Vol. 275, Issue 4, C1048-C1057, October 1998
Cl
transport in an
immortalized human epithelial cell line (NCM460) derived from the
normal transverse colon
Jasminder
Sahi1,2,
Selvaraj G.
Nataraja1,
Thomas J.
Layden2,
Jay L.
Goldstein2,
M. P.
Moyer3, and
Mrinalini C.
Rao1
1 Department of Physiology and
Biophysics and 2 Department of
Medicine, University of Illinois at Chicago, Chicago, Illinois
60612; and 3 Department of
Surgery, University of Texas Health Science Center at San Antonio,
San Antonio, Texas 78284
 |
ABSTRACT |
Cells of a newly
described, immortalized, epithelial, human transverse colonic cell
line, NCM460, reach ~90% confluence on plastic and develop
transepithelial resistances of 120-250
· cm2 on
porous substrates. Its utility as a model for the transverse human
colon was validated by comparing second messenger-mediated Cl
transport, using the
fluorescent probe 6-methoxy-quinolyl acetoethyl ester, in NCM460 cells
and colonocytes isolated from human transverse crypts. Basal
Cl influx was increased
(P < 0.01) by
PGE1 (1 µM), forskolin (1 µM),
8-bromoadenosine 3'5'-cyclic monophosphate (100 µM),
heat-stable Escherichia coli
enterotoxin (STa; 1 µM), 8-bromoguanosine 3'5'-cyclic monophosphate (100 µM), histamine (1 µM), and phorbol
12,13-dibutyrate (1 µM) in both cell types. The
Cl channel blocker
diphenylamine 2-carboxylic acid (50 µM) and the Na+-K+-2Cl
cotransport inhibitor furosemide (1 µM), but not the
K+ channel blocker
Ba2+ (3 mM), inhibited these
Cl permeabilities. These
cells possess transcripts for cystic fibrosis transmembrane conductance
regulator,
Na+-K+-2Cl
cotransporter, STa receptor, and intestine-specific cGMP-dependent protein kinase II. Thus cAMP-, cGMP-, and
Ca2+-dependent secretagogues act
on NCM460 and primary colonocytes to stimulate
Cl transport. This
validates the utility of NCM460 as a model for transverse colonic
crypts and is the first demonstration of a colonic cell line whose
origin is known.
colonocytes; primary cultures; 6-methoxy-quinolyl acetoethyl ester; second messenger regulation; resistance
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INTRODUCTION |
CHLORIDE TRANSPORT IN THE human colon is regulated by a
variety of intracellular messengers, including cAMP, cGMP,
Ca2+, and protein kinase C (PKC).
An understanding of human colonic ion transport has been derived from
in vivo perfusion models (6), intact colonic epithelial sheets (21),
isolated colonocytes (8), primary colonocyte cultures (16, 19, 8),
vesicles derived from colonic tissues (4) and transformed cell lines (10). Each of these has limitations. Thus, although in vivo perfusion
studies in the human colon are the most physiological, they at best
represent a composite picture of net transport. To delineate the
individual ionic components of transport, in vitro studies have been
performed using intact colonic epithelial sheets. However, even when
stripped of underlying muscle layers, these tissues remain a
heterogenous preparation comprised of surface and crypt epithelial
cells and the underlying lamina propria and submucosal layers. Primary
cultures of isolated colonocytes, a preparation we have characterized
in some detail, have proven to be useful models (16). However, they are
limited in their long-term (>72 h) viability in culture and are
subject to the vagaries of tissue availability. Apical and basolateral
membrane vesicle preparations of tissues obtained at the time of
autopsy are good models (4) for delineating events at the membrane level but require larger amounts of tissue and are not amenable to
study of the signaling cascades underlying neurohumoral regulation of
ion transport. Extensive studies evaluating ion transport have also
been performed using animal models with the assumption that they have
applicability to humans. However, there are species-specific differences in colonic transport mechanisms. Human colonic cell lines
are good models for study of epithelial ion transport in general, but
there are some limitations to the available cell lines. First, all but
one of the ~40 commercially available human colon cell lines are
transformed, having been derived from carcinomas. Second, some cell
lines, such as T84, develop transepithelial resistances
(Rt; ~1,000
· cm2)
much greater than does normal human colon (~140-160
· cm2).
Third, some cell lines, such as HCT-EB and Caco-2 cells, exhibit characteristics more representative of fetal and small intestinal tissues; e.g., they exhibit sucrase activity and display intercellular cysts (7a). Fourth, there are differences between
Cl transport regulation in
normal human colonocytes and in colonic cell lines. For example,
neither 8-Br-cGMP (4b), a cGMP analog, nor phorbol esters (20), which
are PKC activators, stimulate Cl secretion in T84 cells,
but both stimulate Cl
transport in isolated primary human colonocytes (7, 16). Fifth, and of
greatest relevance to the present study, the segmental origin of most
colonic cell lines is not known, and therefore they cannot be used to
study differences along the cephalocaudal axis.
We have now established a new model to study ion transport in
colonocytes, using an immortalized, nontransformed, human colonic cell
line (NCM460) that negates some of the inadequacies of the earlier
models. The NCM460 line is derived from the normal human transverse
colonic mucosa (13). These cells do not form tumors in nude mice (13)
and have tested negative for the colonic neoplasm markers (MDM2, DCC,
K-ras, and CEA; Moyer, unpublished observations). These cells are
epithelial in origin, since they stain positively for cytokeratin (17),
human secretory component, villin, and the colon-specific glycoprotein
5E113 (13). Because these cells are not of tumor origin, they better
represent the normal human colon than the transformed cell lines. In
addition, because NCM460 cells are immortalized, their availability is
not a limiting factor. NCM460 cells grow in culture as an attached
population (attached cells) and a floating population
("floaters"), and we have concentrated on delineating the
characteristics of the attached cells. In a recent study, we
demonstrated that these cells exhibit
Na+/H+
exchange activity with characteristics of the NHE-1 and NHE-2 isoforms
but not of the NHE-3 isoform (18). This indicated that NCM460 cells are
crypt in origin, as in situ hybridization studies in human transverse
colon depict NHE-1 and NHE-2 isoforms in the crypts, whereas surface
cells possess all three isoforms (4a). Another ion transport
characteristic ascribed to crypts is
Cl transport, and little is
known about this process in NCM460 cells.
