Human colonic epithelial cells express galanin-1 receptors, which when activated cause Clminus secretion

Richard V. Benya, Jorge A. Marrero, Denis A. Ostrovskiy, Athanasia Koutsouris, and Gail Hecht

Department of Medicine, University of Illinois and Chicago Veterans Affairs Medical Center, West Side Division, Chicago, Illinois 60612


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Galanin is a peptide hormone widely expressed in the central nervous system and gastrointestinal (GI) tract. Within the GI tract galanin is present in enteric nerve terminals where it is known to modulate intestinal motility by altering smooth muscle contraction. Recent studies also show that galanin can alter intestinal short-circuit current (Isc) but with differing results observed in rats, rabbits, guinea pigs, and pigs. In contrast, nothing is known about the ability of galanin to alter ion transport in human intestinal epithelial tissues. By RT-PCR, we determined that these tissues express only the galanin-1 receptor (Gal1-R) subtype. To evaluate Gal1-R pharmacology and physiology, we studied T84 cells. Gal1-R expressed by these cells bound galanin rapidly (half time 1-2 min) and with high affinity (inhibitor constant 0.7 ± 0.2 nM). T84 cells were then studied in a modified Ussing chamber and alterations in Isc, a measure of all ion movement across the tissue, were determined. Maximal increases in Isc were observed in a concentration-dependent manner around 2 min after stimulation with peptide, with 1 µM galanin causing Isc to rise more than eightfold and return to baseline occurring within 10 min. The increase in galanin-induced Isc was shown by 125I efflux studies to be due to Cl- secretion, which occurred independently of alterations in cAMP and phospholipase C. Rather, Cl- secretion is mediated via a Ca2+-dependent, pertussis toxin-sensitive mechanism. These data suggest that galanin released by enteric nerves may act as a secretagogue in the human colon by activating Gal1-R.

diarrhea; pharmacology


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

GALANIN IS A NEUROPEPTIDE originally isolated from porcine intestine (37), now known to be widely distributed in the central nervous system (CNS) (3) and gastrointestinal (GI) tract (26). Within the GI tract galanin is secreted by enteric nerves (5, 20), acting to inhibit pancreatic exocrine and endocrine secretions, cause smooth muscle contraction as well as relaxation, and modulate the actions of other peptide hormones (reviewed in Ref. 33). More recently, a role for galanin released by enteric nerves in altering intestinal ion flux also has been proposed (9, 19, 21, 25).

A total of four studies have explored the role of galanin in modulating intestinal secretion in rats, rabbits, guinea pigs, and pigs (9, 19, 21, 25). These electrophysiological studies demonstrate that galanin has variable effects on short-circuit current (Isc), a measure of net ion flux. For example, galanin increases Isc in rat colon (21) but has no effect in rat jejunum (21) or guinea pig colon (25). In contrast, galanin decreases Isc in pig jejunum (9) and rabbit ileum (19). Thus galanin has markedly different effects in different species, as well as in different locations within the GI tract of the same species. Yet nothing is known about the effects of galanin in human GI tissues, nor is anything known about how this peptide hormone alters Isc in any species studied, including the receptor subtype activated, the signal transduction pathway(s) utilized, or the particular ion(s) involved.

Recent molecular studies indicate that galanin acts by binding to one of three different receptor subtypes, which in humans have been identified as galanin-1 (Gal1-R) (17), galanin-2 (Gal2-R) (7), and galanin-3 (Gal3-R) (A. Pearse, unpublished data; GenBank accession no. Z79630) receptors. Before the molecular identification of Gal2-R and Gal3-R, we had shown that Gal1-R mRNA was ubiquitously expressed in low amounts by epithelial cells lining the human GI tract, including the colon (22). In this study, therefore, we set out to 1) determine whether epithelial cells lining the human colon express other galanin receptor subtypes in addition to Gal1-R and 2) elucidate the specific effect of activating Gal1-R expressed by human colonocytes. To do this we first performed RT-PCR on RNA isolated from endoscopic biopsies obtained during elective colonoscopy, as well as on RNA obtained from selected human colon cancer cell lines of epithelial origin, using primers that allowed us to differentiate between the three receptor subtypes. After establishing that human colonic epithelial tissues express only the Gal1-R, we elucidated physiological effects of galanin by studying the well-characterized human colon cancer cell line T84 (12). Our studies show that galanin activation of Gal1-R expressed by T84 cells results in a rapid and transient increase in Isc that is due to Cl- secretion. These data therefore suggest the novel possibility that galanin secreted by enteric nerves lining the GI tract may act as a secretagogue in the human colon.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. T84 cells were graciously provided by Dr. K. Barrett (University of California, San Diego); all other cell lines were obtained from the American Type Culture Collection (Rockville, MD). All tissue culture supplies including Transwells were from Costar (Cambridge, MA); galanin and other galanin analogs were from either Bachem (Torrance, CA) or Peninsula (Belmont, CA). 125I-galanin, 125I, and the cAMP RIA kit were from Amersham (Arlington Heights, IL). Taq polymerase was obtained from Perkin Elmer (Foster City, CA), Pfu polymerase was from Stratagene (La Jolla, CA), and RNA Stat-60 was from Tel-Test (Friendswood, TX). All endoscopic supplies were from Wilson-Cooke (Winston-Salem, NC). Unless otherwise indicated all other supplies were from Sigma Chemical (St. Louis, MO).

