner, T. Huang, D. D. Tang, and S. W. Watts
A new signaling paradigm for serotonin: use of Crk-associated substrate in arterial contraction
Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2857 - H2863.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
B. Callaghan, J. Zhong, and K. D. Keef
Signaling pathway underlying stimulation of L-type Ca2+ channels in rabbit portal vein myocytes by recombinant Gbeta{gamma} subunits
Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2541 - H2546.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11859    most recent
M411553200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saksena, S.
Right arrow Articles by Dudeja, P. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saksena, S.
Right arrow Articles by Dudeja, P. K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement