Clminus 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 Omega  · 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

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 Omega  · cm2) much greater than does normal human colon (~140-160 Omega  · 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 Omega  · 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Growth pattern of NCM460 colonocytes in culture. NCM460 cells were grown in 12-well plastic tissue culture clusters. Both nonattached (black-triangle) and attached (open circle ) 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.

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 Omega  · cm2). The lowest resistance was with cells grown on the uncoated filters (95 ± 7.1 Omega  · 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 Omega  · cm2; P < 0.001) or on complex fibrillar collagen (218.2 ± 17.8 Omega · cm2; P < 0.001), respectively.


View larger version (21K):
[in this window]
[in a new window]
 
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 Omega  · 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 Omega  · cm2. All values are from n = 4 experiments performed in triplicate.

Ion transporters: activity and transcripts. To study ion transport, we first determined the KCl in NCM460 cells and found it to be 20 M-1. In earlier studies, the KCl for human colonocytes was determined to be 24 M-1 (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.


View larger version (29K):
[in this window]
[in a new window]
 
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.

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.


View larger version (28K):
[in this window]
[in a new window]
 
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).

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.


View larger version (24K):
[in this window]
[in a new window]
 
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.


View larger version (30K):
[in this window]
[in a new window]
 
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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of PDB on NCM460 cells and primary cultures of human transverse colonocytes

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of Ca2+-mediated agents in presence and absence of inhibitors on NCM460 colonocytes and primary cultures of human transverse colonocytes

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Effect of Ba2+ on action of Ca2+-dependent secretagogues in NCM460 cells

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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 Omega  · 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 Omega  · cm2; cells on coated filters, 120-252 Omega  · cm2) set them apart from transformed cell lines like T84, which can grow on plastic and establish high Rt of >= 1,000 Omega  · cm2 (9).

In the human colon, Cl- absorption is via an electroneutral, Na+-independent, HCO-3-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-/HCO-3 exchange is prevalent in colonic crypts, its contributions were presumed to be minimal, since the perfusion buffer was HCO-3 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.

    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.   Benya, R. V., L. N. Schmidt, J. Sahi, T. J. Layden, and M. C. Rao. Isolation, characterization and attachments of rabbit distal colon epithelial cells. Gastroenterology 101: 692-702, 1991[Medline].

3.   Cohen, M. B., A. Guarino, and R. A. Gianella. Age-related differences in receptors for Escherichia coli heat stable enterotoxin in the small and large intestine of children. Gastroenterology 94: 367-373, 1988[Medline].

3a.   Desai, G. N., J. Sahi, P. M. Reddy, D. Vidyasagar, and M. C. Rao. Chloride transport in primary cultures of mammalian colonocytes at different developmental stages. Gastroenterology 111: 1541-1550, 1996[Medline].

4.   Dudeja, P. K., J. M. Harig, M. L. Baldwin, E. J. Cragoe, K. Ramaswamy, and T. A. Brasitus. Na+ transport in human proximal colonic apical membrane vesicles. Gastroenterology 106: 125-133, 1994[Medline].

4a.   Dudeja, P. K., D. D. Rao, I. Syed, V. Joshi, R. Y. Dahdal, C. Gardner, M. C. Risk, L. Schmidt, D. Bavishi, K. E. Kim, J. M. Harig, J. L. Goldstein, T. J. Layden, and K. Ramaswamy. Intestinal distribution of human Na+/H+ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G483-G493, 1996[Abstract/Free Full Text].

4b.   Forte, L. R., P. K. Thorne, S. L. Eber, W. J. Krause, R. H. Freeman, S. H. Francis, and J. D. Corbin. Stimulation of intestinal Cl- transport by heat stable enterotoxin: activation of cAMP-dependent protein kinase by cGMP. Am. J. Physiol. 263 (Cell Physiol. 32): C607-C615, 1992[Abstract/Free Full Text].

5.   Gianella, R. A. Escherichia coli heat stable enerotoxins, guanylins, and their receptors: what are they and what do they do? J. Lab. Clin. Med. 125: 173-181, 1995[Medline].

6.   Goldstein, J. L., N. T. Nash, F. Al-Bazaaz, T. J. Layden, and M. C. Rao. Rectum has abnormal ion transport but normal cAMP binding proteins in cystic fibrosis. Am. J. Physiol. 255 (Cell Physiol. 24): C719-C724, 1988[Abstract/Free Full Text].

7.   Goldstein, J. L., J. Sahi, M. Bhuva, T. J. Layden, and M. C. Rao. Escherichia coli heat stable enterotoxin-mediated colonic Cl- secretion is absent in cystic fibrosis. Gastroenterology 107: 950-956, 1994[Medline].

7a.   Grasset, E., J. Bernabeu, and M. Pinto. Epithelial properties of human colonic carcinoma cell line Caco-2: effect of secretagogues. Am. J. Physiol. 248 (Cell Physiol. 17): C410-C418, 1985[Abstract].

8.   Halm, D. R., S. T. Halm, D. R. DiBona, R. A. Frizzell, and R. D. Johnson. Selective stimulation of epithelial cells in colonic crypts: relation to active chloride secretion. Am. J. Physiol. 269 (Cell Physiol. 38): C929-C942, 1995[Abstract/Free Full Text].