Endoscopic biopsy and RT-PCR. Patients seen for nonemergent colonoscopy at the Chicago Veterans Administration West Side Medical Center (CVAWSMC) were asked if additional mucosal biopsies could be obtained for research purposes at the time of the scheduled endoscopy. The CVAWSMC and University of Illinois Institutional Review Boards approved this study, and signed consent was obtained from all individuals. Patients with tumors or obvious mucosal abnormalities were not subjected to biopsy. Thus all biopsies were obtained from patients with grossly normal mucosa at the time of endoscopy. Colonoscopy was performed using an Olympus videoendoscope (Lake Success, NY). Two separate double biopsies were obtained at the indicated locations and placed directly into sterile 15-ml Falcon polypropylene tubes (Becton-Dickinson Laboratories, Lincoln Park, NJ) containing 2 ml RNA Stat-60 prepared as directed by the manufacturer. Immediately after procurement tissue samples were placed at -20°C, and the RNA was extracted within 24 h.

RNA from cell lines was isolated from confluent cultured cells in a similar manner. Cells were washed with PBS and removed by scraping. The dislodged cells were then pelleted by centrifugation and solubilized in 2 ml RNA Stat-60. The RNA was extracted from the cells within 24 h according to the manufacturer's instructions.

PCR was performed using primers unique to each specific receptor cDNA, with minimal overlap with the other two receptor subtypes (Table 1). In all instances, conditions included denaturing at 94°C for 30 s, annealing at 60°C for 30 s, and extending at 72°C for 1 min for 35 cycles using a reaction mixture containing Taq/Pfu (16:1) in glycerol/DMSO as previously described (14). PCR reactions were resolved on a 1.2% low-melt agarose gel and the reaction product subcloned into pCR2.1 (Invitrogen, Carlsbad, CA). The identity of the PCR product was confirmed by Sanger sequencing.

                              
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Table 1.   Specifications of primers used to differentiate between galanin receptor subtypes

Binding studies. Binding studies were performed on confluent T84 cells grown in six-well plates. After washing in binding buffer (98 mM NaCl, 6 mM KCl, 25 mM HEPES, 5 mM fumarate, 5 mM pyruvate, 5 mM glutamate, 11 mM glucose, and 0.1% soybean trypsin inhibitor, 1.0 mM MgCl2, 0.5 mM CaCl2, 2.2 mM KHPO4, 2 mM glutamine, 0.2% bovine serum albumin, and 0.1% bacitracin), cells were exposed to 125I-galanin alone or with the indicated concentration of unlabeled peptide. Nonsaturable binding of either radiolabeled peptide was the amount of radioactivity associated with cells when the incubation mixture contained 1 µM galanin. Nonsaturable binding was <15% of total binding in all experiments, with all values in this paper reported as saturable binding.

Electrophysiological assays After the presence of appropriate basal resistances consistent with the presence of a confluent monolayer (i.e., >1,000 Omega  · cm2) was established, cells were stimulated with the indicated agent, and potential difference was determined every 15 s. Electrical current (25 µA) was applied across the tissue using Ag-AgCl electrodes, and the subsequent potential difference was measured using calomel electrodes connected via salt bridges using a simplified apparatus as previously described (10). The transepithelial resistance was calculated using Ohm's law (R = V/I). Isc was measured under voltage-clamped conditions.

125I efflux measurements To determine if galanin-induced alterations in Isc were due to Cl- secretion from T84 cells, we studied 125I efflux as a measure of Cl- secretion as previously described (38). Briefly, T84 cells grown to confluence in Transwells (12-mm diam) were loaded with 2 µCi 125I/ml for 180 min at 37°C and then washed four times in HEPES-phosphate-buffered Ringer solution (HPBR) (in mM: 135 NaCl, 5 KCl, 3.3 NaH2PO4, 0.83 Na2HPO4, 1 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose; pH 7.4). After being washed, cells were exposed to 1 µM galanin in HPBR, 1-ml aliquots were removed from the apical reservoir and replaced each minute, and radioactivity was counted as previously described (38). Residual intracellular radioactivity was determined after extraction with 1 ml 0.1 N NaOH, with the efflux rate constant (min-1) calculated as previously described (38).

Evaluation of intracellular signaling pathways. Alterations in cAMP were determined in unstimulated T84 cells and in T84 cells exposed to 1 µM galanin for the indicated lengths of time by commercially available RIA (Amersham). In all instances cells were cultured to confluence in 24-well plates and treated in situ with 1 µM galanin for the indicated time at 37°C. Total cellular cyclic nucleotides were determined as directed by the manufacturer, with all values obtained on the flat portion of the standard curve.

Ability to activate phospholipase C was determined by measuring changes in total cellular phosphoinositides as described previously (6). T84 cells were grown to confluence in 24-well plates in regular medium and then were loaded for 24 h at 37°C with 100 µCi/ml myo-[2-3H]inositol in DMEM containing 2% fetal bovine serum. Cells were washed and incubated in phosphoinositide buffer (binding buffer additionally containing 10 mM LiCl2) for 15 min and then for the indicated time at 37°C with 1 µM galanin. Reactions were stopped by adding 1% HCl in methanol, and total [3H]inositol phosphates were isolated by anion exchange chromatography.

