GASTROJZNTEROLOGYlSSo;89A675-1687
Polarized Secretion of Diamine Oxidase bY Intestinal Epithelial Cells and Its Stimulation by Heparin BRUNO
DANIELE
and ANDREA
QUARONI
Section of Physiology, Division of Biological Sciences, Cornell University, Ithaca, New York
The Caco-% cells have been used as a model system to study the pathways of diamine oxidase secretion hy the intestinal epithelium. When grown in Transwell illter chamber devices, the polarized cell monolayers released the enzyme preferentially into the basal chamber. Heparin (l-10 USP U/mL) rapidly induced a marked stimulation of enzyme secretion only when in contact with the basolateral cell membrane, where high affinity binding sites for [3H]heparin were also exclusively located. Among the other glycosaminoglycans tested, only heparan sulfate (150 mg/mL) was able to induce enzyme release; chondroitin sulfate (150 mg/mL) and dermatan sulfate (150 mg/mL) were without effect. Four monoclonal antibodies speciiic for human diamine oxidase were produced and found to immunoprecipitate a single protein with a molecular weight of g!i,ggg (under reducing conditions) from the culture medium of Caco-t cells. Immunofluorescence staining of cryostat sections of human small intestine with these four antibodies localized diamine oxidase at the lateral and basal sides of the villus cells. Staining was markedly reduced in specimens obtained from patients who received doses of heparin in vivo. This study concludes that release of diamine oxidase by intestinal cells occurs specifically at the basolateral aspect of the cells, most likely through the constitutive secretory pathway. Heparin may induce its marked stimulation of enzyme release by complexing with diamine oxidase bound to the cell surface or through interaction with specific binding sites also located in the basolateral membrane. In the intestinal mucosa in viva, the basal aspect of the villus cells represents the main site of diamine oxidase storage in the presence of normal circulating levels of heparin. he enzyme diamine oxidase (DAO; diamine: oxygen oxidoreductase, EC 1.4.3.6) catalyzes the deamination of histamine and the oxidation of putrescine (1). Although it has been detected in several
T
tissues, more than 90% of the total enzyme activity in the body is located and synthesized in the villus absorptive cells of the small intestine (2). Biochemical analysis based on subcellular fractionation techniques has suggested that DA0 is predominantly present inside the villus cells, with about 60% linked to organelles and the rest free in the cytosol; only a negligible activity was associated with the brushborder membrane (3). In nonpregnant humans and several animal species, the basal blood levels of DA0 are very low but are markedly increased after an IV injection of heparin, which induces release of the enzyme into the circulation (4-6). In the rat, a high dose of intraperitoneal heparin resulted in a rapid depletion of DA0 from the enterocytes, while the enzyme linked to organelles was released more slowly (7). Diamine oxidase has been shown to represent a sensitive plasma marker of intestinal injury in experimental animals (8-10). The ability of heparin to induce DA0 release from the small bowel has been used in clinical studies to assess the degree of small intestinal mucosal damage in patients with celiac disease (11~2). small bowel lymphoma (ll), and small bowel Crohn’s disease (13). It has been suggested that the effects of heparin are caused by displacement of DA0 from microvascular endothelial binding sites (14) where the enzyme may be transported and bound after synthesis by the villus cells. However, the mechanisms regulating secretion of the enzyme by the enterocytes and its transport from the epithelial to the endothelial cells have not been elucidated.
Abbreviations used in this paper: AP, epic& BL, basolateral; BSA, bovine serum albumin: DAO, diamina oxkkwq DME, Dulbecco’s modified Eagle madium; DlT, dithiothr&ol; LP& lipoprotein lipase; OCT, optimum cutting temperahue; SDS, sodium dodecyl sulfate. 0 1990bytheAmericanGaetroenterologicalArwodation OOM-5085/90/$3.00
1676
DANIELE AND QUARONI
To investigate the heparin-sensitive pathways of DA0 secretion by the intestinal cells, this study shows its release into the culture medium by Caco-2 cells, a human colon carcinoma cell line that, in the confluent state, expresses some differentiated features typical of the mature small bowel enterocytes (15). The cells were grown on membrane filters to have independent access to both apical (AP) and basolateral (BL) aspects of the cells. The ability of heparin and other glycosaminoglycans to induce DA0 secretion was tested, and the presence of heparin receptors on the cell membrane was investigated. Four monoclonal antibodies specific for human DA0 were prepared and characterized and used to localize the enzyme in the small intestine of adult humans. The results obtained in this study suggest that release of DA0 by intestinal epitheha1 cells is a specific process occurring selectively through the BL aspect of the cells. Heparin and, to a lesser degree, other glycosaminoglycans may induce release of DA0 from the cells by interacting with high-affinity binding sites located in the BL membrane of the cells.
Materials and Methods
GASTROENTEROLOGY Vol. 99, No. 6
mond, CA); sequenal grade SDS, from Pierce Chemical Co. (Rockfort, IL): polyethylene-glycol 1540 from J. T. Baker Chemical Co. (Phillipsburg, NJ]; and Bacto Freund’s adjuvant (complete and incomplete) from Difco Laboratories Inc. (Detroit, MI).
Cell Culture The Caco-2 cells were routinely cultured in loo-mm plastic petri dishes [Falcon, Becton, Dickinson Labware, Oxnard, CA], at 37’C in DME supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 10 mmol/L HEPES. 50 U/mL penicillin, and 50 pg/mL streptomycin in an atmosphere of 5% CO, and 95% air. They were reefed every other day with 10 mL of fresh medium and were subcultured serially when approximately 80% confluent. Cells to be used for measurement of DA0 release, or binding of heparin, were seeded at a density of 8 x lo6 cells per filter in Transwell clusters; 2.8 mL medium per well was added to the cluster plates (lower chamber volume], and 1.5 mL medium was added to the Transwells [upper chamber volume); the cells were used 8-10 days after reaching confluence. Before all experiments were performed the cells were rinsed twice with serum-free medium to eliminate proteins with a high affinity for heparin (i.e., antithrombin III] potentially present in the fetal bovine serum.
