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Institute of Human Physiology, University of Pavia, Pavia, Italy;
*
National Research Institute for Food and Nutrition, Rome, Italy;
Department of General Physiology and Biochemistry, University of Milan, Milan, Italy
2To whom correspondence and reprint requests should be addressed. E-mail: emanuela{at}unipv.it.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • guanidine • transepithelial transport • Caco-2 cells • apical and basolateral membranes • efflux
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–5
). The following transporters have been identified in the apical intestinal membrane: 1) a potential-dependent mechanism of organic cation (tyramine, tryptamine) absorption; 2) an electroneutral transport of choline, assumed to be engaged in absorption and secretion; 3) an antiport H+/G, responsible for G secretion; 4) an antiport H+/thiamine that is assumed to mediate the uptake; 5) a P-glycoprotein transporter that is assumed to mediate the secretion of certain organic cations; and, finally, 6) one or more transporters for the absorption and secretion of different polyamines (1
,2
, 6
). Limited information is available on basolateral transport mechanisms obtained from studies of membrane vesicles (7
,8
) and cell lines (5
,9
).
The mechanisms responsible for the secretion and absorption of organic cations have recently received considerable attention since a family of organic cation transporters, named OCT, was cloned (10
–18
). Several studies have investigated the characterization of the functional properties of these transporters by expressing the proteins in oocytes and cell lines (10
–16
,18
22
). These studies have shown that each transporter is selective for some substances, suggesting the existence of multiple transporters in the same tissue. There is still limited information on the physiological mechanisms responsible for organic cation secretion and absorption in human tissue through OCT, given that organic cation transporters from different species (21
) show different properties and tissue distribution (12
,19
). Moreover, functional studies performed on transfected cells do not indicate the localization of the transporters on the membrane (i.e., whether they are apical or basolateral) (10
,11
,13
,15
,16
,22
). Until now only rat OCT1, human and rat OCT2 and human OCTN2 have been localized (12
, 23
–26
).
The aim of our work was to investigate the mechanisms of small intestinal absorption and secretion of the endogenous organic cation G, a primary amine that exists almost exclusively as the positively charged guanidinium ion at physiological pH (pKa = 12.5). This substance, found in some foods such as mushrooms, corn germ, mussels characterized by antiviral and antifungal properties, was selected as a model of nutritionally and metabolically essential organic cations. For this purpose, the transepithelial transport and accumulation of G were studied in human intestinal Caco-2 cells, grown on permeable filter supports. These cells, derived from a human colon adenocarcinoma (27
), develop apical tight junctions and a polarized distribution of membrane proteins similar to those found in enterocytes in vivo (28
–32
). The Caco-2 cell line grown and differentiated on permeable filter supports has widely been used as a model for the study of the intestinal transport of nutrients (33
), bile acids (34
), amino acids (35
), vitamins (3
, 36
), drugs (37
–40
) and, recently, organic cations (3
–5
,9
). Moreover, in this study, the apical transport mechanism of G was also characterized by using apical membrane vesicles isolated from Caco-2 cells.
G transport in the small intestine has only been investigated using brush border membrane vesicle preparations by Miyamoto and co-workers (41
), who identified an H+/G antiport, distinct from the organic cation/H+ antiport, which is probably engaged in G secretion from the apical membrane.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Labeled compounds. [14C]-G (specific activity, 2.07 GBq/mmol) was from Amersham International plc (Buckinghamshire, UK). [Carboxyl-14C]-dextran-carboxyl (specific activity: 30 MBq/mmol) was from DuPont NEN Research Products (Boston, MA).
Decynium-22 (1,1'-diethyl-2,2'-cyanine iodide), EDTA, MES hydrate (ß-morpholine-ethane sulfonic acid), cimetidine, TEA (tetraethylammonium), guanidine and ouabain were purchased from Sigma Chemical (St. Louis, MO). Quinine sulfate was from BDH (Poole, Dorset, UK). Trypsin was supplied by SERVA (Heidelberg, Germany). Culture reagents were from Biochrom KG (Berlin, Germany). All other reagents were of analytical grade.