Alterations in intracellular calcium ([Ca2+]i) were determined as previously described (6). Briefly, confluent cells were washed in binding buffer and then loaded in situ with 2 µM fura 2-AM containing 0.2% Pluronic F-127 for 120 min at 37°C. After being loaded with fura 2, cells were washed in binding buffer, mechanically disaggregated, and rapidly transferred at a concentration of 5 × 106 cells/ml into quartz cuvettes placed in a Delta PTI scan-1 spectrophotometer (PTI Instruments, Gaithersburg, MD). This instrument was modified so as to maintain an incubation temperature of 37°C while continuously mixing the cuvette contents by means of a magnetic stirrer. Fluorescence was measured at 500 nm after excitation at 340 nm and at 380 nm. Autofluorescence of the unloaded cells was subtracted from all measurements, and [Ca2+]i was calculated as previously described (6).


    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Human colonic epithelia express only Gal1-R. Initial studies were carried out to determine the expression of galanin receptor subtypes by human colonic epithelial cells. RT-PCR was performed with six different primers (as shown in Table 1) in a single reaction (Fig. 1) or in three separate reactions (data not shown), designed to detect the presence or absence of the three known galanin receptor subtypes. Epithelial biopsies from both the proximal and distal colon expressed mRNA for Gal1-R but not Gal2-R or Gal3-R (Fig. 1). Direct sequencing of the PCR product revealed 100% identity with the appropriate region of only Gal1-R (data not shown). Similarly, RT-PCR performed on RNA extracted from DLD, LoVo, Caco-2, and T84 cells likewise revealed the presence of message for Gal1-R but not the other two galanin receptor subtypes (Fig. 1). Thus human colon epithelial cells express only mRNA for Gal1-R.


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Fig. 1.   Six-primer RT-PCR performed on RNA isolated from indicated tissues to determine presence or absence of galanin receptor subtype expression. As described in METHODS, 5 µg total RNA isolated from indicated tissues were subjected to RT using random hexamers and PCR was performed using 6 primers as described in Table 1, allowing for specific detection of 3 galanin receptor subtypes. Proximal and distal refer to RNA isolated from epithelial biopsies obtained from ascending and descending colon of representative patient. Expected locations of PCR products for galanin-1 receptor (Gal1-R, 373 bp), galanin-2 receptor (Gal2-R, 610 bp), and galanin-3 receptor (Gal3-R, 144 bp) are indicated by arrows. LM, 1-kb lane marker (GIBCO BRL, Gaithersburg, MD).

Pharmacology of Gal1-R expressed by T84 cells. T84 cells are a well-established model for the study of chloride secretion from human colonic epithelial cells. Specifically, previous studies have shown that alterations in Isc are primarily if not exclusively due to changes in Cl- secretion (reviewed in Ref. 1). We therefore restricted our subsequent studies to this cell line.

Binding studies were initially performed to determine the kinetics of 125I-galanin binding to T84 cells (Fig. 2, left). Ligand binding was rapid at 37°C but not at 4°C. Half-maximal binding at 37°C was between 1 and 2 min, with maximal binding observed within 10 min of exposure to 125I-galanin. Binding remained stable for up to 90 min. Reduction of the temperature to 4°C or the addition of 1 µM galanin, decreased 125I-galanin binding to <5% of total added radioactivity (Fig. 2, left).


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Fig. 2.   Pharmacology of Gal1-R expressed by T84 cells. Left: time and temperature dependence of 125I-labeled galanin binding to T84 cells. Approximately 5 × 106 cells/ml were incubated with 75 pM 125I-galanin for indicated times at 37°C alone (), or with 1 µM galanin (bullet ), or at 4°C alone (black-down-triangle ). Results are expressed as means ± SE of at least 3 separate experiments with each experiment performed in duplicate. Middle: ability of unlabeled galanin to inhibit binding of 125I-galanin to T84 cells. Approximately 5 × 106 cells/ml were incubated with 75 pM 125I-galanin for 30 min with indicated concentration of galanin. Results are expressed as means ± SE of at least 3 separate experiments of saturably bound radioactivity in absence of nonradioactive peptide, with each value determined in duplicate. Right: Scatchard analysis of binding data shown in middle. Data are for representative experiment, with each data point evaluated in duplicate.

We next determined the ability of T84 cells to interact with galanin by performing dose-inhibition studies (Fig. 2, middle). Galanin was potent at inhibiting the binding of 125I-galanin, with half-maximal inhibition observed between 0.1 and 1.0 nM and complete inhibition seen with 1 µM galanin (Fig. 2, middle). Scatchard analysis of the binding data using the least-squares curve-fitting program LIGAND (29) demonstrated that the binding data were best fit using a single-site model (Fig. 2, right). Specifically, galanin bound with high affinity [inhibitor constant (Ki) 0.7 ± 0.2 nM] to the receptors present [maximal binding (Bmax) 55.3 ± 1.1 fmol/mg protein, n = 5]. This binding affinity is similar to what has been previously shown for cells expressing only Gal1-R (18, 19, 31, 39, 42).

Stimulation of Gal1-R expressed by T84 cells results in Cl- secretion. T84 cells cultured to confluence in Transwells were used for these studies (23, 35), with only monolayers exhibiting transepithelial resistances >1,000 Omega  · cm2 used for evaluation. Overall, unstimulated T84 cells generated an Isc of 2.2 ± 0.3 µA/cm2 for all experiments. Application of 1 nM galanin, the approximate dose at which half-maximal binding was observed (Fig. 2, left), resulted in a sharp increase in Isc (Fig. 3, left). Specifically, Isc increased from 1.5 ± 0.7 to 7.5 ± 0.2 µA/cm2 within 2 min of exposure to 1 nM galanin (n = 38). Application of pharmacological doses of galanin (i.e., 1 µM) caused Isc to increase from 2.1 ± 0.3 to 17.9 ± 1.1 µA/cm2. In contrast, the smallest dose capable of reliably increasing Isc was 10 pM, causing Isc to increase from a baseline value of 2.1 ± 0.2 to 4.2 ± 0.2 µA/cm2 (n = 38). For all doses the increase in Isc was transient so that the return to basal levels was observed within 5-10 min (Fig. 3, left). Interestingly, we observed identical increases in Isc when galanin was applied to either the apical or basolateral side, suggesting that Gal1-R are functionally present on both sides of T84 cells.


