A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway

Vitamin B₆ is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B₆ and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B₆ homeostasis. Due to the water-soluble nature of vitamin B₆ and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B₆ in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na⁺ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (K_m) of 11.99 ± 1.41 µM and a maximal velocity (V_max) of 67.63 ± 3.87 pmol · mg protein^-1 · 3 min^-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (K_i) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca⁺²/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the V_max, but not the apparent K_m, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH dependent and amiloride sensitive and appears to be under the regulation of an intracellular PKA-mediated pathway. vitamin B₆; intestinal transport; transport regulation; Caco-2 cell     

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.

     

Mechanism and regulation of folate uptake by human pancreatic epithelial MIA PaCa-2 cells

After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1) temperature and energy dependent; 2) pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3) Na⁺ independent; 4) saturable as a function of substrate concentration (apparent K_m = 0.762 ± 0.10 µM); 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake. human reduced folate carrier; small interfering RNA; transport regulation     

Riboflavin uptake by human-derived colonic epithelial NCM460 cells

Normal microflora ofthe large intestine synthesize a number of water-soluble vitaminsincluding riboflavin (RF). Recent studies have shown that colonicepithelial cells posses an efficient carrier-mediated mechanism forabsorbing some of these micronutrients. The aim of the present studywas to determine whether colonic cells also posses a carrier-mediatedmechanism for RF uptake and, if so, to characterize this mechanism andstudy its cellular regulation. Confluent monolayers of thehuman-derived nontransformed colonic epithelial cells NCM460 and[³H]RF were used in the study. Uptake of RF wasfound to be 1) appreciable and temperature and energydependent; 2) Na⁺ independent; 3) saturableas a function of concentration with an apparent K_mof 0.14 µM and V_max of 3.29 pmol · mgprotein¹ · 3 min¹; 4) inhibited by the structural analogslumiflavin and lumichrome (K_i of 1.8 and 14.1 µM,respectively) but not by the unrelated biotin; 5) inhibited ina competitive manner by the membrane transport inhibitor amiloride(K_i = 0.86 mM) but not by furosemide, DIDS, orprobenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RFuptake in RF-deficient and oversupplemented conditions, respectively;and 7) modulated by an intracellularCa²⁺/calmodulin-mediated pathway. These studies demonstratefor the first time the existence of a specialized carrier-mediatedmechanism for RF uptake in an in vitro cellular model system of humancolonocytes. This mechanism appears to be regulated by extracellularsubstrate level and by an intracellularCa²⁺/calmodulin-mediated pathway. It is suggested that theidentified transport system may be involved in the absorption ofbacterially synthesized RF in the large intestine and that this sourceof RF may contribute toward RF homeostasis, especially that of colonocytes.

     

Tin protoporphyrin induces intestinal chloride secretion by inducing light oxidation processes

Heme induces Cl^– secretion in intestinal epithelial cells, most likely via carbon monoxide (CO) generation. The major source of endogenous CO comes from the degradation of heme via heme oxygenase (HO). We hypothesized that an inhibitor of HO activity, tin protoporphyrin (SnPP), may inhibit the stimulatory effect of heme on Cl^– secretion. To test this hypothesis, we treated an intestinal epithelial cell line (Caco-2 cells) with SnPP. In contrast to our expectations, Caco-2 cells treated with SnPP had an increase in their short-circuit currents (I_sc) in Ussing chambers. This effect was observed only when the system was exposed to ambient light. SnPP-induced I_sc was caused by Cl^– secretion because it was inhibited in Cl^–-free medium, with ouabain or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The Cl^– secretion was not via activation of the CFTR, because a specific inhibitor had no effect. Likewise, inhibitors of adenylate cyclase and guanylate cyclase had no effect on the enhanced I_sc. SnPP-induced I_sc was inhibited by the antioxidant vitamins, -tocopherol and ascorbic acid. Electron paramagnetic resonance experiments confirmed that oxidative reactions were initiated with light in cells loaded with SnPP. These data suggest that SnPP-induced effects may not be entirely due to the inhibition of HO activity but rather to light-induced oxidative processes. These novel effects of SnPP-photosensitized oxidation may also lead to a new understanding of how intestinal Cl^– secretion can be regulated by the redox environment of the cell. heme oxygenase; electrolyte transport; carbon monoxide; cGMP; reactive oxygen species     

Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles

Thiamine, a water-soluble vitamin, is essential fornormal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety ofconditions. To date, however, little is known about the mechanism ofthiamine absorption in the native human small intestine. The objectiveof this study was, therefore, to characterize the mechanism of thiaminetransport across the brush-border membrane (BBM) of human smallintestine. With the use of purified BBM vesicles (BBMV) isolated fromthe jejunum of organ donors, thiamine uptake was found to be1) independent of Na⁺ but markedly stimulated byan outwardly directed H⁺ gradient (pH 5.5_in/pH7.5_out); 2) competitively inhibited by thecation transport inhibitor amiloride (inhibitor constant of 0.12 mM);3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structuralanalogs (amprolium, oxythiamine, and pyrithiamine), but not byunrelated organic cations (tetraethylammonium,N-methylnicotinamide, or choline); 5) notaffected by the addition of ATP to the inside and outside of the BBMV;6) potential insensitive; and 7) saturable as afunction of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 µM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein¹ · 10 s¹. Carrier-mediated thiamine uptake was also found inBBMV of human ileum. These data demonstrate the existence of aNa⁺-independent, pH-dependent, amiloride-sensitive,electroneutral carrier-mediated mechanism for thiamine absorption innative human small intestinal BBMV.

     

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells

The reabsorption of filtered di- andtripeptides as well as certain peptide mimetics from the tubular lumeninto renal epithelial cells is mediated by anH⁺-coupledhigh-affinity transport process. Here we demonstrate for the first timeH⁺-coupled uptake of dipeptidesinto the renal proximal tubule cell lineLLC-PK₁. Transport was assessed1) by uptake studies using theradiolabeled dipeptideD-[³H]Phe-L-Ala,2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and3) by measurement of intracellularpH (pH_i) changes as aconsequence of H⁺-coupleddipeptide transport. Uptake ofD-Phe-L-Alaincreased linearly over 11 days postconfluency and showed all thecharacteristics of the kidney cortex high-affinity peptide transporter,e.g., a pH optimum for transport ofD-Phe-L-Alaof 6.0, an apparent K_m value forinflux of 25.8 ± 3.6 µM, and affinities of differently chargeddipeptides or the -lactam antibiotic cefadroxil to the binding sitein the range of 20-80 µM.pH_i measurements established thepeptide transporter to induce pronounced intracellular acidification inLLC-PK₁ cells and confirm itspostulated role as a cellular acid loader.

     

Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO₃ exchange as the mechanism for ³⁶Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of ³⁶Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO₃ exchange. In the absence of voltage clamping, both outward OH gradient (pH_out/pH_in: 7.5/5.5) and outward HCO₃ gradient stimulated transient ³⁶Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven ³⁶Cl uptake, whereas HCO₃ gradient-driven ³⁶Cl uptake was only partially inhibited (38%). Both electroneutral HCO₃ and OH gradient-driven ³⁶Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant (K_i) of 26.9 and 30.6 µM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a K_m for Cl of 12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pH_i) was also determined with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pH_i both in HCO₃-buffered and in the nominally HCO₃-free buffered solutions (0.28 ± 0.02 and 0.11 ± 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO₃ exchange is present in basolateral membranes and that, in the absence of HCO₃, Cl-HCO₃ exchange can function as a Cl-OH exchange and regulate pH_i across basolateral membranes of rat distal colon. crypt glands; superfusion; intracellular pH; membrane vesicles; ³⁶Cl uptake; Cl-anion exchange     

Common mechanisms of monoacylglycerol and fatty acid uptake by human intestinal Caco-2 cells