Cell culture
The Caco-2 cell line, obtained from Professor Alain Zweibaum (Institut National de la Sanité et de la Recherche Medicale, Villejuif, Paris), was used between passages 80 and 100. Cells were routinely grown as previously described (35
) in plastic tissue culture flasks using Dulbecco’s modified minimal essential medium containing 25 mmol/L glucose and supplemented with 10% fetal calf serum, 4 mmol/L L-glutamine, 10 g/L nonessential amino acids, 105 U/L penicillin and 100 mg/L streptomycin. Caco-2 cells were seeded at high density (2 x 106 cells/filter) into polycarbonate filter supports (Transwell, 24-mm diameter, 0.45-µm pore diameter; Costar Europe, Badhoevedorp, The Netherlands). At this density, the cells reached confluence within 48 h and were left to differentiate for 15–19 d. Experiments were performed after checking for the development of functional tight junctions monitored by measuring the transepithelial electrical resistance (TEER) across the monolayer with a Millipore Millicell ERS apparatus (Millipore, Bedford, MA) as previously described (35
). The integrity of the monolayer was also assayed by determining the transepithelial passage of the extracellular marker dextran. Briefly, [carboxyl-14C]-dextran-carboxyl (molecular mass: 50–70 kDa; specific activity: 30 MBq/mmol) was added to the apical medium and after 90-min incubation the radioactivity in the basolateral compartment and inside the cells was determined. [14C]-Dextran passage was expressed as the percentage of radioactivity transferred from the apical to the basolateral compartments after 90 min.
Cells were routinely screened for mycoplasma contamination by using the fluorescent dye bisbenzimide (H33258; Boehringer Mannheim, Milan, Italy) (42
).
All the experiments on cell monolayers were performed in a phosphate buffer saline solution supplemented with 1 mmol/L CaCl2 and 1 mmol/L MgCl2 (PBS+) and containing 10 mmol/L HEPES or MES to adjust the pH to 7.4 or 6.5, respectively. Unless otherwise stated, all experiments were performed by incubating cells with 2 mL of PBS+, pH 6.5 in the apical compartment and pH 7.4 in the basolateral compartment, to reproduce the acidic microclimate existing in vivo on the small intestinal surface. During the experiments, culture plates were maintained at 37°C in a water bath.
Caco-2 cells used for the preparation of apical membrane vesicles were cultured on 75-cm2 plastic flasks. Cells seeded at 4.2 x 105 cells/cm2 density reached confluence within 48 h and were left to differentiate for 15–19 d.
Membrane isolation
Apical membrane vesicles were prepared by using a method already used in our laboratory and modified for Caco-2 cells according to Mohrmann et al. (43
). The purity of apical membranes was estimated by assessing the enrichment in alkaline phosphatase of vesicles compared to the initial cell homogenate according to Murer et al. (44
). The enrichment of apical membrane vesicles was 8.14 ± 1.03 (mean ± SEM, n = 3).
Transport experiments
To evaluate bidirectional transepithelial transport, the monolayers were washed twice and preincubated for 10 min at 37°C with prewarmed PBS+. The time course of [14C]-guanidine ([14C]-G) (specific activity: 2.07 GBq/mmol) transepithelial transport was studied in both apical-to-basolateral and basolateral-to-apical directions by adding 15 µmol/L [14C]-G to the apical or basolateral side, respectively. A similar [14C]-G concentration was previously adopted in transport experiments using a human cell line (45
). The incubation medium (50 µL) was aspirated at each time point (5
,46
) and the radioactivity measured in a liquid scintillation counter (Tri-Carb 2000 CA; Packard Instruments, Downers Grove, IL). The amount of G inside the cells (accumulation) at the end of incubation was evaluated as previously described (35
). Transport experiments were also performed in the absence of an initial transepithelial gradient. To this end, the monolayers were incubated with 15 µmol/L [14C]-G in PBS+ added to both apical and basolateral media (starting equilibrium conditions), and, after set times, 50-µL samples of apical and basolateral incubation media were aspirated and the radioactivity was measured. The possible existence of an uphill process and the direction of a guanidine net flux was inferred from the apical/basolateral ratio. Transepithelial transport and accumulation of G were expressed as nmol/mg protein.