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Fig. 3.   Change in short-circuit current (Isc) in T84 cells exposed to galanin and other known secretagogues. Left: alterations in Isc over time in response to 1 µM (), 1 nM (black-diamond ), or 10 pM (bullet ) galanin. T84 cells were cultured to confluence in Transwells as described in METHODS, with only cells exhibiting resistances >1,000 Omega  · cm2 used for further study and which generated a basal Isc of 2.1 ± 0.1 µA/cm2. Results are expressed as means ± SE for between 20 and 80 separate experiments. Middle: relative increase in Isc as function of galanin concentration. Maximal increases observed in Isc at any point in time for all experiments are shown as percentage of that observed using 1 µM galanin. Dashed line indicates galanin concentration necessary to cause a half-maximal increase in Isc. Results are expressed as means ± SE for between 5 and 80 separate experiments. Right: maximal increases in Isc observed for galanin (1 µM) or known secretagogues carbachol (100 µM) and forskolin (1 µM) 2 min after application of indicated secretagogue. Results expressed are means ± SE for minimum of 12 separate experiments.

We next determined the maximal and half-maximal increases in Isc caused by galanin. We converted our electrophysiological data so that it was expressed as a percentage of the response observed with 1 µM galanin. These data were then plotted versus the log galanin concentration (Fig. 3, middle, n = 5-80 separate experiments per data point). Maximal increases in Isc are observed between 100 nM and 1 µM galanin, and half-maximal increases detected at 0.8 ± 0.2 nM galanin (Fig. 3, middle). Thus the electrophysiological potency of galanin is similar to the affinity with which it binds to Gal1-R (i.e., Ki 0.7 ± 0.2 nM; see Fig. 2, middle)

To correlate the increase in Isc with that observed for other well-established secretagogues, we also exposed T84 cells to the muscarinic cholinergic agonist carbachol, an activator of [Ca2+]i and to forskolin, a direct activator of adenylyl cyclase (Fig. 3, right). In matched experiments, 100 µM carbachol increased Isc in T84 cells from 2.1 ± 0.4 to 21.7 ± 1.2 µA/cm2, whereas 1 µM forskolin increased Isc from 1.7 ± 0.2 to 25.1 ± 2.0 µA/cm2, when determined 2 min after the application of either agent (n = 12). In contrast, 1 µM galanin was ~70% as potent as carbachol and ~50% as potent as forskolin (Fig. 3, right). Thus galanin is nearly as potent as these two well-known secretagogues, known to maximally increase Cl- secretion in T84 cells by two different signal transduction pathways. Finally, we tested the effects of 1 nM and 1 µM galanin on T84 cells after stimulating with 1 µM and 100 µM carbachol and evaluated the effects of both concentrations of carbachol after stimulating T84 cells with the two indicated concentrations of galanin. In neither case did we detect any increase in Isc, once maximal increases had been detected, subsequent to the addition of galanin or carbachol after prestimulating with the other compound (data not shown). Similarly, 1 µM galanin did not cause an additional increase in Isc after T84 cells were preexposed to forskolin, whereas the addition of forskolin after exposure to even 1 µM galanin did not increase Isc beyond that which was observed when stimulated with forskolin alone (data not shown).

Previous studies have shown that Isc is primarily altered in T84 cells by changes in Cl- secretion (1), which we confirmed was the case in response to stimulation with galanin. To do this we studied 125I efflux as an analog for Cl-, previously demonstrated as appropriate in T84 cells (38). Basal efflux rate (or "leak") was 0.068 ± 0.005/min, similar to what has been previously described for this cell line (38). In contrast, exposure to 1 µM galanin markedly increased 125I efflux >10-fold (Fig. 4). Maximal efflux rates were detected between 1 and 2 min after exposure to 1 µM galanin, similar to that observed when studying alterations in Isc (Fig. 3, left). 125I efflux rates returned to normal within 6-8 min (Fig. 4), again similar to what was observed with alterations in Isc (Fig 3, left).


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Fig. 4.   125I efflux from T84 cells over time in response to stimulation with 1 µM galanin (shaded bars). T84 cells were loaded with 125I as described in METHODS, and efflux was recorded as measure of chloride secretion as described previously (38). Open bars, control. Results are expressed as means ± SE for 10 separate experiments.