Materials The Caco-2 cell line was obtained from the American Type Culture Collection (Rockville, MD). Dulbecco’s modified Eagle’s medium (DME), fetal bovine serum, and penicillin-streptomycin mixture were from Whittaker Bioproducts, Inc. (Walkersville, MD]. L-Glutamine, a-methylmannopyranoside, semicarbazide hydrochloride, iproniazid phosphate, histamine dihydrochloride, trans-2-phenylcyclopropylamine (tranylcypromine) hydrochloride, aminoguanidine bicarbonate, tryptamine hydrochloride, and benzylamine were from Aldrich Chemical Company, Inc. [Milwaukee, WI]. Heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, dextran sulfate, and putrescine dihydrochloride were obtained- from Sigma Chemical Co. [St. Louis, MO]. [G-3H]Heparin (0.49 mCi/mg), methylated 14C-labeled molecular weight markers [carbonic anhydrase, bovine serum albumin (BSA), phosphorylase B, globulins, and myosin], r.-[35S]methionine (1083-1151 Ci/mmol), [side chain-2-14C] tryptamine bisuccinate (37 mCi/mmol), and [ring, methylenes-‘HIhistamine dihydrochloride (43.5 Ci/ mmol) were obtained from Du Pont-New England Nuclear (Boston, MA]. [1,4-14C]Putrescine dihydrochloride (100-120 mCi/mmol) and [7-‘4C]benzylamine hydrochloride (58 mCi/ mmol) were obtained from Amersham (Arlington Heights, IL). Transwell clusters, 24.5 mm in diameter (surface area, 4.71 cm’) with a 3.0-Km pore size, were from Costar (Cambridge, MA). Sephadex G-200, cyanogen bromide-activated Sepharose 4B, Concanavalin A coupled to CL-Sepharose 4B, and Protein A-CL Sepharose 4B were obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Balb/c mice (15-17 g] were obtained from Charles River Breeding Laboratory (Wilmington, MA); acrylamide, bisacrylamide, and Dowex AG 5OW-X4 resin were from Bio-Rad Laboratories (Rich-
Diamine Oxidase Assay Diamine oxidase activity was measured according to the method of Okuyama and Kobayashi (16). In a final volume of 1 mL, the assay mixture consisted of (a] 0.7 mL of 0.1 mol/L sodium phosphate buffer, pH 7.2; (b) 0.2 mL of test-sample solution (usually culture medium]; and [c] 0.1 mL of substrate solution [containing 2.5 mmol/L putrescine dihydrochloride and 5 uCi/mL of [1,4-‘4C]putrescine dihydrochloride]. Incubation was for 60 minutes at 37’C. The labeled reaction product, [‘4C]-Al-pyrroline, was directly extracted into 10 mL of scintillation fluid (toluene containing 0.1 g/L of POPOP and 6 g/L of PPO). The radioactivity present in 7 mL of scintillation fluid was measured using a Beckman LS-3800 scintillation counter. Assay blanks were prepared by boiling the samples for 10 minutes before incubation with the substrate solution. 1 mU of enzyme activity was defined as the amount of DA0 oxidizing 1 nmol of putrescine dihydrochloride per hour at 37°C and pH 7.2.
Movement of Heparin Membranes
Through Transwell
Radioactive heparin was added to the basal chamber of Transwell filters (containing fresh culture medium without cells in both chambers) to determine its rate of passage through the membrane pores, and the time required for establishment of an equal concentration in the two chambers. Radioactive heparin concentration in the upper chamber increased linearly during the first 35 minutes, reached half-maximal concentration in 25 minutes, and equilibrated by 1 hour.
DA0 SECRETION
December 1990
Heparin Binding Assay
r
[3H]Heparin with a specific activity of 0.49 mCi/mg and an anticoagulant activity of 145.8 U/mg was used. Before each experiment the cells (in the Transwell plates) were washed twice with serum-free medium. The radioactive heparin in serum-free culture medium was added to either the lower or upper chambers, and the plates were then placed in the incubator at 37%. For measurement of nonspecific binding, the cells were incubated under the same conditions with a 500-fold excess of cold heparin. After the indicated periods of time, the cells were washed three times with phosphate-buffered saline (PBS): then the filters were separated from the inserts and the cells were detached from the filters by gentle scraping and solubilized in 1 mL of 1N NaOH. After neutralization with 1 mol/L acetic acid, the extracts were added to 10 mL Acquasol (Du Pont/NEN, Boston, MA) and counted in a Beckman LS-3800 scintillation counter.
Preparation
of MonocJonaJ Antibodies Diamine Oxidase
Specific for Human
Diamine oxidase was partially purified from the culture medium of confluent Caco-2 cells maintained in serum-free medium containing heparin (10 USP U/mL) for 2 days before harvesting. The entire purification procedure was monitored by measuring DA0 activity in aliquots of samples taken before and after each step and from all chromatographic fractions obtained. Total protein in the samples was determined by the method of Lowry et al. (17). Three hundred milliliters of conditioned medium were spun in a Sorvall RC-5B centrifuge at 15,000 rpm for 20 minutes (at 4”C), and the supernatant was dialyzed against 10 mmol/L Tris-HCl buffer, pH 7.80.4 mol/L NaCI, 1 mmol/L CaCI,, 1 mmol/L MgCl,. The solution was then applied to a column (1.4 x 20 cm) of Concanavalin-A coupled with CLSepharose 4B, equilibrated with the same buffer, at a flow rate of 25 mL/h at 4%; after extensive washing with equilibration buffer (until the absorbance at 280 nm of the eluate dropped to below 0.05 OD units], DA0 was eluted with the same buffer containing 500 mmol/L a-methylmannopyranoside (18). Fractions positive for DA0 activity were combined, dialyzed against 30 mmol/L Tris-HCI buffer, pH 7.6,30 mmol/L NaCl, and then concentrated by surrounding the dialysis bag with dry Sephadex G-200 at 4°C. This concentration step was repeated until the total volume was reduced to about 10 mL. Diamine oxidase was further purified by high-pressure liquid chromatography using a Beckman Spherogel TM TSK DEAE-5PW column (7.5 mm x 7.5 cm] and a Beckman System Gold high-pressure liquid chromatography (Palo Alto, CA). The column was equilibrated with 30 mmol/L Tris-HCl, pH 7.6, 30 mmol/L NaCl, and eluted with a continuous gradient of NaCI, up to 400 mmol/L. Fractions of 0.8 mL were collected and tested for DA0 activity. The active fractions were combined and aliquots were used for measurement of total protein and DA0 activity, and for analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis [SDS-PAGE] under reducing condition [50 mmol/L dithiothreitol (D’IT)]. Approximately 10 protein bands were observed on the gels stained
FROM INTESTINAL
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1677
with Coomassie.blua. .Densitometric scanning [using an LKB laser densitometer (Pharmacia LKB Biotechnology, Piscataway, NJ]] showed that each of the 5-8 most prominent bands (among them one of approximate molecular weight of 90,000, tentatively assumed to represent DA0 subunits) accounted for, at most, 10% of the total protein in the sample. This protein mixture had a specific activity of 1.44 DA0 U/mg protein (representing a go-fold purification of DA0 with respect to the original culture medium) and was used for immunization of Balb-c mice (Charles River Breeding Laboratory; Wilmington, MA]. Each mouse was administered SC 500 pg protein per injection mixed with an equal volume (500 pL) of complete (primary immunization) or incomplete (booster injections) Freund’s adjuvant. The last boost consisted of antigen alone and was administered IP. Three days later, spleen cells were obtained and fused with NSI myeloma cells as previously described (19). Hybridomas were selected with HAT (hypoxanthine, aminopterin, thymidine) medium (20) and screened for antibody production as follows. Hybridoma conditioned media (10 mL) were incubated overnight at 4% with 300 WL of Sepharose-4B beads with covalently coupled affinity-purified rabbit-anti-mouse immunoglobulin G (IgG) (1 mg/mL wet beads). The beads were then spun at 1000 rpm in an IEC centrifuge (International Equipment Co., Needham, MA], the culture media were aspirated, and the beads were washed three times with 10 mL PBS: they were then incubated for 4 hours at 4% with 5 mL conditioned medium obtained from confluent Caco-2 cells. The beads were again washed three times with 10 mL PBS and then tested for DA0 activity using the assay described above. Negative controIs included unused complete culture medium [instead of hybridoma-conditioned medium) and nonimmune mouse IgG coupled to Sepharose 4B.