The current paper therefore characterizes the growth and attachment of
NCM460 cells and the Cl
transport characteristics of the attached cell population. To validate
whether these cells are indeed representative of colonic crypts, we
compared Cl transport in
NCM460 cells with those in primary isolates from the transverse human
colonic crypts. As in our earlier studies with human and rabbit
colonocyte primary cultures (3a, 7, 16, 19), we used the
Cl-sensitive fluorescent
probe 6-methoxy-quinolyl acetoethyl ester (MQAE). Our studies show that
NCM460 cells form resistive monolayers with
Rt akin to those
of human colonic epithelial sheets. The NCM460 cells exhibit
Cl permeabilities that are
regulated by agents acting via the cAMP, cGMP,
Ca2+, and PKC pathways. The basal
and stimulated Cl
permeabilities are partially, although significantly, decreased by
inhibitors of the Cl
channel and the
Na+-K+-2Cl
cotransport pathways but not by inhibitors of the
K+ channel. Equally importantly,
these responses are qualitatively similar to those of primary cultures
of human transverse crypt colonocytes. Both NCM460 cells and primary
transverse colonocytes possess transcripts for the cystic fibrosis
transmembrane conductance regulator (CFTR), the secretory form of the
Na+-K+-2Cl
cotransporter, the heat-stable Escherichia
coli enterotoxin (STa) receptor, guanylate cyclase C
(GCC), and the cGMP-dependent protein kinase (PKG) II isoform.
| |
METHODS |
Materials.
NCM460 cells were obtained from In Cell (San Antonio, TX). M3:10
culture medium (In Cell) for NCM460 colonocytes was provided through
the University of Texas Health Science Center (San Antonio, TX) Center
for Human Cell Biotechnology, and Ham's F-12 nutrient mixture and FCS
for the primary cultures of human transverse colonocytes were from
GIBCO Laboratories (Grand Island, NY). Sterile lactated Ringer was from
Baxter Health Care (Deerfield, IL). Biocoat cell culture inserts were
from Collaborative Research (Bedford, MA), and all other supplies for
cell culture were from Costar (Cambridge, MA). MQAE was purchased from
Molecular Probes (Junction City, OR). Diphenylamine-2-carboxylate (DPC)
was purchased from Aldrich (Milwaukee, WI). All other reagents were of
analytical grade and were purchased from Sigma Chemical (St. Louis,
MO).
NCM460 cell culture.
The NCM460 colonocytes were counted and plated at a density of 2 × 104 cells/ml in Costar
75-cm2 flasks at 37°C with 6%
CO2. The tissue culture medium
used was M3:10 nutrient mix containing 10% fetal bovine serum and
antibiotics. The cells were passaged by using a cell scraper and
splitting them 1:2. The epithelial origin of the cells was confirmed by intermediate filament immunofluorescence, using the method of Yang et
al. (27).
Human colonocyte isolation and culture.
Human transverse colonic tissue was obtained from individuals
undergoing colonic resection at the University of Illinois Hospital and
Clinics for benign or malignant tumors. Donors had not received preoperative irradiation or chemotherapy, and use of human tissue was
approved by the Institutional Review Board (University of Illinois at
Chicago, Chicago, IL). The tissue pieces used were taken
from transverse colon of normal appearance, at sites at least 2 cm away
from the tumor. For the transport studies, human transverse colonic
epithelial cells were isolated as described previously (16). The
transverse colonic tissues were transported on ice in oxygenated
lactated Ringer containing 5 mM dextrose and antibiotics (in µg/ml:
25 ampicillin, 120 penicillin, 270 streptomycin, and 1.25 amphotericin
B). The colonic mucosa was stripped off the underlying muscle and
digested (0.1% pronase, 0.03% collagenase) for 90 min at 37°C, in
the presence of 5 mM dithiothreitol. The cells were filtered to remove
residual tissue and serially centrifuged to enrich for crypt cells as
described previously (16). The colonocytes were plated at 2 × 104 cells/ml in tissue culture
medium (Ham's F-12 nutrient mix) supplemented with 20% FCS, 0.5 U/ml
insulin, 4 mM
L-glutamine, 1 µM
hydrocortisone, 10.5 mM selenium, 0.5 mM sodium butyrate, and
antibiotics for 24 h. Isolated, nonattached colonocytes were used for
the transport studies.
Resistance measurements.
NCM460 cells were grown on Millipore filters precoated with different
extracellular matrix proteins. Resistance measurements were made using
an ohmmeter (World Precision Instruments, Sarasota, Florida).
Background resistance (coated and uncoated filters not plated with
cells) was ~30
· cm2 and
was deducted from each value. Resistance was measured over an 8-day
period.
Ion transport.
MQAE fluorescence is quenched by all halides in a dose-dependent
fashion (19, 25). NCM460 cells were grown on plastic Leighton tubes
(Costar) until they reached 95% confluence. Primary human colonocytes
were used 24 h postplating and used in suspension. The cells were
washed free of the tissue culture medium and dye loaded for 90 min on
ice in buffer A, which contained (in
mM) 5 MQAE, 110 NaCl, 1 MgCl2, 1 CaCl2, 5 dextrose, 50 mannitol,
and 1 KCl. The cells were then resuspended in a
Cl-free solution
(buffer B) containing (in mM) 110 sodium isethionate, 1 MgSO4, 5 dextrose, 50 mannitol, 1 K2SO4,
and 1 CaSO4. Fluorescence was
measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm in a PTI Alphascan spectrofluorometer (Princeton, NJ). The rate of change of fluorescence was monitored in the NCM460 cells as buffers
A and
B were alternately perfused in the
presence and absence of different agents and inhibitors. For the
nonattached human colonocytes, initial fluorescence was observed in
buffer B, and the rate of change of
fluorescence was monitored as 5 mM Cl was added to the cells
under different conditions.