9.   Kachintorn, U., M. Vajanaphanich, A. E. Traynor-Kaplan, K. Dharmsathaphorn, and K. E. Barrett. Activation by calcium alone of chloride secretion in T84 epithelial cells. Br. J. Pharmacol. 109: 510-517, 1993[Abstract].

10.   Mandel, K. G. Characterization of a cAMP-activated Cl- transport pathway in the apical membrane of a human colonic epithelial cell line. J. Biol. Chem. 261: 704-712, 1986[Abstract/Free Full Text].

11.   Matthews, J. B., C. S. Awtrey, G. Hecht, K. J. Tally, R. S. Thompson, and J. L. Madara. Phorbol ester sequentially downregulates cAMP-regulated basolateral and apical Cl- transport pathways in T84 cells. Am. J. Physiol. 265 (Cell Physiol. 34): C1109-C1117, 1993[Abstract/Free Full Text].

12.   Metcalfe, D. D., M. Kaliner, and M. A. Donlon. The mast cell. CRC Crit. Rev. Immunol. 3: 23-74, 1981.

13.   Moyer, M. P., J. Stauffer, L. Manzano, Lee Tanzer, and R. Merriman. NCM460, a normal human colon mucosal epithelial cell line. In Vitro Cell. Dev. Biol. Anim. 32: 315-317, 1996[Medline].

14.   Nilsson, O., J. Cassuto, P.-A. Larsson, M. Jodal, P. Lidberg, H. Ahlman, A. Dahlstrom, and O. Lundgren. 5-Hydroxytryptamine and cholera secretion: a histochemical and physiological study in cats. Gut 24: 542-548, 1983[Abstract].

15.   Sahi, J., G. Bissonnette, J. L. Goldstein, T. J. Layden, and M. C. Rao. Effect of Ca2+-dependent regulators on Cl- transport and protein phosphorylation in human colon (Abstract). FASEB J. 10: A544, 1996.

16.   Sahi, J., J. L. Goldstein, T. J. Layden, and M. C. Rao. Cyclic AMP- and phorbol ester-regulated Cl- permeabilities in primary cultures of human and rabbit colonocytes. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G846-G855, 1994[Abstract/Free Full Text].

17.   Sahi, J., J. S. Stauffer, T. J. Layden, M. P. Moyer, and M. C. Rao. Chloride transport characterisics of an immortalized human epithelial cell line (N3) derived from the normal transverse colon (Abstract). Gastroenterology 108: A321, 1995.

18.   Sahi, J., J. S. Stauffer, T. J. Layden, M. P. Moyer, and M. C. Rao. Na+/H+ exchange activity of an immortalized human epithelial colonic cell line (Abstract). Gastroenterology 110: A356, 1996.

19.   Sahi, J., M. P. Wiggins, G. B. Gibori, T. J. Layden, and M. C. Rao. Calcium regulated chloride permeabilities in primary cultures of rabbit colonocytes. J. Cell. Physiol. 168: 276-283, 1996[Medline].

20.   Saj, M. M., R. V. Benya, G. A. Hecht, and M. C. Rao. Down regulation of phorbol ester effects on cAMP-activated Cl secretion in T-84 cells (Abstract). Gastroenterology 88: A666, 1990.

21.   Sellin, J. H., and R. De Soignie. Ion transport in human colon in vitro. Gastroenterology 93: 441-448, 1987[Medline].

22.   Suvitayavat, W., P. B. Dunham, M. Haas, and M. C. Rao. Characterization of the proteins of the intestinal Na+-K+-2Cl- cotransporter. Am. J. Physiol. 267 (Cell Physiol. 36): C375-C384, 1994[Abstract/Free Full Text].

23.   Vaandrager, A. B., N. Van Den Berghe, A. G. M. Bot, and H. R. De Jonge. Phorbol esters stimulate and inhibit Cl- secretion by different mechanisms in a colonic cell line. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G249-G256, 1992[Abstract/Free Full Text].

24.   Van Den Berghe, N., A. B. Vaandrager, A. G. M. Bot, P. J. Parker, and H. R. De Jonge. Dual role for protein kinase Calpha as a regulator of ion secretion in the HT29cl.19A human colonic cell line. Biochem. J. 285: 673-679, 1992[Medline].

25.   Verkman, A. S. Development of biological applications of chloride-sensitive fluorescent indicators. Am. J. Physiol. 259 (Cell Physiol. 28): C375-C388, 1990[Abstract/Free Full Text].

26.   Wasserman, S. I., K. E. Barrett, P. A. Huott, G. Beuerlein, M. F. Kagnoff, and K. Dharmsathaphorn. Immune-related intestinal Cl- secretion. I. Effect of histamine on the T84 cell line. Am. J. Physiol. 254 (Cell Physiol. 23): C53-C62, 1988[Abstract/Free Full Text].

27.   Yang, H. Y., N. Lieska, A. E. Goldman, and R. D. Goldman. A 300,000-mol. wt. intermediate filament-associated protein in baby hamster kidney (BHK-21) cells. J. Cell Biol. 100: 620-631, 1985[Abstract].


Am J Physiol Cell Physiol 275(4):C1048-C1057
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society