Cultures of interest were cloned twice by dilution plating in the presence of mitomycin-C-treated 3T3 cells. Doublecloned hybridomas were used for antibody characterization and large-scale antibody production in ascites form (19). Characterization
of Monoclonal
Antibodies
Hybridoma-conditioned media were used for determination of Ig subtype that was performed with a Mouse Immunoglobulin Subtype Identification Kit (Boehringer Mannheim Biochemical, Indianapolis, IN] following the protocol suggested by the manufacturer. All four monoclonal antibodies specific for DA0 were found to be of the IgG, subclass and were purified from culture media and ascitic fluids by affinity chromatography on a Protein A-CL Sepharose-4B column (21) as previously described (19). Affinity-purified monoclonal antibodies were bound to cyanogen bromide-activated Sepharose 4B (19).
Determination of the Antigen Specificity of the Diamine Oxidase Antibodies Analysis of antigens by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Proteins released into the culture medium by Caco-2 cells cultured on Transwell filters were labeled metabolically with [“S]methionine: cells
1676
DANIELE AND QLJARONI
confluent since 8 days were rinsed twice with methioninefree medium (DME methionine-free; Gihco Laboratories Inc., Grand Island, NY, containing 5% dialyzed fetal calf serum, 10 mmol/L HEPES, 2 mmol/L glutamine. 50 U/mL penicillin, 50 pg/mL streptomycin) and incubated 1 hour with the same medium. Then, the medium was aspirated and replaced with methionine-free medium containing 250 &i/mL of [35S]methionine and 10 USP U/mL of heparin; incubation was for 10 hours in the incubator. The media present in the lower and upper chambers were harvested separately, cleared of any suspended cells and cell debris by centrifugation at 50,000 rpm for 1 hour in a Beckman L8-70M centrifuge (FA-70 rotor], and dialyzed against PBS. An aliquot of each solution was dialyzed against water and then dried down in a Speed Vat evaporator (Savant Instruments, Farmingdale, NY); the pellets obtained were dissolved directly in SDS-PAGE sample solution (total protein samples]. The rest was supplemented with 1% Triton X-100 and protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 50 pg/mL leupeptin, 50 pg/mL antipain, 0.1 mg/mL aprotinin), divided into aliquots that were sequentially incubated with 4-5 aliquots of nonimmune mouse IgGSepharose-4B beads [to eliminate nonspecific binding, in particular of the molecular weight 250,000 protein produced by Caco-2 cells which is known to stick avidly to mouse Ig bound to Sepharose beads (22,23); the presence of 100 mmol/L galactose during incubation with antibodies-beads conjugates also markedly reduced unspecific binding of the molecular weight 250,000 glycoprotein], and then incubated with monoclonal antibodies bound to Sepharose-4B beads for 4 hours. Specifically bound antigens were eluted from the beads as previously described (24,25), separated by SDS-PAGE under reducing conditions and detected by fluorography (see below]. A monoclonal antibody specific for rat maltase (BBC3/88) (25) and known not to react with the corresponding human antigen, also affinity-purified and bound to Sepharose 4B, was used as a negative control. Determination of substrate specificity and inhibitors of afinity-purified diamine oxidase. The enzyme was purified from the culture medium of confluent CaCo-2 cells using the four DA0 monoclonal antibodies bound to Sepharose-4B beads as described above. The beads with the bound DA0 were washed extensively with 0.1 mol/L sodium phosphate buffer, pH 7.0, and then used in all enzyme assays. Assay blanks were prepared by boiling the beads for 10 minutes before incubation with the substrate solutions. Diamine oxidase activity was determined with putrescine (0.1mmol/L) as substrate as described above. Inhibitors (tranylcypromine. iproniazid, semicarbazide, aminoguanidine, NaCl) were added to the incubation mixture at the indicated concentrations (Table 1). Histaminase (with radioactive histamine, 0.025 mmol/L as substrate) and monoamine oxidase (with radioactive benzylamine or tryptamine. both 0.05 mmol/L, as substrates) activities were determined as described by Baylin and Margolis (26). The assays were performed at 87°C in 1.5-mL Eppendorf tubes (Brinkmann Instruments, Westbury, NY), each containing 200 PL of washed beads (with bound DAO) in a total volume of 1 mL.
GASTROENTEROLOGY
Table 1. Effects of Inhibitors Oxidase
Inhibitor None Aminoguanidine Semicarbazide Iproniazid Tranylcypromine NaCl
on Afinity-Purified
Inhibitor concentration (moVL)
Enzyme activity 50,000
2 x lo-5 1x1o-5 1x10-4 1 x 1o-4 1x1o-3 lx 1o-4 lx 10-a
0.2 0.5
0 a047 780 41,677 12,377 48,265 41,662
35,264 23,473
Vol. 99. No. 6
Diamine
% Inhibition 100.0 82.4 98.4 16.6 75.2 3.4 16.6
29.4 53.0
NOTE. I&mine oxidase was purified from the culture medium of confluent CaCo-2 cells using the four DA0 monoclonal antibodies bound to Sepharose-4B beads (see Methods). The beads with the bound DA0 were used in the enzyme assays (200 pL/tube]. Diamine oxidase activity was determined with putrescine (0.1 mmol/L) as substrate (see Methods). The inhibitors were added to the incubation mixture [final volume I mL) and the assays (in triplicate] were performed for 60 min at 37°C in 1.5-mL Eppendorf tubes. “Diamine oxidase activity expressed as cpm of labeled reaction product extracted into 10 mL of scintillation fluid minus the radioactivity extracted from assay blanks, prepared by boiling the beads for 10 minutes before incubation with the substrate solution.
Immunofluorescence
Staining
Segments of human jejunum and ileum were rinsed with 0.155 mol/L NaCl, cut into small fragments (0.5-l-cm long], embedded in optimum cutting temperature (OCT) compound, and quickly frozen in liquid nitrogen. Sections 4-8-Nrn thick were spread on glass slides and allowed to dry at room temperature for at least 1 hour, fixed with formaldehyde, and stained by the double-antibody fluorescence technique as described previously (19,25,27). The sections were examined with a Nikon Optiphot microscope equipped with epifluorescence illumination (Nikon Inc., Garden City, NY), or with a Zeiss Confocal Laser microscope (Rainin Instrument Co., Woburn, MA). Monoclonal antibodies were used in the form of straight conditioned culture media or ascites fluids diluted 1:lOO to 1:lOOO in PBS. Negative controls used in all experiments included fresh hybridoma culture medium, nonimmune mouse serum, and the monoclonal antibody BBC3/88, specific for rat maltase (25).
Gel Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detection of labeled proteins by fluorography were performed as previously described (25) using 7.5% acrylamide gels. Tissue Specimens Samples of normal small intestine were a generous gift of Dr. Lemuel Herrera, Surgical Developmental Oncology, Roswell Park Memorial Institute, Buffalo, NY, and
DA0 SECRETION PROM INTESTINAL CELLS
December 1990
were obtained from patients undergoing surgical resection. The protocol for tissue procurement was approved by the human research review committee of the Roswell Park Memorial Institute. The specimens were immediately embedded in OCT compound and frozen in liquid nitrogen.