Cl influx was calculated as
previously described (16), using the formula
JCl = (Fo/KClF2)(dF/dt),
where JCl is the
Cl influx rate (mM/s),
dF/dt is the slope of the initial rate
of change of fluorescence upon addition of
Cl (fluorescence units/s),
KCl is the Stern-Volmer
constant for quenching of intracellular MQAE by
Cl, and
Fo and F are absolute fluorescence
units in the absence and presence of
Cl, respectively.
Background fluorescence was obtained by adding 150 mM KSCN and 5 µM
valinomycin to the cells. This value was deducted from both F and
Fo.
Determination of KCl.
MQAE fluorescence is quenched strongly by nonphysiological anions such
as thiocyanate and nitrite and weakly by other intracellular anions
(25). To account for this, the
KCl has to be
determined for each cell type. The constant for human primary
colonocytes had been determined previously (16).
KCl was
determined for the NCM460 cells as the slope of the equation
Fo/F = 1 + KCl[Cl],
where [Cl] is
Cl concentration.
Regulation of Cl transport.
Changes in Cl permeability
in the presence of the Cl
channel blocker DPC (50 µM) and the
Na+-K+-2Cl
cotransport inhibitor furosemide (10 µM) were studied to determine the putative transporters accountable for
Cl permeability. To
investigate the second messenger regulatory pathways in these cells,
agents were selected based on their known effects on intact epithelial
preparations and a lack of interference with MQAE fluorescence. To
study the effects of cAMP, forskolin (1 µM),
PGE1 (1 µM), and
8-bromoadenosine 3'5'-cyclic monophosphate (8-BrcAMP, 100 µM) were used. 8-Bromoguanosine 3'5'-cyclic monophosphate (8-BrcGMP, 100 µM) and STa (1 µM) were used to study cGMP-mediated Cl transport.
The tumor promoter phorbol 12,13-dibutyrate (PDB; 1 µM) was used as a
PKC modulator. Although short-term exposure to phorbol esters activates
PKC, long-term exposures are known to downregulate the enzyme (11).
Both long-term (24 and 48 h) and short-term (5 min) effects of PDB on
Cl secretion in NCM460
cells were studied. For the long-term treatment, 100 nM PDB was added
to cells in the Leighton tubes for 24 or 48 h. Before
Cl influx measurements, and
after MQAE loading and Cl
depletion, an additional 1 µM PDB was added for 5 min. Longer time
periods were not studied, as the cells tended to slough off in the
perfusion chamber 72 h postconfluence, even in the absence of PDB.
Ca2+-mediated
Cl permeability was studied
by using serotonin (1 µM) and histamine (10 µM) in both NCM460
cells attached to the matrix and NCM460 cells in suspension. Cells in
the latter preparation were not floaters but were attached
cells that had been released from the substratum by scraping. The cells
were studied in suspension to address the question of whether there
were qualitative differences between them and attached cells. To study
the effect of inhibiting K+
channels on Ca2+-mediated
Cl transport, we blocked
the K+ channels using
Ba2+ and carried out the
fluorescence study in the presence of various Ca2+ agonists. Cells in suspension
were loaded with MQAE in buffer A for
90 min. One-half of the cells was suspended in the
Cl-free
buffer B, and the rest were
transferred to modified
Cl-free
buffer B containing (in mM) 110 sodium
gluconate, 2 hemimagnesium gluconate, 2 hemicalcium gluconate, 5 dextrose, 50 mannitol, and 5 potassium gluconate (pH 7.4). This
modification of buffer B was needed to
circumvent solubility problems with
Ba2+ salts. The cells in modified
buffer B were exposed to 3 mM barium acetate with or without secretagogues and with or without inhibitors for 5 min, whereas the cells in regular buffer
B were exposed to these agents in the absence of
Ba2+ for 5 min. The rate of change
in fluorescence was monitored after the addition of NaCl to the control
cells or of BCl2 to the cells in
modified buffer B.
RNA extraction and RT-PCR.
Total RNA was isolated from 10-cm culture dishes containing attached
NCM460 cells or from human colonic mucosa (~100 mg wet weight) by a
one-step extraction procedure using the TRIzol isolation kit (Life
Technologies, Rockville, MD) according to the manufacturer's protocol.
Optical densities were measured at 260 and 280 nm using a
Hewlett-Packard 8451 A diode array spectrophotometer (Hewlett-Packard, Palo Alto, CA). The quality of the RNA was assessed by fractionating it
on 1% agarose gel and observing the presence of the typical 28S and
18S rRNA under ultraviolet light.
Total RNA (20 µg) was reverse transcribed to first-strand cDNA with
200 units of Moloney murine leukemia virus RT in the presence of 0.5 mM
dNTPs and 25 µg/ml oligo(dT) in a total volume of 20 µl for 60 min
at 37°C. After completion of the reaction, the tubes were boiled
for 5 min and then transferred to 
20°C. The primers employed
in the PCR were designed from the coding region of the various
sequences using the software Lasergene (DNASTAR, Madison, WI). The
amplified genes, the location and expected fragment sizes of the
amplified products (given in parentheses), and the primers used were as
follows: human secretory
Na+-K+-2Cl
cotransporter (carboxy-terminal region, 468 bp), sense
primer 5'-GAG AGC GAT GGC TAC TTT GC-3', antisense primer
5'-TAC CAT TCT GGA GGG CTG TC-3'; rabbit CFTR (first
transmembrane domain, 530 bp), sense primer 5'-ATC GCG ATT TAC
CTG GGC ATA G-3', antisense primer 5'-AGT TTC AGT TCT GTT
TGT CTT AG-3'; human GCC (cytoplasmic domain, 660 bp), sense
primer 5'-ATA CAA TCC AGA GAC TAC GAC-3', antisense primer
5'-GCT CTT TTT CCT CTG CTG TTT-3'; human PKG II
(amino-terminal region, 323 bp), sense primer 5'-TGG ACA CTC TGG
GAA CCT CA-3', antisense primer 5'-CCT TTG CTC CCC TCC TGC T-3'; and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (371 bp), sense primer 5'-ATG GCA CCG TCA AGC CTG AGA-3',
antisense primer 5'-GGC ATG GAC TGT GGT CAT GAG-3'.
