Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line

We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Proc. Natl. Acad. Sci. USA. 86:9313-9317). While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (greater than 90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other well-characterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.     

Uptake and intracellular fate of L-DOPA in a human intestinal epithelial cell line: Caco-2

The aim of the presentstudy was to examine the kinetic characteristics of theL-3,4-dihydroxyphenylalanine (L-DOPA)transporter and the fate of newly formed dopamine in Caco-2 cells. Inthe presence of 50 µM benserazide (an inhibitor of aromaticL-amino acid decarboxylase), L-DOPA was rapidlyaccumulated in Caco-2 cells. At equilibrium (30 min of incubation) theintracellular L-DOPA concentration was 10.2 ± 0.1 µM ata medium concentration of 0.5 µM. In saturation experiments theaccumulation of L-DOPA was saturable with aMichaelis-Menten constant (K_m) of 60 ± 10 µMand a maximal reaction velocity (V_max) of 6.6 ± 0.3 nmol · mg protein¹ · 6 min¹; at 4°C the amount of L-DOPAaccumulated in the cells was nonsaturable. When cells were incubatedwith increasing concentrations of L-DOPA (10-100 µM)in the absence of benserazide, a substantial amount of theL-DOPA that was taken up was decarboxylated to dopamine, with an apparent K_m of 27.2 µM. In experimentsperformed in cells cultured in polycarbonate filters, theaccumulation of L-DOPA in the presence of benserazide wasgreater when the substrate was applied from the basolateral cell borderthan when it was applied from the apical cell border. In the absence ofbenserazide, L-DOPA applied from the basolateral cellborder resulted in a nonlinear formation of dopamine(K_m = 43 ± 7 µM,V_max = 23.7 ± 1.2 nmol · mgprotein¹ · 6 min¹). Theamount of dopamine leaving the cell through the apical cell border waslower than the amount that escaped through the basolateral cell border,and the process was saturable (K_m = 623 ± 238 µM, V_max = 0.19 ± 0.02 nmol · mgprotein¹ · 6 min¹). Inconclusion, the data presented here show that Caco-2 cells are endowedwith an efficient L-DOPA uptake system, and intracellular L-DOPA was found to be rapidly converted to dopamine, someof which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way tolook at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.

     

The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications

The gastrointestinal tract remains the most popular and acceptable route of administration for drugs. It offers the great advantage of convenience and many compounds are well absorbed and thereby provide acceptable plasma concentration-time profiles. Currently there is considerable interest from the pharmaceutical industry in development of cell culture systems that would mimic the intestinal mucosa in order to evaluate strategies for investigating and/or enhancing drug absorption. The intestinal epithelial cells of primary interest, from the standpoint of drug absorption and metabolism, are the villus cells, which are fully differentiated cells. Anin vitro cell culture system consisting of a monolayer of viable, polarized and fully differentiated villus cells, similar to that found in the small intestine, would be a valuable tool in the study of drug and nutrient transport and metabolism.The Caco-2 cell line, which exhibits a well-differentiated brush border on the apical surface and tight junctions, and expresses typical small-intestinal microvillus hydrolases and nutrient transporters, has proven to be the most popularin vitro model (a) to rapidly assess the cellular permeability of potential drug candidates, (b) to elucidate pathways of drug transport (e.g., passive versus carrier mediated), (c) to assess formulation strategies designed to enhance membrane permeability, (d) to determine the optimal physicochemical characteristics for passive diffusion of drugs, and (e) to assess potential toxic effects of drug candidates or formulation components on this biological barrier. Since differentiated Caco-2 cells express various cytochrome P450 isoforms and phase II enzymes such as UDP-glucuronosyltransferases, sulfotransferases and glutathione-S-transferases, this model could also allow the study of presystemic drug metabolism.     