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Statistical Analysis Student’s t test for unpaired data was usad as a statistical test. Results, expressed as mean + SD, were considered as significant when P < 0.05. 0
Results Spontaneous Secretion Caco-2 Cells
of Diamine Oxidase by
Caco-2 cells, grown in the presence of standard culture medium, were found to release significant amounts of DA0 activity into the lower chambers of the Transwell filters only when they established a confluent monolayer (Figure 1). A high level of unstimulated DA0 secretion was maintained in confluent cultures for at least 2-3 weeks. In both subconfluent and confluent cultures, little or no DA0 activity could be detected in the medium facing the apical side of the cells. Stimulation Heparin
of Diamine Oxidase Secretion
by
Addition of heparin (10 USP U/mLj to the basal medium of confluent Caco-2 cells resulted in a rapid and significant increase in their rate of DA0 release
-6
0 b” 2 0
0
2
4
6
8
10
12
14
16
18
days Figure 1. Unstlmulated secretion of DA0 by s&confluent and confluent Caco-2 cells grown on Transwell polycarbonate filters (3.0~Nrnpore size) The cells were seeded at a density of 8 x 10' cellsper filter (day O]and reached confluence at day 8; samples of medium were taken every other day from both basal I-) and upper (rZ~--_p)chambers and assayed for DA0 activity. J&W& point represents the mean of three different experiments, aud vertical bars represent SD.
lo
20
30
40
min
50
60
70
80
90
Pigure 2. Stimulation of DA0 secretion by heparin added to the basal chamber of Transwell filters. The Caco-2 cells were confluent since 8-10 days when used. At the start of the experiment, the basal medium was replaced with serum-free DME containing 10 USP U/mL of heparin. At the indicated thnes, samples of medium were taken [fkom three separate wells) from both basal Hand upper (O---O)chambers and assayed for DA0 activity. Control cultures received serum-free culture medium without heparin, and DA0 activity was measured in their basal medium (w----8). Each point represents the mean of three different experlmerits, and vertical bars represent SD.
into the same compartment (Figure 2). This stimulation was already apparent 10 minutes after heparin addition and approached a plateau by 60-90 minutes. Diamine oxidase activities in basal and apical media, determined 90 minutes after heparin addition, were 8.1 f 1.9 mU/mL and 0.05 + 0.01 mU/mL, respectively: at the same time, DA0 activity in the basal chamber of control cultures receiving serum-free medium without heparin was 1.8 * 0.3 mU/mL. In several similar experiments the highest dose of BL heparin tested (10 USP U/mL] produced, on the average, a 4.8-fold increase in the rate of DA0 secretion during the first 60 minutes. The magnitude of this increase was dependent on the concentration of BL heparin; it was evident at a dose as low as 1 USP U/mL and increased proportionately up to 10 USP U/mL (Figure 3). The rate of heparin-stimulated secretion of DA0 from Caco-2 cells was reduced by cooling of the cells at 4OC, but the observed differences between secretion rates at 4“C and 37°C did not reach statistical significance. Similar results were obtained with cells incubated in the absence of heparin. These findings, although limited in scope, suggest that heparin may act at extracellular sites in releasing, into the culture medium, DA0 previously bound to the cells’ plasma membrane. In contrast with the above findings, addition of heparin (10 USP U/mL) to the apical medium was without significant effect: the rates of DA0 secretion into both apical and basal chambers of the Transwell
1680 DANIELE AND QUARONI
GASTROENTEROLOGY Vol. 99, No. 8
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0
10
20
30
40
50
60
70
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min Figure 3. Effect of increasing concentrations of heparin, added to the hasal chamber of Transwell Illters, on DA0 secretion by confluent Caco-2 cells. At the start of the experiment, the basal medium was replaced with serum-free DME alone (A-A] or medium containing the following concentrations of heparin: 1 USPU/mL(+-+),5USPU/mL(O-@,lOUSPU/mL(O-O&At the indicated times, samples of basal medium were taken from three separate wells and assayed for DA0 activity. Each point represents the mean of three different experiments, and vertical bars represent SD.
filters were essentially identical to those of control cultures without heparin. Stimulation of DA0 secretion by Caco-2 cells was a specific effect of heparin and structurally related glycosaminoglycans. As shown in Table 2, heparan sulfate (150 mg/mL) added to the BL chambers of the Transwell filters also resulted in a significant increase in the DA0 activity detected in the basal culture medium when compared with controls [cells treated with serum-free medium alone). Under the same conditions, dermatan sulfate (150 mg/mL) and chondroitin sulfate (150 mg/mL) produced no stimulation of DA0 release. Interestingly, low molecular weight (5000) dextran sulfate, which is not a glycosaminoglycan and is structurally unrelated to heparin, also induced a moderate stimulation of enzyme secretion. The high molecular weight (500,000) dextran sulfate was without effect.
Binding of Heparin
to Caco-2 Cells
The results described above showed that heparin could stimulate DA0 secretion only by interacting with the BL aspect of the Caco-2 cells, prompting us to investigate whether binding sites for heparin are present in this region of the cells’ surface membrane. [‘HI-Heparin added to the basal chamber of the Transwell filters demonstrated a rapid, specific, and saturable binding to Caco-2 cells; half-maximal binding at each heparin concentration was achieved in about 20 minutes, and a plateau was observed after 1 hour. This also represented the time required for heparin, added to Transwell filters without cells, to
reach an equal concentration in the basal and apical compartments [see Methods). When the radioactive heparin was added to the apical culture medium, half maximal binding to Caco-2 cells was achieved within 5 minutes of incubation. Scatchard analysis (Figure 4) of the binding data obtained for heparin added to either the basal or apical chambers of the Transwell filters suggested that both low- and high-affinity heparin binding sites are present on the surface membrane of the Caco-2 cells. The high-affinity sites (3700/tell) had a Kd of 9.2 x lop9 mol/L and were detected exclusively on the BL aspect of the cells.
Preparation of Monoclonal Antibodies to Human Intestinal Diamine Oxidase The DA0 present in the culture medium of confluent Caco-2 cells was partially purified by affinity chromatography on a concanavalin A column (18) followed by ion-exchange chromatography (see Methods). The most active fractions represented a go-fold purification of DA0 and, when analyzed by SDSPAGE under reducing conditions, showed the presence of 4-5 major, and several minor, polypeptide bands. This protein mixture was used for immunizations of Balb-c mice, from which spleen cells were obtained and fused to NSI myeloma cells. Eight independent fusions yielded approximately 1650 hybridoma cultures, and four of them were found to produce antibodies immunoprecipitating DA0 activity from the culture medium of confluent Caco-2 cells. They were named DAO-1 through -4, and successfully
Table 2. Effects of Low Molecular Weight Heparin, Glycosaminoglycans, and Dextran Sulfate on Diamine Oxidase Secretion From Caco-2 Cells DA0 activity in BL chamber
(mU/mL) 30 minutes No addition Heparin (10 USP U/mL) Low molecular weight heparin (10 IU/mL) Heparan sulfate (150 mg/mL) Dermatan sulfate (150 mg/mL) Chondroitin sulfate (150 mg/mL) Dextran sulfate [molecular weight 500,000,50 pmol/L) Dextran sulfate (molecular weight 5000.50 fimol/L)
60 minutes
0.8 zt 0.2 4.2zt0.6
1.0+ 0.1 6.8f 0.4
4.3* 0.5 1.7f 0.1 0.9 f 0.1 0.7 * 0.2
5.3* 0.6 4.0* 0.3 0.9 * 0.2 0.8 zt 0.1
0.9 * 0.1
1.0 * 0.1
2.7 f 0.2
3.8 zt 0.4
NOTE. Caco-2 cells were grown in Transwell filters and used when 8-10 days confluent. The various compounds in serum-free medium were added to the basal chamber of the filters, then the cultures were placed in the incubator (at 37’C). At the indicated times, samples of basal medium were taken from six separate wells per compound and assayed for DA0 activity.