The Na+ dependency of the bidirectional transepithelial transport was evaluated by substituting NaCl in the donor compartment with isoosmotic amounts of either KCl or LiCl.
The influence of pH on the apical accumulation of G was evaluated by changing the PBS+ buffered at pH 6.5 of the apical compartment with the PBS+ buffered at pH 7.4 (pH of the basolateral side fixed at 7.4).
The specificity of G transport was assessed by using the possible inhibitors tetraethylammonium, cimetidine, quinine or decynium-22. In these experiments a 10-fold excess (1.5 mmol/L) of tetraethylammonium, cimetidine or quinine or 15 µmol/L decynium-22 was added to the incubation medium with the transepithelial transport and accumulation being evaluated after 30-min incubation.
For kinetic studies, [14C]-G accumulation across apical and basolateral membranes was determined after 30-min incubation by adding increasing concentrations of the organic cation (10–2000 µmol/L) to the apical and basolateral compartments, respectively. The best fit of the curves and the kinetic parameters (the apparent Michaelis–Menten constant Km and maximal velocity Jmax) were obtained by computerized least-squares regression analysis of the data (GraphPad Prism 2.01, San Diego, CA).
Transport experiments across apical membrane vesicles
The effect of changes in the transmembrane electrical potential on [14C]-G apical transport was evaluated by using apical membrane vesicles isolated from Caco-2 cells. To this purpose G uptake was measured in the presence of inorganic anions showing different permeabilities across the apical membrane. Incubation with the more liposoluble I- (about 20-fold compared with liposolubility of Cl- and K+) (47
) created a relatively more negative or positive intravesicular compartment when an inwardly or outwardly directed I- gradient was imposed, respectively. Vesicles were suspended and preequilibrated for 2 h at 4°C and then for 30 min at 25°C in solutions containing (in mmol/L): 2 MgSO4; 20 Tris–Hepes, pH 7.5; 140 KI or KCl. Preincubated vesicles (10 µL) were incubated at 25°C with 90 µL of solution containing 15 µmol/L [14C]-G and (in mmol/L): 2 MgSO4; 20 Tris–Hepes, pH 7.5; 140 KI or KCl. At each time point the incubation was terminated with 3 mL of cold stopping solution (150 mmol/L NaCl and 1 mmol/L Tris–Hepes, pH 7.5). The amount of radioactivity taken up by the vesicles was measured by a rapid filtration procedure (48
) using cellulose nitrate microfilters (Millipore; pore diameter, 0.65 µm). In each experiment, appropriate blanks were prepared to evaluate the radioactivity of [14C]-G nonspecifically absorbed on the microfilters. Radiometric measurements were carried out using the liquid scintillation counter.
Efflux experiments
The efflux of [14C]-G from the cells to the lumen and serosa was evaluated under different experimental conditions. After rinsing the monolayers with PBS+, the cells were loaded by incubating with 15 µmol/L of [14C]-G added to both the apical and the basolateral media (49
). After 30 min, the cells on the filters were washed twice with ice-cold buffer and transferred to a prewarmed incubation media at 37°C. To avoid backflow at each time point (1 to 30 min), the apical and basolateral media were removed and substituted with fresh prewarmed medium. The Na+ dependency of efflux was assayed by replacing NaCl with isoosmotic amounts of either LiCl or mannitol. The membrane potential dependency of apical and basolateral G efflux was also evaluated by following the time course in the presence of: 1) 145 mmol/L NaCl; 2) 145 mmol/L KCl; 3) 135 mmol/L NaCl plus 10 mmol/L BaCl2 (50
). The (Na+-K+)-ATPase involvement was monitored by adding 1 mmol/L ouabain to both apical and basolateral compartments. For specificity, a 10-fold excess (1.5 mmol/L) of tetraethylammonium or cimetidine or 15 µmol/L decynium-22 was added to the apical and basolateral incubating media. The effect of these organic cations was evaluated by measuring the amount of [14C]-G effluxed in the apical and basolateral incubating medium after 30-min incubation.
