Human colonic epithelial cells express galanin-1 receptors,
which when activated cause
Cl
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 |
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 |
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 |
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.
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
· 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 |
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).
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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 ( ), or at
4°C alone ( ). 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.
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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
· 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 ( ), or 10 pM ( ) galanin. T84 cells
were cultured to confluence in Transwells as described in
METHODS, with only cells exhibiting
resistances >1,000
· 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.
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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.
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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.
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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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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.
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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 |
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.
View this table:
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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
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
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-
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.
 |
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