December
DA0
1990
SECRETION
FROM INTESTINAL
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1681
teins Al and E, transferrin, and a-fetoprotein (22) which follow a selective basal secretory pathway. The labeled DA0 was immunoprecipitated from both apical and basal media with the four DAO-series antibodies coupled to Sepharose-4B beads: the specifically bound antigens were eluted by heating the beads at 100°C in a buffer containing 1% SDS and 50 mmol/L dithiotheirol and also analyzed by SDS-PAGE. The results obtained are presented in Figure 5: a protein band with an apparent molecular weight of 95,000 was detected in the immunoprecipitates obtained from the basal medium (lanes 3-6). This molecular weight value agrees well with previous estimates of the
8
6
5 2
m’ 4
BASAL MEDIUM rcYc3Yf kDa r
2
2ooW.
* *
clT 0
5
10
-6 f moles/lo
20
25
cells
Figure 4. Specilfc binding of [tH]heparin to confluent Caco-2 cells (Scatchard analysisj The ceb were washed twice with serum-free medium, then [Vi)heparfn was added at different concentrations to either the basal or upper chambers of the Transwell plates: incubation was for 90 minutes at 37OC. Then the cells were washed three times with PBS and the bound radioactivity was determined as described in the Methods section. Nonspecific binding WM determined by incubating the cells under the same conditions with a stttt-fold excerm of cold heparin and was sub tracted from the Mnding data to obtain the specific binding reported in this figure. 4-4, AP; ?-Q ? BL.
cloned twice by dilution plating. All of them were found to secrete Ig of the IgG, subtype. Purification and Characterization of Caco-2 Cells’ Diamine Oxidase With Monoclonal Antibodies Confluent cultures of Caco-2 cells on Transwell filters were administered radioactive methionine, and the labeled proteins secreted into the basal and apical compartments were analyzed by SDS-PAGE followed by fluorography of the dried gels (Figure 5, lanes 2 and 8). Relatively minor differences were observed between total protein patterns from apical and basal media [see arrowheads to the left of lane 2). This suggests that many major secretory cell products were released into both compartments of the Transwell filters, in contrast with DA0 (see below], apolipopro-
1
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8
9 101112 13
Figure 5. The SDS-PAGE analysis of total proteina secreted into the culture medium by Caco-2 cells, and DA0 immunopurified with the four DAO-series monoclonal antibodies. Confluent cultures on Transwell filters were incubated for 10 hours with medium containing 10 USP U/mL heparin and radioactive methionine; the basal and apical media were collected separately and aliquote of them were dialyzed, freexedrted, dissolved in SDS sample solution, and applied to the gel. The remaining media were supplemented with 1% T&on X-100 and protease inhibitors and then divided into equal aliquots that were incubated with monoclonal antibodies bound to Sepharose-cB beads. Speciflcallybound labeled antigens were eluted from the beads and applied to the gel. The radfoactive proteins were detected by Suorography. Lane 1, molecular weight markers (myosin, 200 kdaltons; phosphorylase h, 97.4 kdaltons; bovine serum albumin, 69 kdaltons; ovalbumin, 43 kdaltonsj Lanes 2 and I), total proteti secreted into the basal and apical compartments, reepectfvely (arrowheads to tbe left of lane 2 point to bands showing a preferential basal location j Lanes 7 and 13, negative controls (BBC, antibody BBC3k8, speci5c for rat maltase and not reacting with human proteins) incubated with basal and apical media, reqectively. Lance 3-6, DA0 p&p Bated from the basal medium with antibodies DAOl-4. Lanes 9-12, DA0 predpitated from the apical medium with the same antibodies.
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subunit size of DA0 purified from human pregnancy plasma (261 and human placenta (281. A band of identical mobility was also observed in immunoprecipitates obtained from the apical medium, but was too faint to reproduce photographically (lanes 9-12); however, it was apparent in autoradiagraphs exposed for longer periods of time (not shown). The relative average intensities of the DA0 bands in apical and basal samples, determined by scanning of the autoradiograph shown in Figure 5 with an LKB laser densitometer, were 2.73 f 0.34 and 0.21 + 0.07 AU x mm, respectively. This suggests that at least 92.8% of the newly synthesized DA0 was secreted in a basal direction. Incubation of Caco-2 conditioned medium with all four DAO-series antibodies covalently bound to Sepharose-4B beads resulted in the complete depletion of DA0 activity from the medium. The enzyme was nearly quantitatively recovered on the washed beads, suggesting that antigen-antibody interaction had little or no effect on DA0 activity. The substrate specificity of the immunoprecipitated DA0 is shown in Table 3: the enzyme was active against both putrescine and histamine, whereas typical substrates of monoamine oxidase (benzylamine and tryptamine) were not oxidized. Its relative activity against histamine was approximately one third of that observed with putrescine, in accordance with a previous report for DA0 partially purified from human intestine (291. The effects of several known inhibitors of plasma DA0 on the enzyme produced by Caco-2 cells are summarized in Table 1. The immunoprecipitated DA0
Table 3. Activity of
Affinity-Purified Diamine Oxidase Against Different Amines Substrate oxidized [nmol]
Putrescine (0.1 mmol/L] Histamine (0.025 mmol/L] Benzylamine (0.05 mmol/L] Tryptamine (0.05 mmol/L]
15 minutes
30 minutes 60 minutes
2.03 + 0.04 0.76 + 0.07 0 0
4.24 f 0.17 1.44 f 0.05 0 0
8.16 + 0.12 3.34 f 0.09 0 0
NOTE. Diamine oxidase was purified from the culture medium of confluent Caco-2 cells using the four DA0 monoclonal antibodies bound to Sepharose-4B beads [see Methods]. The beads with the bound DA0 were washed extensively with 0.1 mol/L sodium phosphate buffer, pH 7.0, and then used in the enzyme assays (200 al/tube]. Diamine oxidase with [l,4-‘4C]putrescine as substrate (0.5 &i/tube], histaminase with [ring, methylenes-3H]histamineas substrate (10 pCi/tube], and monoamine oxidase with either [7-%]benzylamine (1 &i/tube] or [side chain-2-‘4C]tryptamine (1 &i/tube] as substrates were determined as described in the Methods section. All assays were performed in triplicate in a final volume of I mL at 37°C in 1.5.mL Eppendorf tubes. Assay blanks were prepared by boiling the beads for 10 minutes before incubation with the substrate solutions. Enzyme activities are expressed as nanomoles of substrates oxidized/deaminated in the indicated times.