PCR was performed in a total volume of 50 µl with 5 or 10 µl of
reverse-transcribed product, 2.5 units
Taq polymerase, 50 µM dNTP, 0.6 µM
primers in 1× PCR buffer [10 mM Tris (pH 9), 50 mM KCl, and
1 mM MgCl2]. Amplification
was performed using 1 cycle at 95°C for 2 min, followed by 35 or 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s,
and then final extension at 72°C for 5 min (GeneAmp, Perkin-Elmer,
Norwalk, CT). For control, each sample was amplified in the same
condition with GAPDH primers. The PCR product was analyzed on 2%
agarose gel containing 0.5 µg/ml ethidium bromide using TAE buffer
[10 mM Tris (pH 7.5), 5.7% glacial acetic acid, and 1 mM
EDTA].
Statistics.
To determine the statistical significance of differences between
observations, we ran Student's
t-test. ANOVA tests were used to
determine statistical significance when more than two means were
compared. Values of P < 0.05 were
considered statistically significant. In all experiments,
n values represent the number of
experiments, in each of which the measurements were made in triplicate;
n = 5 or more for NCM460 cells, and
n = 3 for the primary cultures, unless
otherwise indicated.
| |
RESULTS |
Growth.
The growth characteristics of only the NCM460 cells were studied, since
we had previously observed that the viability of human colonocytes in
primary culture declines after 48-72 h (16). When grown in 12-well
plastic culture plates, NCM460 cells (7.5 × 105) approximately doubled in 48 h, yielding 10.4 × 105 ± 1.80 × 105 attached cells and 3.03 × 105 free-floating cells
(floaters) (Fig. 1). On
day 4, there was a 2.8-fold increase
in the number of attached cells compared with day
2, and by day 8 postplating these cells had increased to 57.85 × 105 ± 2.15 × 105 and reached ~95%
confluence. No further growth was observed in this cell population up
to day 10. The floaters, in contrast, did not show any significant growth until after day
4. At this stage, these cells grew rapidly, and by
day 6 postplating there was a
threefold increase (P < 0.001) in
the floating cell population. This trend continued through
day 8 postplating, when the numbers of
floaters increased to 21.5 × 105 ± 0.3 × 105. No reasonable quantitation
could be done past day 8, as the medium could not support this large quantity of cells and the rapidly
growing floating cell population had to be passaged.
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Fig. 1.
Growth pattern of NCM460 colonocytes in culture. NCM460 cells were
grown in 12-well plastic tissue culture clusters. Both nonattached
( ) and attached ( ) colonocytes were counted over a 10-day period.
Nonattached cells were harvested by centrifugation, and attached cells
were scraped off tissue culture wells. Viability was assessed by Trypan
blue exclusion; n = 4 cultures, each
counted in triplicate.
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Measurement of Rt.
To determine whether the attached NCM460 cells were capable of
developing Rt, we
grew the cells on a variety of extracellular matrices and
Rt was measured.
Cells were plated either on uncoated Millipore filters or filters
coated with one of the following: laminin, Matrigel, collagens I and
IV, and fibrillar collagen (collagen type 1, which is treated to form
large collagen fibrils with a normal cross-striation pattern). The
Rt was measured
over an 8-day period and, regardless of the matrix used, was found to
plateau by day 6 postplating. An
example in which maximal resistance was developed 5 days postplating in
cells grown on complex fibrillar collagen is shown in Fig.
2, inset.
The differential effects of the extracellular matrices on
Rt are shown in
Fig. 2. The data are derived from culture wells, 7 days postplating,
after subtraction of baseline resistance (~30
· cm2).
The lowest resistance was with cells grown on the uncoated filters (95 ± 7.1 · cm2).
Resistance was significantly increased, 1.25-fold over the basal, when
cells were grown on laminin (P < 0.05), Matrigel (P < 0.05), and
collagen I (P < 0.01). The increase
was even greater (
2-fold) when the cells were grown on collagen IV
(201.52 ± 4.5 · cm2;
P < 0.001) or on complex
fibrillar collagen (218.2 ± 17.8 · cm2;
P < 0.001), respectively.
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Fig. 2.
Resistance measurements in NCM460 colonocytes grown on permeable
supports. NCM460 colonocytes were grown on coated or uncoated
Transwells in 24-well tissue culture clusters. Resistance was measured
over an 8-day period. Values were derived on day
8 postplating. Background values (0-30
· cm2;
coated or uncoated wells not plated with cells) were subtracted from
each reading. Inset: development of
resistance over time in cells plated on Transwells coated with
fibrillar collagen. Y-axis depicts
resistance in
· cm2. All
values are from n = 4 experiments
performed in triplicate.
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Ion transporters: activity and transcripts.
To study ion transport, we first determined the
KCl in NCM460
cells and found it to be 20 M1. In earlier studies, the
KCl for human
colonocytes was determined to be 24 M1 (16). To identify the
pathways involved in Cl
transport, we characterized
Cl permeability based on
DPC (Cl channel) and
furosemide
(Na+-K+-2Cl
cotransport) sensitivity (Fig. 3). We have
previously demonstrated that these inhibitors, unlike bumetanide, do
not interfere with MQAE fluorescence (16). Basal
Cl permeability (0.214 ± 0.02 mM/s) was inhibited 87% by 50 µM DPC (P < 0.001) and 35% by 10 µM
furosemide (P < 0.01). When both inhibitors were added together,
Cl permeability was further
suppressed, to ~95% of the basal value and significantly
(P < 0.05) more than with DPC alone.