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

Effects of capsaicin on human intestinal cell line Caco-2

The influence of capsaicin processing on human intestinal cell line Caco-2 was examined by measuring transepithelial electrical resistance (TER). There was an increase in permeability at high concentration (200 to 500 μM) of capsaicin, and the effect was inhibited by pretreatment of capsazepine, which is a competitive antagonist of the vanilloid receptor 1 (VR1). LDH-activity as well as changes in intracellular Ca²⁺ were determined to know whether or not capsaicin affected TER activity through its influence on the tight junction. We also determined the expression of the VR1-like protein on Caco-2 cells in time-dependent manner by western blotting using vanilloid receptor (VR1) antiserum. Our results showed that the permeability increase by capsaicin was through binding to VR1-like protein of Caco-2 cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transport of a large neutral amino acid in a human intestinal epithelial cell line (Caco-2): uptake and efflux of phenylalanine.

The processes of L-phenylalanine (Phe) uptake and efflux from the apical (AP) and basolateral (BL) sides of an intestinal epithelial cell line (Caco-2) were investigated to further characterize the mechanism of transcellular transport of this amino acid. The results indicated that the initial uptake rates of Phe were saturable with a Km of 2.7 mM for AP uptake and 0.18 mM for BL uptake. Unlike the uptake, the initial efflux rates were shown to be proportional to the intracellular concentrations of Phe. Based on these kinetic studies and determination of other characteristics (e.g., Na+ dependency) of the uptake and efflux processes, it was concluded that AP uptake, BL uptake and BL efflux were distinctly different. This suggests that either different carriers or a different combination of carriers are responsible for the transmembrane transport of this amino acid. When the results of kinetic studies of Phe uptake and efflux were used to determine the rate-limiting step in the AP-to-BL transcellular transport of this amino acid, it was concluded that the BL efflux is the rate-limiting step in the transcellular transport of Phe in the Caco-2 cell monolayers.     

Vitamin A metabolism in the human intestinal Caco-2 cell line

The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox-dependent modulation of lipid synthesis induced by oleic acid in the human intestinal epithelial cell line Caco-2

The absorption, remodeling, and delivery of dietary lipids by intestinal cells are part of a complex multi-step process, the dynamics of which is influenced by the lipid composition of the diet and the physiological state of enterocytes. Emerging data indicate that, among the parameters known to modulate the cell functionality, the internal oxidative balance plays a pivotal role. In this study, we analyzed the effects of varying redox equilibria on the way in which the intestinal Caco-2 cell line utilize an exogenous lipid source such as oleic acid. Firstly, we manipulated the intracellular levels of soluble thiols (glutathione), and the amount of cell-associated products of lipid peroxidation, commonly regarded as two critical parameters characterizing the redox profile of the cells. Two different perturbants having opposite effects on the cell's redox profile were used: the pro-oxidizing agent CuSO4 (2.5 and 10 microM) and the antioxidant and thiol supplier N-acetylcysteine (NAC, 2.5 and 5 mM). The influence of these mild but critical manipulations on the incorporation of oleate (50 and 500 microM) into cholesterol, triacylglycerol, and phospholipid was then evaluated. We found that the emerging pro-oxidant condition induced by CuSO4 pre-exposure was associated with a significant up-regulation of phospholipid synthesis, while minor modifications were detected in that of triacylglycerols. Conversely, when a more reducing state was induced by NAC pre-treatment, there was a significant down-regulation of triacylglycerol synthesis, with minor modifications in that of phospholipids. In addition, the incorporation of oleic acid in the cholesteryl ester fraction appeared to be unmodified under all the redox conditions reported. On the whole, these results indicate that the pre-existing internal redox potential of the enterocytes is a critical factor that is able to differentially modulate lipid synthesis at the intestinal level. Thus, the adoption of a strategy designed to control/buffer the antioxidant capacity of the gastrointestinal tract could have important consequences for the modulation of lipid balance in the body.     

Caco-2 cell line: a system for studying intestinal iron transport across epithelial cell monolayers.