We next studied the effect on Isc of compounds previously described as galanin antagonists before the molecular identification of galanin receptor subtypes including M15-galantide (2), M35 (2, 41), and M40 (4). With the cloning of the three different galanin receptor subtypes and creation of stably transfected cell lines expressing only one receptor subtype, it has come to be appreciated that all previously described "antagonists" act as full agonists at Gal1-R (18, 31, 39, 42). Because T84 cells express only Gal1-R (Fig. 1), we determined the effect of these compounds on Isc (Fig. 5). We found that M35 [Gal(1-13)bradykinin(2-9)] and M40 [Gal(1-12)-(Pro)3-(Ala-Leu)2-Ala-amide] increased Isc approximately the same at physiological concentrations (i.e., 1 nM) as galanin (Fig. 5). Interestingly, the increase in Isc for both ligands was only about 66% of the galanin response when studied using pharmacological concentrations of drug (i.e., 1 µM; Fig. 5), consistent with their acting as partial agonists. In contrast, M15 (galantide), or Gal(1-13)-substance P(5-11) had no effect on Isc in T84 cells (Fig. 5). Preincubation of T84 cells with 1 nM of either M15, M35, or M40 for 30 min, followed by stimulation with 1 nM or 1 µM galanin, had no effect on the subsequent increase in Isc compared with T84 cells not preexposed to these drugs (data not shown). These data show that previously identified galanin antagonists either do not antagonize the ability of galanin to increase Isc and have either no effect or act as partial agonists on Gal1-R expressed by T84 cells.


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Fig. 5.   Alteration in Isc in response to exposure to galanin and to purported galanin antagonists M15, M35, and M40. T84 cells were cultured to confluence in Transwells and evaluated in modified Ussing chamber as described in METHODS. Peak increases in Isc ~2 min after exposure to indicated concentration of peptide were recorded. Data represent means ± SE of minimum of 12 separate experiments.

Galanin increases in Cl- secretion are associated with increases in [Ca2+]i. Prior studies have indicated that Gal1-R activation acts primarily to decrease cellular cAMP by activating Gi and inhibiting adenylyl cyclase activity (40). However, studies have also shown that galanin can activate cellular phospholipase C and/or [Ca2+]i (36). To study the signal transduction mechanism(s) activated by Gal1-R expressed by T84 cells, we systematically evaluated the ability of galanin to alter these three different signal transduction pathways. We studied cells at variable time points around the time of peak increase in Isc. We did not find any significant alteration in cellular cAMP 1, 2, or 5 min after stimulation with galanin (Table 2). However, we did see a significant decrease in cellular cAMP levels 60 min after stimulation with galanin (Table 2). Thus similar to other systems, galanin inhibits cellular cAMP concentrations in T84 cells but at time points that do not correspond to the observed increase in Cl- secretion 1-10 min after exposure to this peptide hormone (as shown in Fig. 3, left). In contrast, no alteration in cellular [3H]inositol phosphate production was observed at any time point up to 60 min after stimulation with galanin (Table 2). Rather, we observed a temporally associated increase in [Ca2+]i within 30 s of galanin administration (Fig. 6, left). Maximal increases in [Ca2+]i were observed ~2 min after stimulation with peptide in a dose-dependent manner (Fig. 6, left). Previous studies have indicated that other Gi-coupled receptors can increase [Ca2+]i by a pertussis toxin-sensitive mechanism (27). When T84 cells were preincubated with pertussis toxin, the ability of galanin to increase [Ca2+]i was completely ablated (Fig. 6, left).

                              
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Table 2.   Summary of second messengers evaluated in T84 cells in response to galanin stimulation



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Fig. 6.   Left: increase in intracellular calcium in T84 cells subsequent to stimulation with galanin. Confluent T84 cells were loaded with 2 mM fura 2-AM in presence of Pluronic in binding buffer for 2 h, washed, and disaggregated and change in fluorescence was determined immediately after stimulation with indicated concentration of galanin. These tracings are representative of at least 3 separate experiments. Right: change in Isc in T84 cells exposed to galanin with or without preincubating with pertussis toxin for 90 min. Cells were exposed to 1 nM or 1 µM galanin alone or after preincubating with pertussis toxin (PT), and alterations in Isc were recorded. T84 cells were prepared as described in Fig. 3 legend.

Because pertussis toxin ablated galanin-induced increases in [Ca2+]i, we next studied the effect of this compound on altering Isc. Preincubating with pertussis toxin alone for 90 min had no effect on basal Isc (2.1 ± 0.1 vs. 2.1 ± 0.2 µA/cm2, n = 8). However, pertussis toxin almost completely eliminated the ability of either 1 nM or 1 µM galanin to increase Isc (Fig. 6, right). Thus these studies show that galanin acts to increase Isc by causing Cl- secretion via a pertussis toxin-sensitive, [Ca2+]i-dependent, cAMP- and phospholipase C-independent mechanism.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In this study we demonstrate that epithelial cells lining the human colon express galanin-1 receptors and not other galanin receptor subtypes. Using T84 cells as a model for the study of colonocyte ion transport, we show that Gal1-R activation causes Cl- secretion by a calcium-dependent mechanism. Thus these data indicate for the first time that galanin, ubiquitously present in nerve terminals lining the human colon (20), may function as a potential colonic secretagogue. Galanin is well known to alter the contraction in vitro of smooth muscle cells lining the GI tract of all species studied (8, 15, 34, 37). Consequently this peptide hormone is presumed to be important in regulating intestinal motility. Our data now suggest that in the human colon, galanin also may be important in causing fluid secretion.

Prior studies of galanin as a modulator of intestinal secretion are surprisingly limited (Table 3). These studies performed in rats (21), rabbits (19), guinea pigs (25), and pigs (9) show that the effects of galanin are variable as well as species and location specific. For example, galanin acts to increase Isc in rat colon while having no effect in rat jejunum or guinea pig colon. Only two of these studies investigated the effects of galanin on ion transport. In rabbit ileum galanin inhibition of Isc is due to its promotion of both Na+ and Cl- absorption (19). In contrast, galanin-induced increases in Isc in rat colon are due to decreased Na+ and Cl- absorption (21). Because the decrease in net Cl- absorption was greater than the net Na+ absorption, Kiyohara et al. (21) suggested but did not prove that in rat colon galanin likely acts to increase Cl- secretion. In the present study we show that galanin increases Isc in T84 cells by causing Cl- secretion. Thus these data show for the first time that galanin can act as a secretagogue in human colonic epithelial cells specifically by activating Gal1-R.