The influence of pH on the guanidine efflux was determined by changing the PBS+ buffered at pH 6.5 of the apical compartment with PBS+ buffered at pH 7.4, whereas the pH of the basolateral compartment was maintained at pH 7.4.
The diffusional component of the guanidine efflux from the cell to the apical and basolateral side was also evaluated by lowering the incubating temperature to 4°C.
Protein assay
The protein content was determined according to Lowry et al. (51
) after dissolving the filter-grown Caco-2 cells in 1 mol/L NaOH.
Statistics
All data were expressed as means ± SEM. The significance of the differences of the means in Na+- and membrane potential dependency and in inhibition experiments was evaluated by using the one-way ANOVA followed by Newman–Keuls’s Q test. Student’s t test for unpaired data was used to compare the apical-to-basolateral and the basolateral-to-apical transepithelial transport and accumulation, the apical vs. the basolateral efflux and the efflux at 37°C vs. 4°C. All statistical tests were carried out with a computerized program (52
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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/cm2 and the [14C]-dextran passage through the paracellular pathway was < 2% at 90 min, indicating the presence of well-formed tight junctions between the cells. Guanidine transport
After incubation with 15 µmol/L [14C]-G, G transepithelial transport in the basolateral-to-apical direction was greater than that in the opposite direction (apical-to-basolateral) (Fig. 1A
). The differences were statistically significant after 30-min incubation. The accumulation of G in the cells after 90 min was almost sixfold higher from the basolateral than from the apical side (Fig. 1
B).
|
When either KCl or LiCl replaced NaCl in the incubation medium, the transepithelial transport of 15 µmol/L [14C]-G decreased in the apical-to-basolateral direction (Fig. 2A
), becoming significantly different after 30 min, although no change was observed in the basolateral-to-apical direction (Fig. 2
C). However, G accumulation inside the cells decreased (P < 0.05) in both directions by about 60 and 50% for KCl and LiCl, respectively (Fig. 2
B, D).
|
Effect of transmembrane electrical potential
The experiments performed to differentiate between the effect of Na+ depletion and the related changes in transmembrane electrical potential caused by Na+ substitution showed that there were no differences in G uptake when an electrical negative or positive potential across apical membrane vesicles was imposed (Fig. 3
).
|
Both apical and basolateral accumulation curves showed a dual-transport mechanism, which was saturable at low concentrations with Michaelis–Menten-like kinetics; a nonsaturable mechanism prevailed at high concentrations (Fig. 4
). The saturable component of apical accumulation predominated for G concentrations below 25 µmol/L (Fig. 4
A), whereas that of the basolateral accumulation prevailed for G concentrations below 1800 µmol/L (Fig. 4
B). The kinetic constants of the saturable components were, for the apical transporter: Jmax, 0.05 ± 0.01 nmol/(mg protein·30 min); Km, 20.5 ± 1.5 µmol/L, and, for the basolateral transporter: Jmax, 11.3 ± 0.5 nmol/(mg protein·30 min); Km, 310 ± 11 µmol/L. Both Jmax and Km values were higher for the basolateral transporter (P 
0.01, Student’s t test).