Iron transport across polarized intestinal epithelium was studied by using Caco-2 cells grown in bicameral chambers. When cells were grown under conditions of low, normal, or high iron concentration not only was the iron content of the cells markedly altered but the low iron cells exhibited a nearly 2-fold increase in transepithelial electrical resistance (TEER). 59Fe uptake from the apical surface into cells and transport into the basal chamber was affected both by the valency of the iron and the iron status of the cells. Uptake from 59Fe(II)-ascorbate was about 600 pmol 59Fe/h per mg protein, increased about 2-fold in low iron cells, and was about 13-200-fold greater than uptakes from 59Fe(III) chelated to nitrilotriacetic acid, BSA, or citrate. Transport into the basal chamber from 59Fe(II)-ascorbate was 3.7 +/- 1.7 pmol/h per cm2 for Fe-deficient cells vs. 0.72 +/- 0.1 pmol/h per cm2 for normal-Fe cells and from 59Fe(III)-BSA 1.1 +/- 0.2 pmol/h per cm2 vs. 0.3 +/- 0.03 pmol/h per cm2 for deficient vs. normal iron cells, respectively. The greater transport of iron both from Fe(II) and in iron deficient cells supports the use of the Caco-2 cells as a model for iron transport.     

Copper uptake and intracellular distribution in the human intestinal Caco-2 cell line

The apical uptake of ⁶⁴CuCl₂ was investigated in human differentiated intestinal Caco-2 cells grown on permeable supports. At pH 6.0 in the apical compartment, the uptake of copper was linear over the first 6 min and between 10 and 80 M CuCl₂ exhibited non-saturable transport kinetics. In addition, copper uptake was energy-independent, affected by the valency state of copper, preferring Cu(II) over Cu(I), and not influenced by high (10 mM) extracellular calcium. The intracellular distribution of copper was investigated by FPLC at different times of uptake (`pulse') and of `chase'. Intracellular copper initially bound predominantly to low molecular weight components (i.e., glutathione), and subsequently shifted to higher molecular weight components such as metallothionein and Cu,Zn superoxide dismutase.     

Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2

Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.     

Modulation of human Caco-2 intestinal epithelial cell phenotype by protein kinase C inhibitors

Protein kinase C (PKC) isoforms are altered in colon tumors and upon exposure of intestinal mucosa to nutrients. We evaluated the effects of the PKC inhibitors staurosporine and calphostin C on human Caco-2 intestinal epithelial proliferation, motility, and differentiation. Motility was quantitated by monolayer expansion and the brush border enzymes dipeptidyl dipeptidase (DPDD) and alkaline phosphatase (AP) by synthetic substrate digestion. Staurosporine (0.03-1.0 ng/ml) and calphostin C (10^-12M-10^-4 M) dose-dependently inhibited monolayer expansion and AP but stimulated DPDD. Proliferation was also inhibited but the effects of each inhibitor on motility, AP, and DPDD were preserved after mitomycin C proliferative blockade, suggesting that these effects were proliferation-independent. PKC inhibitors independently inhibit motility, AP and proliferation in human intestinal Caco-2 epithelial cells, but selectively stimulate the small intestinal differentiation marker DPDD. PKC may regulate small intestinal epithelial differentiation.     

Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2

The transport of the orally absorbed cephalosporin, cephalexin, was examined in the human epithelial cell line, Caco-2 that possesses intestinal enterocyte-like properties when cultured. In sodium-free buffer, the cells accumulated 1 mM D-[9-14C]cephalexin against a concentration gradient and obtained a distribution ratio of 3.5 within 180 min. Drug uptake was maximal when the extracellular pH was 6.0. Uptake was reduced by metabolic inhibitors and by protonophores indicating that uptake was energy- and proton-dependent. Kinetic analysis of the concentration dependence of the rate of cephalexin uptake showed that a non-saturable component (Kd of 0.18 +/- 0.01 nmol/min per mg protein per mM) and a transport system with a Km of 7.5 +/- 2.8 mM and a Vmax of 6.5 +/- 0.9 nmol/min per mg protein were responsible for drug uptake. Uptake was competitively inhibited by dipeptides. The transport carrier exhibited stereospecificity for the L-isomer of cephalexin. Drug uptake was not affected by the presence of amino acids, organic anions, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid or 4,4'-diisothiocyano-2,2'-disulfonic stilbene. Therefore, Caco-2 cells take up cephalexin by a proton-dependent dipeptide transport carrier that closely resembles the transporter present in the intestine. Caco-2 cells represent a cellular model for future studies of the dipeptide transporter.     