was totally inhibited by low concentrations of aminoguanidine; semicarbazide at a lo-pmol/L concentration was also a very effective inhibitor of DA0 activity, whereas 0.1 mmol/L iproniazid and tranylcipromine produced an only moderate inhibition. Increasing concentrations of sodium chloride strongly reduced enzyme activity (Table l), as previously observed for histaminase released into circulation by heparin injection (301, pig kidney DA0 (311, and histaminase purified from human pregnancy plasma (26). Immunolocalization of Diamine Oxidase in the Intestinal Mucosa The four DAO-series monoclonal antibodies stained the lateral and basal aspects of the villus cells (Figure 6Bj in cryostat sections of ileum obtained from patients not subjected to systemic heparinization before surgery. The region of the crypts was consistently negative. In cross-sections of the villi (Figure 6C) the fluorescence appeared to be most intense in correspondence with the basal cell membrane, but at this level of resolution we could not exclude staining also of the basement membrane underlying the epithelial cells. Only a weak and diffuse fluorescence was observed in the cytoplasm of the villus cells. Small areas of lamina propria along the villi were occasionally stained (Figure 7A), but muscular layers, blood vessels (Figure 6D), and the serosa were completely negative. Cryostat sections prepared from intestinal specimens obtained from patients treated systemically with heparin before surgery were negative in most cases, using any of the four DAO-series antibodies (Figure 6E). In some cross-sections of villi, a punctuated fluorescence was noted in the lamina propria (Figure 6F], possibly corresponding to microvasculature. These results suggest that heparin can, in vivo, displace DA0 from its location on the basal and lateral aspects of the villus cells, releasing it into circulation. Similar results were obtained by using sections of human jejunum (Figure 7) where DA0 appeared to be absent in the upper portions of the lateral membrane of the villus cells and was predominantly localized in “pockets” at the base of the cells (Figure 7A and C). Discussion Our interest in the physiological role of DA0 stems from the observation that more than 96% of its total body activity is located in the differentiated small bowel enterocytes (2.29). This enzyme has been known to exist since 1929 (32), and has been well characterized in terms of its kinetics, substrate specificity, and susceptibility to specific inhibitors (18,26,28,31,33,34). However, its function in the small intestine and in other tissues like placenta and kidney, where it dis-
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Figure 6. Immunobcalization of DA0 in human small intestine with the monoclonal antibody DAO-1. Frozen sections (&6-m thick] of intestine were &ed with 1% formaldehyde and stained by indirect immuno0uoIvSicence. A. Negative control (antibody BBC3/66, specific for rat maltase, which does not crossreact with the corresponding human antigen) (original magnification x 320; bar = 100 pm). B-D, Ileum obtained from a patient who did not have systemic heparinization before surgery; E and F, ileum obtained from a patient treated with heparln before operation. B. Staining is restricted to the basal and lateral aspects of the villus cells, with no fluorescence detected in the region of the crypts, in the lamina propria, or in the muscular layers (original magnification x 180; bar = 100 pm). C. Cross-sections of the villi demonstrate the presence of fluorescence predominantly at the basal side of the epithelial cells, possibly corresponding to both the basal cell membrane and the underlying basement membrane (original magnification x410; bar = 50pm). D. No staining of the blood vessels is apparent [orlginal magnification x320; bar = 100pm]. E. No positive areas are apparent in this section comprising the upper region of the crypts and the lower two thirds of the vllli [original magnification x 300; bar = 100pm). F. Cross-sections of the villi demonstrate the presence of a punctuated fluorescence localized in the lamina proprla (original magnification x410; bars = 50 pm). Results identical to those shown in this figure were obtained with the other three monoclonal antibodies specific for human DA0 and by using unfixed frozen sections.
plays relatively high total activities, is still essentially a matter of speculation (35). Since putrescine, a polyamine whose levels are related to proliferative rates in intestinal cells and most other cell types examined (36), is an excellent substrate for DAO, it has been suggested that this enzyme is primarily involved in the regulation of intestinal cell proliferation. Evidence in favor of this hypothesis has been obtained (373, but is not supported by the results of other studies (38). The exclusive localization of DA0 in the upper regions of the intestinal villi (2,14,39; see also Figure 6), and its
release into the bloodstream by heparin (2,14), also seem to contradict a possible role of this enzyme in the regulation of polyamine metabolism in the proliferative crypt cells. An alternative hypothesis is that the primary function of intestinal DA0 is to protect the villus enterocytes from exogenous and endogenous amines. Some pathological conditions resulting in histamine release from mast cells present in the lamina propria (i.e., anaphylaxis and intestinal ischemia) have been associated with high blood levels of DA0 (40,41). However,
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Figure 7. Immunolocalizationof DA0 in human jejunum with the monoclonal antibody DAO-1 observed with a Zeiss Confocal Laser microscope. Frozen sections (4-6 wu thick) of MeMinewere fixedwith 1% formaldehydeand stainedby indirectimmunofluorescence. A. Longitudinal section of the upper portion of a villus showing the presence of DA0 at the basal and lateral sides of the epithelial cells. In this section, the fluorescence appears stronger in “pockets” localized at the base of the cells; small positive areas are also present in the lamina propria [original magnification x680: bars = 49 pm). B. Same section as in (A], viewed under rodamine settings to visualize ihe Evans-blue counterstain, (original magnification x680; bars = 49 pm). C. Cross-section of a villus, showing the preferential localization of DA0 in the lower portions of the lateral cell membrane and at the basal aspect of the epithelial cells (original magnification x840; bars = 40 pm).