|
, were, for the apical side, 1.96 ± 0.04 µL/(mg protein·30 min) and, for the basolateral side, 4.8 ± 0.2 µL/(mg protein·30 min), the former being lower (P 0.01, Student’s t test). Effects of some organic cations
Of the organic cations used, only quinine significantly reduced transepithelial transport in both directions (by about 47% in the apical-to-basolateral direction and about 40% in the opposite direction) (Fig. 5A, C
). This effect was first observed with the 15-min incubation (data not shown). Moreover, the apical and the basolateral accumulations of G were also strongly inhibited by quinine 60 and 80%, respectively (Fig. 5
B, D).
|
The basolateral efflux of G was consistently higher than the apical efflux at all times considered (P < 0.001) (Fig. 6
). The G exit from the apical and basolateral membranes was unaffected by NaCl replacement and by the presence of ouabain added to the apical and basolateral media (Fig. 7
). Temperature-dependency experiments showed that at 4°C, the amount of [14C]-G effluxed was markedly lower than that observed at 37°C and accounted for about 50 and 10% of total G effluxed from the apical and the basolateral membranes, respectively (Fig. 7)
.
|
|
). No difference in the apical efflux was observed when the membrane potential was reduced (Fig. 8
A).
|
The effects of some possible inhibitors on the carrier-mediated component of G efflux were evaluated by subtracting from the total G efflux values those measured at 4°C obtained in the presence of the inhibitors. Whereas the apical efflux was unaffected by all the organic cations used (Fig. 9A
), the basolateral efflux was reduced by quinine by about 50% (Fig. 9
B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and thiamine (3
). The transepithelial transport of G from the basolateral to the apical side was higher compared to that from the opposite direction (Fig. 1)
. This suggests that, in vivo, G secretion prevails over G absorption. Moreover, experiments performed in the initial absence of a [14C]-G gradient showed an uphill G net transport from the basolateral to the apical side, and confirmed that, in vivo, G is mainly secreted in the intestinal lumen (41
).
G transport across the apical membrane of Caco-2 monolayers decreased after Na+ replacement with K+ and Li+. The observation that G transport was unaffected by changes in pH suggests that an apical Na+-dependent transport mechanism could be involved (Fig. 2)
. However, the involvement of an electrical potential-dependent mechanism could not be ruled out, given that Na+ substitution with K+ also modifies the membrane potential (10
,54
). Experiments performed by using apical membrane vesicles isolated from Caco-2 cells allowed us to discriminate between Na+-dependent and potential-dependent mechanisms. Changes in the transmembrane electrical potential did not affect G apical uptake, thus confirming the hypothesis of a Na+-dependent mechanism located in the apical membrane (Fig. 3)
. Bleasby and co-workers found that apical uptake of the organic cation, 1-methyl-4-phenilpyridinium (MPP+) in Caco-2 cells is Na+ dependent, reinforcing the assumption of a new Na+-dependent process for the apical transport of organic cations (5
). Among the human OCT (hOCT) that have been cloned, only hOCTN2 mediates the Na+-dependent transport of carnitine, although it apparently does not transport G (55
).
TEA, cimetidine and decynium-22, specific OCT inhibitors, were ineffective in both apical transport and accumulation of G (Fig. 5
A, B), suggesting that the lumen-to-cell flux of G is mediated by a transporter that is different from those recently cloned. Interestingly, the presence of a novel organic cation transporter, distinct from the known members of the OCT family, was recently demonstrated on the apical membrane surface of retinal pigment epithelial cells (56
).
The G efflux of both apical and basolateral membranes has been shown to be carrier mediated because of its temperature dependency. Moreover, the apical efflux of G was membrane potential independent and stimulated by an inwardly directed H+ gradient (Figs. 7
and 8)
. This supports the existence of a G/H+ antiport mechanism responsible for apical secretion, as previously suggested by Miyamoto et al. (41
). Together, these results seem to indicate that two distinct mechanisms responsible for G influx and efflux exist in the apical membrane of Caco-2 cells. This hypothesis is also supported by the different effects of quinine on apical accumulation (Fig. 5)
and efflux (Fig. 9)
of G. Quinine inhibited G transport and accumulation (from the apical and the basolateral side) but did not affect apical efflux.