                              
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Table 3.   Summary of studies evaluating effect of galanin in modulating Cl- secretion from epithelial cells lining the gastrointestinal tract

Our data demonstrate that galanin mediates its physiological effects in T84 cells by interacting with high affinity (Ki 0.7 nM) to Gal1-R (Bmax 55 fmol/mg protein) expressed by T84 cells (Fig. 2). This interaction is similar to what has been previously described for other cell lines expressing only Gal1-R, such as human Bowes melanoma cells [dissociation constant (Kd) 0.4 nM (17)]. Of the few studies evaluating the effects of galanin on GI epithelia, only one performed a pharmacological analysis. In rabbit ileal epithelia (19), galanin bound with high affinity (Kd 0.4 nM) to a similar number of binding sites (Bmax 28 fmol/mg protein) as we detected to T84 cells. Thus both the binding affinity of galanin and the number of binding sites observed in this study are consistent with what has been previously described for this receptor.

The ability of galanin to cause Cl- secretion is consistent with the action of other peptide hormones present in enteric nerve terminals. In the T84 model system alone, bradykinin (28), calcitonin gene-related peptide (32), pituitary adenylate cyclase-activating polypeptide (30), and vasoactive intestinal polypeptide (11, 24) all have been shown to cause Cl- secretion. However, these peptide hormone secretagogues mediate their effects by increasing cellular cAMP (24, 28, 30, 32). Yet we demonstrate that galanin causes Cl- secretion via a cAMP-independent pathway. Our findings are particularly interesting in light of a recent study, using stably transfected CHO cells expressing either Gal1-R or Gal2-R (40). In this study, Gal1-R activation caused decreased cellular cAMP, whereas Gal2-R activation resulted in increased phospholipase C activity (40). In contrast, we show that whereas galanin decreases cAMP, this decrease is not temporally associated with increases in Cl- secretion. Rather, the rapid and transient increase in Cl- secretion is related to changes in [Ca2+]i.

Other Gi-coupled heptaspanning receptors have been shown to increase [Ca2+]i when stimulated. Perhaps the best studied is the alpha 2A-adrenergic receptor, which when activated slowly decreases cellular cAMP and rapidly increases [Ca2+]i in an inositol 1,4,5-trisphosphate-independent manner (27). Similar to what we observed with galanin activation of Gal1-R, increased [Ca2+]i generated by stimulation of the alpha 2A-adrenergic receptor is pertussis toxin sensitive (27). The ability of Gi-coupled heptaspanning receptors to increase [Ca2+]i, which has been suggested to occur via G-beta gamma subunits (13), may represent a cell type-specific property of this receptor class. For instance, Gal1-R expressed by CHO cells act only to inhibit cAMP accumulation (40), whereas preliminary studies indicate that when expressed by HEL cells this receptor increases [Ca2+]i in a manner similar to what we observed in T84 cells (Kenneth Dickinson, Bristol-Meyers Squibb, personal communication).

In this study we confirm that antagonists identified before the molecular cloning of galanin receptor subtypes act as agonists at the Gal1-R (18, 31, 39, 42). It is possible that this altered pharmacology is cell type or organ system specific. A prior study has shown that various galanin analogs that act as antagonists in the CNS are full agonists when physiologically tested on GI smooth muscle cells (16). In this study we likewise show that these compounds, which act as galanin antagonists in the CNS, act as agonists at the Gal1-R expressed by T84 cells (Fig. 5). Although the efficacy of these compounds varies, none was able to inhibit the effects of galanin in terms of either binding or of increasing Isc. Intriguingly, the pharmacology of all galanin receptor subtypes may be both species and location dependent, as appears to be the case in the regulation of intestinal fluid secretion (Table 3).

In conclusion, this study is the first to show that epithelial cells lining the human colon exclusively express galanin-1 receptors and not other galanin receptor subtypes. Using T84 cells as a model for the study of human colon epithelial ion transport, we show that Gal1-R activation causes Cl- secretion by a Ca2+-dependent mechanism. Galanin is thus the first peptide hormone identified to cause Cl- secretion in human colonic epithelium by a cAMP-independent mechanism. Finally, the variability of the effects of galanin on altering ion transport in different species underscores the importance of studying this peptide hormone in human tissues.


    ACKNOWLEDGEMENTS

This work was supported by an American Digestive Health Foundation (ADHF)-American Gastroenterological Association Industry Research Scholar Award, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51168, and a Veterans Affairs Merit Review Award to R. V. Benya; by an ADHF-Astra Merck Advanced Research Fellowship Award to J. A. Marrero; and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50694 and a Veterans Affairs Merit Review Award to G. Hecht.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: R. V. Benya, Dept. of Medicine, Univ. of Illinois, 840 South Wood St., M/C 787, Chicago, IL 60612.

Received 15 July 1998; accepted in final form 10 September 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Barrett, K. E. Positive and negative regulation of chloride secretion in T84 cells. Am. J. Physiol. 265 (Cell Physiol. 34): C859-C868, 1993[Abstract/Free Full Text].