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Fig. 3.
Effect of agents that act via cAMP to increase
Cl influx, in presence and
absence of inhibitors, on NCM460 colonocytes and primary cultures of
human transverse colonocytes. NCM460 cells
(A) were grown to 95% confluence on
Leighton tubes, and isolated transverse colonocytes
(B) were grown in tissue culture
flasks for 24 h as described in
METHODS. Cells were loaded with
6-methoxy-quinolyl acetoethyl ester (MQAE),
Cl depleted, and treated
with agent with or without inhibitor for 5 min at room temperature.
Initial fluorescence was taken before addition of NaCl. Rate of change
of fluorescence was monitored, and
Cl influx was calculated in
mM/s. Open bars, cells in basal state or treated with agent but not
with inhibitor; shaded bars, cells treated with
diphenylamine-2-carboxylate (DPC); hatched bars, cells treated with
furosemide; solid bars, cells treated with both inhibitors. Values are
means ± SE; n = 5 experiments for
NCM460 cells, and n = 3 for primary
cultures. With exception of effect of furosemide on
PGE1-stimulated
Cl permeability, all values
with inhibitors were significantly (P < 0.05) below corresponding baseline value.
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Similar studies were conducted on primary cultures of colonocytes
derived from the transverse human colon. Basal
Cl permeability (0.162 ± 0.03 mM/s) was inhibited 76% by DPC
(P < 0.001) and 52% by furosemide
(P < 0.001). The two inhibitors together decreased Cl
permeability to a greater extent (89%) than that of either inhibitor alone.
The transport results suggest that the NCM460 cells and the primary
colonocytes contain Cl
channels and the
Na+-K+-2Cl
cotransporter. To demonstrate that they contain the transcripts for
such proteins, we designed primers to detect, by RT-PCR amplification, the transcript for CFTR and the secretory form of the
Na+-K+-2Cl
cotransporter. CFTR is highly conserved in the transmembrane domains
across species, and we used primers based on the rabbit sequence to
amplify a 530-bp region in the first transmembrane domain. The primers
for the
Na+-K+-2Cl
cotransporter were based on the known human sequence. As shown in Fig.
4, both NCM460 cells and primary human
colonocytes contain transcripts for CFTR and the
Na+-K+-2Cl
cotransporter.
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Fig. 4.
Detection of cystic fibrosis transmembrane conductance regulator (CFTR)
and
Na+-K+-2Cl
cotransporter (NKCC1) transcripts in NCM460 (NCM) colonocytes and
primary cultures of human transverse colonocytes (PHC). Total RNA was
isolated from attached NCM460 cells or from human colonic mucosa and
reverse transcribed to first-strand cDNA as described in
METHODS. RT product was incubated with
Taq polymerase, nucleotides, and
specific primers to amplify secretory
Na+-K+-2Cl
cotransporter or CFTR or glyceraldehyde-3-phosphate dehydrogenase
(GAPDH; see Fig. 6). Primers were selected to amplify the following
products: a 530-bp sequence in first transmembrane domain of rabbit
CFTR (this region has very high homology to human CFTR), a 468-bp
sequence in carboxy-terminal domain of human secretory
Na+-K+-2Cl
cotransporter, and a 371-bp sequence of GAPDH as control. Negative
controls included samples without RT or without RT product and showed
no specific product (data not shown).
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Cl influx induced by the cAMP
pathway.
To dissect out the effect of cAMP on
Cl permeability,
PGE1, a receptor-G
protein-mediated activator of adenylate cyclase (1 µM),
forskolin, a direct activator of adenylate cyclase (1 µM), and the
cAMP analog 8-BrcAMP (100 µM) were tested (Fig. 3). The secretagogues
were tested in the presence and absence of the inhibitors DPC
and/or furosemide to assess the transport pathways involved.
All three agents caused a significant increase in the influx rate of
Cl in both the NCM460 cells
and the primary cultures. In the NCM460 cells, a 2.3-fold increase in
Cl influx was found with
PGE1, a 1.8-fold increase with
forskolin, and a 2.5-fold increase with 8-BrcAMP
(P < 0.001 for all 3; Fig. 3A). This secretagogue-stimulated
Cl influx was partially and
significantly inhibited by DPC
(PGE1, 65%; forskolin, 83%; and
8-BrcAMP, 66.1%; P < 0.001) and
furosemide (PGE1, 37.6%,
P < 0.05; forskolin, 55.3%,
P < 0.001; and 8-BrcAMP, 47.3%,
P < 0.01). When both inhibitors were
added together along with the secretagogue, the effect was partially
additive. The residual Cl
permeability after addition of both inhibitors in the stimulated cells
is higher (P < 0.05) than that in
the nonstimulated cells (0.012 mM/s). These studies were conducted on
attached NCM460 cells. To determine whether qualitatively similar
results are observed when these cells are not attached to a substratum,
we examined the Cl
permeabilities in suspended NCM460 cells (note that these cells are not
the floater population). Cells were scraped with a rubber policeman,
and Cl transport was
studied with and without PGE1 (1 µM). PGE1 caused a 2.44-fold
increase (inhibitor-sensitive influx in mM/s:
PGE1, 0.44 ± 0.06; basal,
0.18 ± 0.02; n = 5).
Similar results were obtained with the primary colonocytes (Fig.
3B).
PGE1, forskolin, and 8-BrcAMP
significantly (P < 0.001) increased
the Cl influx rates, and
these were inhibited by DPC (PGE1,
72%, forskolin, 83% and 8-BrcAMP, 77%;
P < 0.001) and furosemide
(forskolin, 73%, P < 0.001;
8-BrcAMP, 48%, P < 0.01). Although
furosemide appeared to cause a modest, ~20% decline in
PGE1 stimulation, this was not
statistically significant. As with the NCM460 cells, when both
inhibitors were added together along with the secretagogue, Cl permeability was further
decreased, although not to basal levels. It therefore appears that,
both in NCM460 and in primary colonocytes, the secretagogues stimulate
Cl channels,
Na+-K+-2Cl
cotransport, and some other
Cl permeabilities.