the rationale for such a proposed function is unclear because the proliferative crypt cells should be equally (or more) sensitive to increased levels of histamine in their environment, and intestinal ischemia can effectively deplete the villus cell population (42). As an approach to a better understanding of the cellularand physioldgical functions of DAO, we have studied the mechanisms of its release by Caco-2 cells, the effects of heparin on enzyme secretion, and its detailed localization in the human small intestine in vivo. The Caco-2 cells were chosen for these studies because they represent an excellent in vitro model for some functions of differentiated villus enterocytes (15.22) and form a tight monolayer allowing a clear distinction between apical and basal secretory pathways (22). During their proliferative phase they have been shown to possess low levels of DA0 and a high ornithine decarboxylase activity; both intracellular and extracellular levels of DA0 were found to progressively increase (along with sucrase activity) as the cells reached confluence, and cell proliferation was greatly reduced (43). The DA0 produced by Caco-2 cells appeared as a
single band of molecular weight 95,000 on SDS-PAGE run under reducing conditions (Figure 51, a value consistent with previous estimates for the subunit size of this enzyme purified from other sources (26,28). The four monoclonal antibodies we have obtained independently precipitated the entire DA0 activity present in the conditioned culture medium of Caco-2 cells, demonstrating that it represents the only enzyme with putrescine-oxidizing ability secreted by these cells. The affinity-purified enzyme behaved as a classic DAO/histaminase in its substrate specificity and susceptibility to inhibitors (Tables 2 and 3): it was more active with putrescine than histamine as substrates and was completely inhibited by low concentrations of aminoguanidine. However, semicarbazide (1x 10e5 mol/L) was much more inhibitory, and iproniazid (0.1 mmol/L) less inhibitory than previously reported for DA0 purified from human pregnancy plasma (26) and human placenta (28). These results are suggestive of the existence of different isoforms of the enzyme. In this respect, it is noteworthy that an antiserum prepared against DA0 purified from human placenta was previously found to have identical patterns of immu-
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noreactivity with enzyme preparations from placenta, kidney, and small cell lung carcinoma, but to demonstrate only partial identity with histaminase from human intestine (44). Confluent Caco-2 cells were found to spontaneously release relatively high amounts of DA0 only through their BL membrane (Figure 1).This is consistent with the general physiology of secretion in these cells and in villus enterocytes in vivo (22). By studying several other products of Caco-2 cells, some of them coded for by transfected genes, Rindler and Traber (22) have concluded that this represents the default pathway for constitutively secreted proteins in intestinal cells, and that proteins transported through the BL pathway need not have specific sorting signals. They detected no proteins secreted specifically from the apical surface, but we have found that many major secretory products were present in both apical and basal culture media in comparable amounts (Figure 5). Similarly, alkaline phosphatase was shown to be secreted in both directions by Caco-2 cells grown on filters (45). Heparin at a concentration as low as 1 USP U/mL rapidly increased the rate of DA0 secretion [Figure 2). This effect was dose-dependent [Figure 3) and was only observed when heparin was added to the basal culture medium [data not shown). Among the other glycosaminoglycans tested, only heparan sulfate appeared to share this strong DA0 releasing ability with heparin (Table 2). These two compounds have the same hexosamine composition and are both rich in sulfate groups that are responsible for their high negative charge. Heparan sulfate has also been found to induce a high level of DA0 release upon IV injection in rats (46), although heparin was still more effective. The charge density of these compounds may be the key factor determining their ability to promote release of DA0 both in cultured cells and in the intestine in vivo, and the release mechanism may be centered on the formation of complexes with DA0 through electrostatic interactions. Conceivably the secreted enzyme may remain attached to the cell surface by interaction with a sulfated glycosaminoglycan, such as cell surand be reface-associated heparan sulfate (47,481, leased only by macromolecules that have a comparable or higher negative charge. The presence of relatively high levels of heparin (or structurally related compounds, such as heparan sulfate] has been demonstrated in total membrane fractions from Caco-2 cells, and in jejunal brush border membranes (46). This hypothesis has already been proposed for lipoprotein lipase (LPL), another enzyme released into the circulation by heparin (50). It is supported by the observation that dextran sulfate, which is neither a glycosaminoglycan nor structurally related to heparin, but is highly sulfated, was effective in releasing DA0
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CELLS
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into the culture medium (Table 2). However, it is unclear why the high molecular weight form of dextran sulfate we tested produced little or no effects in these experiments. In cultured mouse adipocytes, heparin was found to mobilize intracellular LPL without changing its rate of export from the endoplasmic reticulum to the cell surface (51). Under heparin stimulation, quantitative secretion of the mature form of LPL took place and intracellular degradation (eliminating up to 80% of newly synthesized LPL in cells not treated with heparin) was arrested. This could represent another mechanism of action of heparin on Caco-2 cells, and account for the presence of a limited number of high-affinity heparin receptors on the lateral-basal membrane of these cells (Figure 4). The existence of cell surface receptors for heparin has also been implied in a study investigating its antiproliferative effect on 3T3 cells involving inhibition of a protein kinase C-dependent pathway (52). To better understand the significance of the results we have obtained by using the Caco-2 cell model with respect to synthesis, release, and possible functions of DA0 in vivo, it was important to determine the precise location of this enzyme in the intestinal mucosa. To date, relatively few studies have attempted to localize DA0 in any of the tissues where it is known to be present in relatively large amounts, and many of them have been hampered by the limited specificity of the antisera used. In human placenta (53)and in the proximal convoluted tubuli (541,DAO/histaminase has been localized mainly in the cytoplasm of the epithelial cells. In pig kidney some staining of the basal portion of the proximal tubule cells and of regions of basal membrane was also observed in one study (55). The fluorescence associated with the basal membrane was later attributed to the presence of an impurity in the antigen preparation (56). The cellular and subcellular distribution of DA0 in the intestinal mucosa has been previously investigated only by relatively indirect cell fractionation and biochemical techniques (2). In this study, the four monoclonal antibodies we have produced showed the presence of detectable amounts of DA0 mainly at the BL aspects of the villus cells (Figures 6 and 7).In this location, small regions of accumulation were apparent in jejunal sections examined by confocal laser microscopy [Figure 7). These results confirm that the differentiated enterocytes are the primary (or only) source of this enzyme in the intestine, and are consistent with a BL pathway of DA0 secretion also in vivo. Little or no staining was observed in the lamina propria, the muscular layers, and in the serosa, suggesting that the base of the villus cells, and not the microvasculature, represents the primary site of storage of DA0 in the intestinal mucosa.
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In conclusion, the results obtained in this study indicate that DA0 is exclusively synthesized by the differentiated villus cells and secreted through their BL membrane, with which it may remain associated until heparin, or other highly negatively charged molecules, induce its release into circulation. The existence of high-affinity binding sites for heparin in the same location is suggestive of other, as yet uncharacterized functions and mechanisms of action of this compound on the villus cells. Our results are consistent with an extracellular function of DA0 in the intestine, unrelated to regulation of polyamines metabolism in the crypt cells. This may involve the previously suggested protection of the villus enterocytes from exogenous amines or induction and modulation of differentiated functions in the intestinal epithelial cells.
References 1. Buffoni F. Histaminase
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14.Robinson-White A, Baylin SB, Olivecrona T, Beaven MA. Binding of diamine oxidase activity to rat and guinea pig microvascular endothelial cells. J Clin Invest 1985;76:93-100. 15.Pinto M, Robine-Leon S, Appay MD, Kedinger M, Triadou N, Dussaulx E, Lacroix B, Simon-Assmann P, Haffen K, Fogh J, Zweibaum A. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell 1983;47:323-330. 16.Okuyama T, Kobayashi Y. Determination of diamine oxidase activity by liquid scintillation counting. Arch Biochem Biophys 1961;95:242-250. 17.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 18.Crabbe MJC, Waight RD, Bardsley WG, Barker RW, Kelly ID, Knowles PF. Human placental diamine oxidase. Improved purification and characterization of a copper- and manganesecontaining amine oxidase with novel substrate specificity. Biothem J 1976;155:679-687. 19.Quaroni A. Crypt cell development in newborn rat small intestine. J Cell Bio11985;100:1601-1610. 20.Littlefield JW. Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 1964;145:709710. 21.Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IBGI, IllGZAand I,G,, immunoglobulins from mouse using protein A-sepharose. Immunochemistry 1978;15:429-436. 22.Rindler MJ, Traber MG. A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells. J Cell Biol 1988;107:471-479. 23.Simon-Assmann P. Bouziges F, Vigny M, Kedinger M. Origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine. J Cell Biol 1989;109:18371848. 24.Quaroni A. Crypt cell antigen expression in human tumor colonic cell lines. Analysis with a panel of monoclonal antibodies to CaCo-2 luminal membrane components. J Nat1 Cancer Inst 1986;76:571-585. 25.Quaroni A, Isselbacher KJ. Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface components. Dev Biol1985;111:267-279. 26.Baylin SB, Margolis S. Purification of histaminase [diamine oxidase) from human pregnancy plasma by affinity chromatography. Biochim Biophys Acta 1975;397:294-306. 27.Quaroni A. Development of fetal rat intestine in organ and monolayer culture. J Cell Bio11985;100:1611-1622. 28.Bardsley WG, Crabbe MJC, Scott IV. The amine oxidases of human placenta and pregnancy plasma. Biochem J 1974;139:169181. 29.Bieganski T, Kusche J, Lorenz W, Hesterberg R, Stahlknecht CD, Feussner KD. Distribution and properties of human intestinal diamine oxidase and its relevance for the histamine catabolism. Biochim Biophys Acta 1983;756:196-203. 30.Baylin SB. Beaven MA, Krauss RM, Keiser HR. Response of plasma histaminase activity to small doses of heparin in normal subjects and patients with hyperlipoproteinemia. J Clin Invest 1973;52:1985-1993. 31.Bardsley WG, Crabbe MJC, Shindler JS. Kinetics of the diamine oxidase reaction. Biochem J 1973;131:459-469. 32.Best CH. The disappearance of histamine from autolysing lung tissue. J Physiol1929;67:256-263. 33.Bardsley WG, Ashford JS. Inhibition of pig kidney diamine oxidase by substrate analogues. Biochem J 1972;128:253-263. 34.Bardsley WG, Childs RE, Crabbe MJC. Inhibition of enzymes by metal ion-chelating reagents. The action of copper-chelating reagents on diamine oxidase. Biochem J 1974;137:61-66.