2.   Bartfai, T., K. Bedecs, T. Land, U. Langel, R. Bertorelli, P. Girotti, S. Consolo, Y.-J. Yu, Z. Wiesenfeld-Hallin, S. Nilsson, V. Pieribone, and T. Hokfelt. M-15: high affinity chimeric peptide that blocks the neuronal actions of galanin in the hippocampus, locus caeruleus, and spinal cord. Proc. Natl. Acad. Sci. USA 88: 10961-10965, 1991[Abstract].

3.   Bartfai, T., G. Fisone, and U. Langel. Galanin and galanin antagonists: molecular and biochemical perspectives. Trends Pharmacol. Sci. 13: 312-317, 1992[Medline].

4.   Bartfai, T., U. Langel, K. Bedecs, S. Andell, T. Land, S. Gregersen, B. Ahren, P. Girotti, S. Consolo, R. Corwin, J. Crawley, X. Xu, Z. Wiesenfeld-Hallin, and T. Hokfelt. Galanin-receptor ligand M40 peptide distinguishes between putative galanin-receptor subtypes. Proc. Natl. Acad. Sci. USA 90: 11287-11291, 1993[Abstract].

5.   Bauer, F. E., T. E. Adrian, N. D. Christofides, G. L. Ferri, N. Yanaihara, J. M. Polak, and S. R. Bloom. Distribution and molecular heterogeneity of galanin in human, pig, guinea pig, and rat gastrointestinal tracts. Gastroenterology 91: 877-883, 1986[Medline].

6.   Benya, R. V., T. Kusui, T. K. Pradhan, J. F. Battey, and R. T. Jensen. Expression and characterization of cloned human bombesin receptors. Mol. Pharmacol. 47: 10-20, 1995[Abstract].

7.   Bloomquist, B. T., M. R. Beauchamp, L. Zhelnin, S. E. Brown, A. R. Gore-Willse, P. Gregor, and L. J. Cornfield. Cloning and expression of the human galanin receptor GalR2. Biochem. Biophys. Res. Commun. 243: 474-479, 1998[Medline].

8.   Botella, A., M. Delvaux, J. Fioramonti, J. Frexinos, and L. Bueno. Galanin induces opposite effects via different intracellular pathways in smooth muscle cells from dog colon. Peptides 15: 637-643, 1994[Medline].

9.   Brown, D. R., K. R. Hildebrand, A. M. Parsons, and G. Soldani. Effects of galanin on smooth muscle and mucosa of porcine jejunum. Peptides 11: 497-500, 1990[Medline].

10.   Colgan, S. P., A. Nusrat, C. Delp, and C. A. Parkos. A simple approach to measurement of electrical parameters of cultured epithelial monolayers. Use in assessing neutrophil epithelial interactions. J. Tissue Cult. Res. 14: 209-216, 1992.

11.   Dharmsathaphorn, K., K. G. Mandel, H. Masui, and J. A. McRoberts. Vasoactive intestinal polypeptide-induced chloride secretion by a colonic epithelial cell line. J. Clin. Invest. 75: 462-471, 1985[Medline].

12.   Dharmsathaphorn, K., J. A. McRoberts, K. G. Mandel, L. D. Tisdale, and H. Masui. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G204-G208, 1984[Abstract/Free Full Text].

13.   Dorn, G. W., K. J. Oswald, T. S. McCluskey, D. G. Kuhel, and S. B. Liggett. alpha 2A-Adrenergic receptor stimulated calcium release is transduced by Gi-associated Gbeta gamma -mediated activation of phospholipase C. Biochemistry 36: 6415-6423, 1997[Medline].

14.   Ferris, H. A., R. E. Carroll, M. M. Rasenick, and R. V. Benya. Constitutive activation of the gastrin-releasing peptide receptor expressed by the non-malignant human colon epithelial cell line NCM460. J. Clin. Invest. 100: 2530-2537, 1997[Abstract/Free Full Text].

15.   Gu, Z.-F., T. K. Pradhan, D. H. Coy, and R. T. Jensen. Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP. Peptides 15: 1425-1430, 1994[Medline].

16.   Gu, Z.-F., W. J. Rossowski, D. H. Coy, T. K. Pradhan, and R. T. Jensen. Chimeric galanin analogs that function as antagonists in the CNS are full agonists in gastrointestinal smooth muscle. J. Pharmacol. Exp. Ther. 266: 912-918, 1993[Abstract].

17.   Habert-Ortoli, E., B. Amiranoff, I. Loquet, M. Laburthe, and J.-F. Mayaux. Molecular cloning of a functional human galanin receptor. Proc. Natl. Acad. Sci. USA 91: 9780-9783, 1994[Abstract/Free Full Text].

18.   Heuillet, E., Z. Bouaiche, J. Menager, P. Dugay, N. Munoz, H. Dubois, B. Amiranoff, A. Crespo, J. Lavayre, J.-C. Blanchard, and A. Doble. The human galanin receptor: ligand-binding and functional characteristics in the Bowes melanoma cell line. Eur. J. Pharmacol. 269: 139-147, 1994[Medline].

19.   Homaidan, F. R., S. H. Tang, M. Donowitz, and G. W. Sharp. Effects on galanin on short-circuit current and electrolyte transport in rabbit ileum. Peptides 15: 1431-1436, 1994[Medline].

20.   Hoyle, C. H., and G. Brunstock. Galanin-like immunoreactivity in enteric neurons of the human colon. J. Anat. 166: 23-33, 1989[Medline].