Cl influx induced by the cGMP
pathway.
Studies in the distal human colon, in human colonic cell lines, and in
colonic membranes indicate that STa acting via its specific membrane
guanylate cyclase receptor increases cGMP and stimulates
Cl secretion (3, 5, 7). The
effects of cGMP on the transverse colon per se are not known. The
effects of both STa (1 µM) and the cGMP analog 8-BrcGMP (100 µM) in
the presence and absence of inhibitors were studied. As shown in Fig.
5A, in
NCM460 cells, baseline Cl
influx was significantly increased by STa (2.3-fold,
P < 0.001) and to a lesser extent by
cGMP (1.3-fold, P < 0.01). The
STa-induced Cl influx was
mainly via the DPC-sensitive
Cl channels (67.2%,
P < 0.001) and to a lesser extent
through the cotransporter (34%, P < 0.01). However, furosemide inhibited the 8-BrcGMP-stimulated
Cl influx by 86.1%
(P < 0.001), and DPC blocked it by
53.9% (P < 0.01). The combined
effect of the two inhibitors was greater than either inhibitor alone.
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|
Fig. 5.
Effect of agents that act via cGMP to increase
Cl influx, in presence and
absence of inhibitors, on NCM460 colonocytes and primary cultures of
human transverse colonocytes. NCM460 cells
(A) were grown to 95% confluence on
Leighton tubes, and isolated colonocytes
(B) were grown in tissue culture
flasks for 24 h as described in
METHODS. Cells were loaded with MQAE,
Cl depleted, and treated
with agent with or without inhibitor for 5 min at room temperature.
Initial fluorescence was taken before addition of NaCl. Rate of change
of fluorescence was monitored, and
Cl influx was calculated in
mM/s. STa, heat-stable Escherichia
coli enterotoxin. Open bars, cells in basal state or
treated with agent but not with inhibitor; shaded bars, cells treated
with DPC; hatched bars, cells treated with furosemide; solid bars,
cells treated with both inhibitors. Values are means ± SE;
n = 5 experiments for NCM460 cells,
and n = 3 for primary cultures. All
values after treatment with inhibitors were significantly
(P < 0.05) below corresponding
baseline value.
|
|
In contrast to the NCM460 cells, in the primary cultures (Fig.
5B), basal
Cl influx (0.16 ± 0.03 mM/s) was increased equally by STa (0.74 ± 0.12 mM/s) and 8-BrcGMP
(0.64 ± 0.11 mM/s). As in NCM460 cells, STa stimulated
Cl permeability largely via
the DPC-sensitive Cl
channels (DPC, 66%; furosemide, 50%), whereas 8-BrcGMP did so mainly
through the cotransporter (furosemide, 81%; DPC, 43%).
Their responses to STa and to 8-BrcGMP suggest that both NCM460 cells
and primary human colonocytes possess receptors for STa as well as a
cGMP-specific signaling mechanism. To determine whether they express
the transcripts for STa receptors (i.e., GCC) and the
intestine-specific isoform PKG II, RNA from NCM460 cells and primary
human colonocytes was amplified by RT-PCR using human-specific primers.
As shown in Fig. 6, both cell preparations possess transcripts for GCC and PKG II. As a positive control, the
"housekeeping" gene GAPDH was amplified in every RT-PCR
experiment. A representative amplification is shown in Fig. 6.
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Fig. 6.
Detection of guanylate cyclase C (GCC) and PKG II transcripts in NCM460
colonocytes and primary cultures of human transverse colonocytes. Total
RNA was isolated from attached NCM460 cells or from human colonic
mucosa and reverse transcribed to first-strand complementary DNA (cDNA)
as described in METHODS. RT product
was incubated with Taq polymerase,
nucleotides, and specific primers to amplify GCC or PKG II or GAPDH.
Primers were selected to amplify the following products: a 660-bp
sequence in cytoplasmic domain of human GCC, a 323-bp sequence in
amino-terminal domain of human PKG II, and a 371-bp sequence of GAPDH
as control. Negative controls included samples without RT or without RT
product and showed no specific product (data not shown).
|
|
Cl influx induced by the PKC
pathway.
It is well recognized that short-term phorbol ester treatment
stimulates the PKC cascade, whereas long-term treatment downregulates the enzyme. The effects of short-term exposure to phorbol esters on
Cl secretion in human
colonocytes have varied with the source of the cells. Thus, although
PDB does not increase short-circuit current in naive T84
monolayers (1, 20), it stimulates
Cl secretion in primary
isolates of human colonocytes (16) and in the colon carcinoma cell line
HT-29.cl19a (24). To determine whether similar differences exist
between primary isolates and a cell line of the transverse colon, we
studied the effects of PDB. As shown in Table
1, short-term treatment with PDB caused an
approximately sixfold (P < 0.05)
increase in Cl transport in
primary colonocytes and an approximately twofold (P < 0.05) increase in NCM460 cells.
In both cell types, either DPC or furosemide inhibited
Cl influx to
~60-67% (P < 0.05, data not
shown). We also examined the long-term effects of PDB on
Cl secretion in the NCM460
cells; again due to viability and availability problems, similar
studies could not be carried out in the primary colonocytes.
Twenty-four-hour treatment with 0.1 µM PDB resulted in
Cl influx rates no
different than those of untreated controls. Addition of PDB to a final
concentration of 1 µM for 5 min to these cells also did not stimulate
Cl influx rates. Although
Cl transport rates in cells
exposed to 0.1 µM PDB for 48 h were also similar to those in
untreated cells, exposure to 1 µM PDB for an additional 5 min caused
a small 1.3-fold increase (P < 0.05, by paired analysis) in Cl
influx.
Cl influx induced by the
Ca2+ pathway.