Factors affecting Caco-2 intestinal epithelial cell interleukin-6 secretion

Summary Intestinal epithelial cells (IEC) have previously been shown to produce several cytokines including interleukin-6 (IL-6). However, many factors which may regulate IL-6 secretion by human IEC still remain a mystery due in part to the lack of appropriate model cell lines and the difficulty of culturing human IEC over long periods of time. We have determined that the human colonic carcinoma cell line Caco-2 is capable of secreting IL-6 when stimulated by the inflammatory cytokines IL-1β or tumor necrosis factor-α (TNF-α), and stimulation of these cells with IL-1β plus TNF-α induced a synergistic enhancement of IL-6 secretion. The inflammatory cytokine-induced enhancement in IL-6 secretion was greatest when the cells were cultured in a 10% CO₂ atmosphere as compared to cells grown in 5% CO₂, suggesting that environmental CO₂ levels may affect IEC cytokine secretion. Finally, long-term culture of the Caco-2 cells to induce cellular differentiation had no effect on the capacity of these cells to produce IL-6, indicating that the regulation of IL-6 secretion was not affected by differentiation. Taken together, these studies provide important information on the factors which regulate IL-6 secretion by human IEC as they may contribute to the cytokine network during a mucosal inflammation. The results also suggest that the Caco-2 cell line is an appropriate model for further studies on the regulation of cytokine secretion by human IEC.     

Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line

Lipoprotein secretion by Caco-2 cells, a human intestinal cell line, was studied in cells grown on inserts containing a Millipore filter (0.45 micron), separating secretory products from the apical and basolateral membranes into separate chambers. Under these conditions, as observed by electron microscopy, the cells formed a monolayer of columnar epithelial cells with microvilli on the apical surface and tight junctions between cells. The electrical resistances of the cell monolayers were 250-500 ohms/cm2. Both 14C-labeled lipids and 35S-labeled proteins were used to assess lipoprotein secretion. After a 24-hr incubation with [14C]oleic acid, 60-80% of the secreted triglyceride (TG) was in the basolateral chamber; 40% of the TG was present in the d less than 1.006 g/ml (chylomicron + VLDL) fraction and 50% in the 1.006 less than d less than 1.063 g/ml (LDL) fraction. After a 4-hr incubation with [35S]methionine, apolipoproteins were found to be major secretory products with 75-100% secreted to the basolateral chamber. Apolipoproteins B-100, B-48, E, A-I, A-IV, and C-III were identified by immunoprecipitation. The d less than 1.006 g/ml fraction was found to contain all of the major apolipoproteins, while the LDL fraction contained primarily apoB-100 and apoE; the HDL (1.063 less than d less than 1.21 g/ml) fraction principally contained apoA-I and apoA-IV. Mn-heparin precipitated all of the [35S]methionine-labeled apoB-100 and B-48 and a majority of the other apolipoproteins, and 80% of the [14C]oleic acid-labeled triglyceride, but only 15% of the phospholipid, demonstrating that Caco-2 cells secrete triglyceride-rich lipoproteins containing apoB. Secretion of lipoproteins was dependent on the lipid content of the medium; prior incubation with lipoprotein-depleted serum specifically reduced the secretion of lipoproteins, while addition of both LDL and oleic acid to the medium maintained the level of apoB-100, B-48, and A-IV secretion to that observed in the control cultures.     

1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line

Background  

The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line.