21.   Kiyohara, T., M. Okura, and H. Ishikawa. Galanin-induced alteration of electrolyte transport in the rat intestine. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G502-G507, 1992[Abstract/Free Full Text].

22.   Lorimer, D. D., and R. V. Benya. Cloning and quantification of human galanin-1 receptor expression by mucosal cells lining the gastrointestinal tract. Biochem. Biophys. Res. Commun. 222: 379-385, 1996[Medline].

23.   Madara, J. L., and G. Hecht. Tight junctions in cultured epithelial cells. In: Functional Epithelial Cells in Culture. New York: Liss, 1991, p. 131-163.

24.   Mandel, K. G., J. A. McRoberts, G. Beuerlein, E. S. Foster, and K. Dharmsathaphorn. Ba2+ inhibition of VIP- and A-23187-stimulated Cl- secretion by T84 cell monolayers. Am. J. Physiol. 250 (Cell Physiol. 19): C486-C494, 1986[Abstract/Free Full Text].

25.   McCulloch, C. R., A. Kuwahara, C. D. Condon, and H. J. Cooke. Neuropeptide modification of chloride secretion in guinea pig distal colon. Regul. Pept. 19: 35-43, 1987[Medline].

26.   Melander, T., T. Hokfelt, A. Rokaeus, J. Fahrenkrug, K. Tatemoto, and V. Mutt. Distribution of galanin-like immunoreactivity in the gastrointestinal tract of several mammalian species. Cell Tissue 239: 253-270, 1985.

27.   Michel, M. C., L. F. Brass, A. Williams, G. M. Bokoch, V. J. LaMorte, and H. J. Motulsky. alpha 2-Adrenergic receptor stimulation mobilizes intracellular Ca2+ in human erythroleukemia cells. J. Biol. Chem. 264: 4986-4991, 1989[Abstract/Free Full Text].

28.   Miller, D. H., A. W. Baird, S. Bennet, M. Halushka, M. Sasaguri, H. Schomer, and H. S. Margolius. Regulation of bradykinin-induced chloride secretion in a human epithelial cell line. Agents Actions Suppl. 38: 81-86, 1992[Medline].

29.   Muson, P. J., and D. Robard. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107: 220-229, 1980[Medline].

30.   Nguyen, T. D., G. G. Heintz, and J. A. Cohn. Pituitary adenylate cyclase-activating polypeptide stimulates secretion in T84 cells. Gastroenterology 103: 539-544, 1992[Medline].

31.   Patterson, J., D. Conklin, K. Murphy, R. Horlick, B. L. Largent, and L. W. Fitzgerald. Pharmacological characterization of a recombinant human galanin receptor (GalR1) in HEK293 cells (Abstract). Soc. Neurosci. Abstr. 23: 392, 1997.

32.  Poyner, D. R., E. A. Tomlinson, M. Gosling, I. R. Tough, and H. M. Cox. Stimulation of choride secretion and adenylate cyclase secretion in human colonic derived cell lines by calcitonin gene-related peptide. Biochem. Soc. Trans. 21, Suppl.: 434S, 1993.

33.   Rattan, S. Role of galanin in the gut. Gastroenterology 100: 1762-1768, 1991[Medline].

34.   Rossowski, W. J., T. M. Rossowski, S. Zacharia, A. Ertan, and D. H. Coy. Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations. Peptides 11: 333-338, 1990[Medline].

35.   Savakovic, S. D., A. Koutsouris, and G. Hecht. Activation of NF-kappa B in intestinal epithelial cells by enteropathogenic Escherichia coli. Am. J. Physiol. 273 (Cell Physiol. 42): C1160-C1167, 1997[Medline].

36.   Sethi, T., and E. Rozengurt. Galanin stimulates Ca2+ mobilization, inositol phosphate accumulation, and clonal growth in small cell lung cancer cells. Cancer Res. 51: 1674-1679, 1991[Abstract].

37.   Tatemoto, K., A. Rokaeus, H. Jornvall, T. J. McDonald, and V. Mutt. Galanin, a novel biologically active peptide from porcine intestine. FEBS Lett. 164: 124-128, 1983[Medline].

38.   Vlengarik, C. J., R. J. Bridges, and R. A. Frizzell. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells. Am. J. Physiol. 259 (Cell Physiol. 28): C358-C364, 1990[Abstract/Free Full Text].

39.   Walker, M. W., K. E. Smithe, B. Borowsky, R. Zhou, Z. Shaposhnick, R. Nagorny, P. J.-J. Vaysse, C. Gerald, and T. A. Brancheck. Cloned galanin receptors: pharmacology of GalR1 and GalR2 receptor subtypes (Abstract). Soc. Neurosci. Abstr. 23: 962, 1997.

40.   Wang, S., T. Hashemi, S. Fried, A. L. Clemmons, and B. E. Hawes. Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes. Biochemistry 37: 6711-6717, 1998[Medline].

41.   Wiesenfeld-Hallin, Z., X. J. Xu, U. Langel, K. Bedecs, T. Hokfelt, and T. Bartfai. Galanin-mediated control of pain: enhanced role after nerve injury. Proc. Natl. Acad. Sci. USA 89: 3334-3337, 1992[Abstract].

42.   Yu, J., D. G. Harden, T. A. Pitler, and D. W. Gallager. Agonist properties of chimeric galanin peptides (Abstract). Soc. Neurosci. Abstr. 23: 393, 1997.


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