To determine whether the transverse colon was responsive to
Ca2+-dependent secretagogues, we
determined the effects of histamine, serotonin, and neurotensin (Tables
2 and 3). Two
different types of NCM460 preparations were used, the attached cells
(Table 2) and attached cells that had been scraped and studied in
suspension (Table 3; note that these cells are not the floater
population). Due to limited availability of primary colonocytes, the
effects of only one secretagogue could be tested in that preparation. Studies in human and rabbit distal colonocytes had indicated
interspecies differences in the actions of histamine, and therefore
this agent was tested in primary colonocytes (15, 19).
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Table 2.
Effect of Ca2+-mediated agents in presence and absence
of inhibitors on NCM460 colonocytes and primary cultures of human
transverse colonocytes
|
|
There was no qualitative difference in the responsiveness of the three
different preparations used. However, the relative degree of
stimulation varied. Histamine caused significant 1.34-fold and 2.3-fold
increases in Cl influx in
attached and suspended NCM460 cells, respectively, while causing a
2.66-fold increase in that of primary colonocytes. Serotonin and
neurotensin caused 1.6- to 1.7-fold increases in Cl permeabilities in NCM460
cells. In contrast to the effects of the cyclic nucleotide-dependent
secretagogues, those of the
Ca2+-dependent secretagogues were
not inhibited by furosemide. However, DPC caused a 70%
(P < 0.05) decrease in
serotonin-stimulated Cl
influx and a 40-57% (P < 0.05)
decrease in histamine-stimulated transport. The fact that there is no
qualitative difference in the responses of NCM460 cells whether they
are studied attached to a matrix (Table 2) or in suspension (Table 3)
suggests that these two preparations are similar with respect to
detection of Cl
permeabilities. In addition, there is no major qualitative difference between the NCM460 cells and the primary cultures.
In T84 cells, the secretagogue actions of
Ca2+-dependent agents are
Ba2+ sensitive, and these agents
are known to activate K+ channels
(1). To determine whether this is also true for the NCM460 cells, we
examined the effects of Ba2+ on
basal and Ca2+-stimulated
Cl permeabilities. The
cells were preincubated with 3 mM barium acetate. Influx was measured
by the addition of 5 mM NaCl or
BaCl2 as described in
METHODS. As shown in Table 3,
preincubation with Ba2+ affected
neither basal nor secretagogue-stimulated
Cl transport. Equally
importantly, there was no difference in the actions of
Ca2+-dependent secretagogues with
and without Ba2+.
The studies with the
Ca2+-dependent secretagogues
suggest that, in both NCM460 cells and primary colonocytes,
Cl permeability induced by
the Ca2+-mediated pathway is via
DPC-sensitive and DPC-insensitive
Cl channels but does not
appear to activate the
Na+-K+-2Cl
cotransporter. In addition, activation of
Ba2+-sensitive
K+ channels does not appear to
play a major role in NCM460 cells.
| |
DISCUSSION |
In this study, we characterized a new human colonic epithelial cell
line of known segmental origin. We demonstrate that the immortalized
NCM460 colonocytes derived from the transverse colon (13) are similar
to primary cultures of cells isolated from the same region.
The NCM460 cells grow as two populations, attached cells and floaters.
Viability is 90% in the floating cell population, and these cells
appear to require paracrine factors for division, as they grow faster
in conditioned medium. We have not characterized the floating cell
population further. The attached cells grow actively in fresh medium
and reach 90-95% confluence on nonporous substrates like plastic
or plastic coated with collagen type IV. On porous membranes, NCM460
cells form monolayers with
Rt that increase
steadily over time, indicating the ability of these cells to form tight
junctions. The Rt
developed by the NCM460 cells are within the range reported for intact
sheets of human colonic mucosa (~99
· cm2)
(21). In addition, the requirement for being bathed with medium along
both the apical and basolateral surface for optimal growth and the
increase in Rt in
the presence of extracellular matrix proteins (cells on uncoated
filters 85 · cm2; cells
on coated filters, 120-252
· cm2) set
them apart from transformed cell lines like T84, which can grow on
plastic and establish high
Rt of 1,000
· cm2 (9).
In the human colon, Cl
absorption is via an electroneutral,
Na+-independent,
HCO3-dependent process, and
Cl secretion is via apical
membrane Cl channels acting
in conjunction with the basolateral membrane Na+-K+-2Cl
cotransporter (21). To determine whether NCM460 cells are
representatives of transverse colonic crypts, we compared their
Cl transport and its
regulation with those of primary cultures of human transverse crypt
colonocytes. Our results with inhibitors suggest that, in both cell
types, in the resting state,
Cl channels such as CFTR
are the predominant Cl
permeability, although
Na+-K+-2Cl
cotransporter is also present. This is borne out by the presence of
transcripts for CFTR and the
Na+-K+-2Cl
cotransporter in both cell types (Fig. 3). The small residual Cl permeability seen in the
presence of both DPC and furosemide is presumed to be due to
DPC-insensitive Cl
channels. Although
Cl/HCO3
exchange is prevalent in colonic crypts, its contributions were
presumed to be minimal, since the perfusion buffer was
HCO3 free.
Forskolin, PGE1, and 8-BrcAMP
enhanced Cl permeabilities
via the Cl channels and the
Na+-K+-2Cl
cotransporter in both cell types. This demonstrated the presence of
hormone-specific receptors, an activatable adenylate cyclase, and a
cAMP-sensitive Cl transport
mechanism. This is consistent with previous findings in primary human
distal colonocyte cultures (16) and in T84 cells (1). As in the case of
basal Cl transport,
combined addition of DPC and furosemide inhibited forskolin-stimulated
Cl permeabilities
89-95% in both cell types, suggesting that
Cl channels and the
Na+-K+-2Cl
cotransporter are the major routes. In contrast, combined addition of
the inhibitors caused a significantly smaller (72-74%;
P < 0.05) decline in
PGE1-stimulated
Cl transport. Thus
PGE1 appears to activate DPC and
furosemide-insensitive Cl
transport processes, perhaps via a cAMP-independent pathway.