Kinetic experiments showed well-defined saturable components on both the apical and the basolateral membrane, whose kinetic constants were much higher for basolateral transport (Fig. 4)
. The basolateral maximal flux (Jmax) of G was about 220 times higher than that of the apical one, but the affinity of the transporters for G was 15-fold lower (higher Km). The passive permeability coefficient KD, although significantly higher on the basolateral side, was only doubled.
Although the G transepithelial transport from the basolateral-to-apical side was unaffected by isoosmotic Na+ substitution with K+ and Li+, G accumulation decreased (Fig. 2)
. This was probably attributable to the alteration of membrane potential caused by Na+ substitution, as observed by Martel et al. (4
), who studied MPP+ apical transport in Caco-2 cells. Because the organic cation transporters are mainly basolaterally located and functionally Na+- and pH independent but voltage dependent (1
,2
), the involvement of a member of the OCT family is likely.
In Caco-2 cells, the presence of hOCT1 (5
,57
), hOCT2 (57
), hOCT3 (58
) and hOCTN2 (18
) has been demonstrated by RT-PCR or Northern blot techniques. However, we found that the well-known inhibitors of hOCT1 (13
), hOCT2 (12
), hOCT3 (58
), hOCTN1 (59
) and hOCTN2 (60
), cimetidine and decynium-22, did not affect basolateral to apical transport and efflux (Fig. 5
, 9)
. Furthermore, both basolateral transport and accumulation of G were also unaffected by TEA, which has been shown to be a substrate of these transporters (12
,18
,19
,57
–59
), suggesting that basolateral transport could occur by a separate mechanism. It was previously demonstrated that hOCTN1 and hOCTN2 (55
) do not transport G and that hOCT3 can transport G but with a lower affinity than that of TEA (58
). However, as previously reported for the other substrates (12
,13
,19
,57
,61
–63
), we observed that quinine on the basolateral side trans-inhibited G efflux and cis-inhibited G influx (Figs. 5
and 9)
. Interestingly, quinine has been shown to freely permeate through plasma membranes and inhibit rat OCT2 from the intracellular side (63
).
Moreover, G efflux at the basolateral side was voltage dependent, given that a reduction of the membrane potential significantly increased G efflux (Fig. 8
B). Thus, as in the kidney and liver, the influx and efflux across the basolateral membrane of intestinal cells might occur through a member of the OCT family, apparently with high specificity for G.
The possible involvement of a mechanism responsible for organic cation efflux from the enterocyte directly coupled with the (Na+-K+)-ATPase, as found for thiamine (7
), can be excluded here because ouabain was ineffective in blocking G efflux (Fig. 7)
. A similar (Na+-K+)-ATPase independence in the basolateral transport of polyamines was also recently demonstrated (8
). Up to now, no organic cation, except for thiamin, has been shown to use a (Na+-K+)-ATPase–mediated mechanism for transmembrane exit from enterocytes (7
).
In conclusion, these results indicate that in Caco-2 cells, G uptake across the apical membrane is mediated by a Na+-dependent mechanism, whereas G efflux into the apical compartment uses a H+/organic cation exchanger. On the basolateral side, G transport in both directions probably occurs through the mediation of a new member of the OCT family transporter that is highly specific for G.
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FOOTNOTES |
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3 Abbreviations used: G, guanidine; MES, ß-morpholine-ethane sulfonic acid; OCT, organic cation transporter; PBS+, phosphate buffer saline solution added with 1 mmol/L CaCl2 and 1 mmol/L MgCl2; TEER, transepithelial electrical resistance.
Manuscript received 23 January 2002. Initial review completed 21 February 2002. Revision accepted 17 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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