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35. Bieganski T. Biochemical, physiological and pathophysiological aspects of intestinal diamine oxidase. Acta Physiol Pol 1983;34: 139-153. 36. Luk GD, Yang P. Distribution of polyamines and their biosynthetic enzymes in intestinal adaptation. Am J Physiol 1988;254: G194-G200. 37. Erdman SH, Park JHY, Thompson JS, Grandjean CJ. Hart MH, Vanderhoof JA. Suppression of diamine oxidase activity enhances postresection ileal proliferation in the rat. Gastroenterology 1989;96:1533-1538. 38. D’Agostino L, Daniele B, Pignata S, Barone MV, D’Argenio G, Mazzacca G. Modifications in ornithine decarboxylase and diamine oxidase in small bowel mucosa of starved and reefed rats. Gut 1987;28(S1]:135-138. 39. Baylin SB, Stevens SA, Shakir KM. Association of diamine oxidase and ornithine decarboxylase with maturing cells in rapidly proliferating epithelium. Biochim Biophys Acta 1978541: 415-419. 40. Wollin A, Navert H, Bounous G. Effect of intestinal ischemia on diamine oxidase activity in rat intestinal tissue and blood. Gastroenterology 1981;80:349-355. 41. Bounous G, Echave’ V, Vobecky SJ, Navert H, Wollin A. Acute necrosis of the intestinal mucosa with high serum levels of diamine oxidase. Dig Dis Sci 1984:29:872-874. 42. Rijke RPC, Hanson WR, Plaiser HM. The effect of ischemic villas damage on crypt cell proliferation in the small intestine: evidence for a feedback control mechanism. Gastroenterology 1976;71:786-792. 43. D’Agostino L, Daniele B, Pignata S, Gentile R, Tagliaferri P, Contegiacomo A, Silvestro G. Polistina C, Bianco AR, Mazzacca G. Ornithine decarboxylase and diamine oxidase in human colon carcinoma cell line Caco-2 in culture. Gastroenterology 1989;97:888-894. 44. Baylin SB. Histaminase (diamine oxidase) activity in human tumors: An expression of a mature genome. Proc Nat1 Acad Sci USA 1977;74:883-887. 45. Sussman NL, Eliakim R, Rubin D, Perlmutter DH, DeSchryverKecskemeti K, Alpers DH. Intestinal alkaline phosphatase is secreted bidirectionally from villous enterocytes. Am J Physiol 1989;257:G14-G23. 46. D’Agostino L, Pignata S. Daniele B, Ventriglia R, Ferrari G, Ferraro C, Spagnuolo S, Lucchelli PE, Mazzacca G. Release of diamine oxidase into plasma by glycosaminoglycans in rats. Biochim Biophys Acta 1989;993:228-232. 47. Kremer PM. Heparan sulphate of cultured cells. I. Membrane associated and cell-sap species in Chinese hamster cells. Biochemistry 1971;10:1437-1445.
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48. Kremer PM. Heparan sulphate of cultured cells. II. Acid soluble and precipitable species of different cell lines. Biochemistry 1971;10:1445-1451. 49. Bosner MS, Gulick T, Riley DJS, Spilburg CA, Lange LG. Receptor-like function of heparin in the binding and uptake of neutral lipids. Proc Nat1 Acad Sci USA 1988;85:7438-7442. 50. Olivecrona T, Bengtsson G, Marklund SE, Lindahl U. Hook M. Heparin-lipoprotein lipase interactions. Fed Proc 1977;36:60-65. 51. Vannier C, Ailhaud G. Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport. J Biol Chem 1989;264:13206-13216. 52. Wright TC. Pukac LA, Castellot JJ, Karnovsky MJ, Levine RA, Kim-Park HY, Campisi J. Heparin suppresses the induction of c-fos and c-myc mRNA in mu&e fibroblasts by selective inhibition of a protein kinase C-dependent pathway. Proc Nat1 Acad Sci USA 1989;66:3199-3203. 53. Weisburger WR, Mendelsohn G, Eggleston JC, Baylin SB. Immunohistochemical localization of histaminase (diamine oxidase) in decidual cells of human placenta. Lab Invest 1978;38: 703-706. 54. Takano K, Suzuki T, Yasuda K. Localization of diamine oxidase and D-amino acid oxidase in kidney, demonstrated by means of immunohistochemical method. Acta Histochem Cytochem 1970; 3:105-113. 55. Argento-Ceru MP, Oratore A, Mondovi B, Finazzi-Agro A. Localization of diamine oxidase in pig kidney: immunofluorescence method. Cell Mol Biol1981;27:359-362. 56. Argento-Ceru MP, Oratore A, Beninati S, Finazzi-Agro A. Localization of diamine oxidase in pig kidney: immunoperoxidase method. Cell Mol Biol1981:27:363-367.
Received December 6.1989. Accepted June 20.1990. Address requests for reprints to: Andrea Quaroni, Ph.D., Section of Physiology, Cornell University, 820 Veterinary Research Tower, Ithaca, New York 14653. This study was supported by Grant No. DK-32656 from the National Institutes of Health, U.S.P.H.S. Dr. Daniele was the recipient of a fellowship from the Dottorato di Ricerca in Scienze Gastroenterologiche, Universita’ di Roma “La Sapienza,” Roma, Italy. Dr. Daniele’s present address is: Divisione di Gastroenterologia, 2a Facolta di Medicina, via S. Pansini 5,60131 Naples, Italy. The authors thank Dr. Lemuel Herrera for his invaluable help in the procurement of human intestinal specimens and gratefully acknowledge the technical assistance of Elaine Quaroni and Kelley Hurst.