In the small intestine, cGMP and 8-BrcGMP act via PKG II to stimulate
secretion. However, the cGMP signal transduction cascade in the large
intestine appears to vary with cell type. In T84 cells, STa stimulates
cGMP and Cl secretion, but
8-BrcGMP has no effect (4b). This has been demonstrated to be due to
the lack of PKG II and the fact that cGMP, but not its analog, can
cross-activate protein kinase A and thereby
Cl secretion (4b). In
contrast, our studies demonstrate that both STa and 8-BrcGMP can
stimulate Cl transport in
distal colonocytes (7) as well as in NCM460 cells and transverse human
colonocytes (Fig. 5), suggesting that these cells have an active PKG
II. The data in Fig. 6 confirm that these cells possess transcripts
both for PKG II and for the STa receptor GCC. There are two noteworthy
differences in the responses elicited by STa and 8-BrcGMP. First,
STa-stimulated transport, like those of cAMP- and
Ca2+-dependent secretagogues,
appears to be largely via DPC-sensitive channels, whereas 8-BrcGMP acts
mainly via the
Na+-K+-2Cl
cotransporter. This is true for both NCM460 cells and the primary colonocytes. It could be speculated that activation of PKG II (action
of 8-BrcGMP) affects the cotransporter, whereas activation of PKG II
and protein kinase A (PKA) (action of STa) activate both
Cl channels and the
cotransporter. We have previously demonstrated that, in the flounder
intestine, 8-BrcGMP regulates
Na+-K+-2Cl
cotransporter phosphorylation and activity (22). The second difference
between STa and 8-BrcGMP is that, whereas they elicit responses of
similar magnitude in the primary colonocytes, STa has a significantly
greater effect than 8-BrcGMP in the NCM460 cells. This interesting
difference between these cells and the primary cultures implies that
STa is acting via multiple (PKA and PKG II?) pathways in NCM460 cells.
The Ca2+-mediated and PKC pathways
are other examples of the similarity between primary cultures and the
immortalized colonocytes. The PKC cascade is utilized by several
secretagogues in the intestine. In primary human colonocytes (16) and
HT-29.cl19A cells (23, 24), short-term exposure to phorbol esters alone
stimulates Cl
permeabilities. In marked contrast, we and others have found that
short-term exposure to phorbol esters alone had no direct effects on
Cl secretion in T84 cells
but attenuated cAMP-activated
Cl secretion (11, 20).
Furthermore, long-term treatment with PDB induced downregulation of PKC
activity in T84 cells (20). Both NCM460 cells and the primary
transverse colonocyte cultures exhibited enhanced
Cl permeability with
short-term PDB treatment, similar to the results of our studies on the
distal human colonocytes. However, long-term treatment with submaximal
doses of PDB shows desensitization to further PDB stimulation at 24 h.
This is most probably due to downregulation of PKC as reported for T84
cells by Matthews et al. (11) and our own unpublished observations. The
desensitizing effect of prolonged exposure to PDB begins to dissipate
by 48 h both in NCM460 cells (Table 1) and T84 cells (20). This
observation remains to be correlated with PKC activity.
Histamine and serotonin are present in large amounts in the colon (12,
14), and histamine and cholinergic agonists are known to
act by releasing Ca2+ from
intracellular stores in T84 cells (26). We had previously demonstrated
that serotonin and neurotensin stimulate
Cl channels and the
Na+-K+-2Cl
cotransporter in rabbit (19) and distal human (15) colonocytes. In
contrast, although histamine largely activates DPC-sensitive Cl transport in human
distal colonocytes (15), it activates only the
Na+-K+-2Cl
cotransporter in rabbit distal colonocytes (19), suggesting species-specific differences. In the present study, the responses of
NCM460 cells to serotonin and histamine are very similar to those seen
in primary cultures of transverse human colonocytes (Table 2),
emphasizing the advantage of the NCM460 cells as a model of the human
transverse colon. Another striking feature of NCM460 cells is that,
unlike T84 cells, the activation of
Cl permeabilities by
Ca2+-dependent secretagogues is
not Ba2+ sensitive. These results
imply that activation of K+
channels may not be required for
Ca2+-dependent
Cl transport in all
colonocyte preparations and reinforce the advantage of studying
nontransformed colonic cell lines, such as NCM460.
The present studies demonstrate that the immortalized NCM460
colonocytes establish resistance and demonstrate second
messenger-mediated Cl
permeabilities very similar to human transverse colonic crypt cells in
primary culture. With the exception of the striking quantitative differences in response to STa, we found no major differences between
these two cell types in the responses to second messengers. Together
with our recent findings that NCM460 colonocytes, like the transverse
colonic crypts, possess transcripts for the NHE-1 and NHE-2 isoforms,
but not for NHE-3 isoform, these studies validate the NCM460
colonocytes as a good model of the transverse colonic crypts. The fact
that the origin of these cells is known makes NCM460 cells an
invaluable tool for dissecting the cellular basis of ion transport in
the human colon.
| |
ACKNOWLEDGEMENTS |
These studies were supported in part by National Institutes of
Health (NIH) Grants DK-38510 and DK-46910 (to M.C. Rao), by a
Department of Veterans Affairs merit review grant (to T. J. Layden),
and by NIH Grant HL-48497 and Smokeless Tobacco Research Council Grant
A200 (to M. P. Moyer). J. Sahi was supported by NIH Research Service
Award F32-DK-08849.
| |
FOOTNOTES |
Present address of J. Sahi: Pharmocokinetics and Drug Metabolism Dept.,
Parke Davis, 2800 Plymouth Rd., Ann Arbor, MI 48105.
Address for reprint requests: M. C. Rao, Dept. of Physiology and
Biophysics, University of Illinois at Chicago, 835 S. Wolcott, m/c 901, Chicago, IL 60612-7342.
Received 31 October 1996; accepted in final form 15 July 1998.
| |
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Abstract
Introduction