Free fatty acids (FFA) andsn-2-monoacylglycerol (sn-2-MG), the twohydrolysis products of dietary triacylglycerol, are absorbed from thelumen into polarized enterocytes that line the small intestine.Intensive studies regarding FFA transport across the brush-bordermembrane of the enterocyte are available; however, little is knownabout sn-2-MG transport. We therefore studied the kineticsof sn-2-MG transport, compared with those of long-chain FFA(LCFA), by human intestinal Caco-2 cells. To mimic postprandial luminaland plasma environments, we examined the uptake of taurocholate-mixed lipids and albumin-bound lipids at the apical (AP) and basolateral (BL)surfaces of Caco-2 cells, respectively. The results demonstrate thatthe uptake of sn-2-monoolein at both the AP and BL membranes appears to be a saturable function of the monomer concentration ofsn-2-monoolein. Furthermore, trypsin preincubation inhibits sn-2-monoolein uptake at both AP and BL poles of cells.These results suggest that sn-2-monoolein uptake may be aprotein-mediated process. Competition studies also support aprotein-mediated mechanism and indicate that LCFA and LCMG may competethrough the same membrane protein(s) at the AP surface of Caco-2 cells.The plasma membrane fatty acid-binding protein (FABP_pm) isknown to be expressed in Caco-2, and here we demonstrate that fattyacid transport protein (FATP) is also expressed. These putative plasmamembrane LCFA transporters may be involved in the uptake ofsn-2-monoolein into Caco-2 cells.

     

Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K⁺, Cl^–, and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl^– current (I_Cl,swell) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl^– or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied I_Cl,swell and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted I_Cl,swell, but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl^–, taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane. caveolae; osmolyte; epithelial cell; chloride channel     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells

Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular K⁺, the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca²⁺ signal, and removal of external Ca²⁺ promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca²⁺ did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na⁺-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na⁺-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but Ca²⁺ signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport. membrane potential; Na⁺-alanine cotransport; epithelium; phosphatidylinositol 3-kinase; LLC-PK₁ cells     

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes

We recently identified a cDNA clone frommouse small intestine, which appears to be involved in folate transportwhen expressed in Xenopus oocytes. Theopen reading frame of this clone is identical to that of the reducedfolate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley,and K. H. Cowan. J. Biol. Chem. 269: 17-20,1994). The characteristics of this cDNA clone [previously referred toas intestinal folate carrier 1 (IFC-1)] expressed inXenopus oocytes, however, were foundto be different from the characteristics of folate transport in nativesmall intestinal epithelial cells. To further study these differences,we determined the characteristics of RFC when expressed in anintestinal epithelial cell line, IEC-6, and compared the findings toits characteristics when expressed inXenopus oocytes. RFC was stablytransfected into IEC-6 cells by electroporation; its cRNA wasmicroinjected into Xenopus oocytes.Northern blot analysis of poly(A)⁺RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFCwas found to be fourfold higher than uptake in control sublines. Thisincrease in folic acid and 5-MTHF uptake was inhibited by treatingIEC-6/RFC cells with cholesterol-modified antisense DNAoligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity(V_max) of theuptake process [the apparent Michaelis-Menten constant(K_m) alsochanged (range was 0.31 to 1.56 µM), but no specific trend wasseen]. In both IEC-6/RFC and control sublines, the uptake of bothfolic acid and 5-MTHF displayed 1)pH dependency, with a higher uptake at acidic pH 5.5 compared with pH7.5, and 2) inhibition to the sameextent by both reduced and oxidized folate derivatives. Thesecharacteristics are very similar to those seen in native intestinalepithelial cells. In contrast, RFC expressed inXenopus oocytes showed1) higher uptake at neutral andalkaline pH 7.5 compared with acidic pH 5.5 and2) higher sensitivity to reducedcompared with oxidized folate derivatives. Results of these studiesdemonstrate that the characteristics of RFC vary depending on the cellsystem in which it is expressed. Furthermore, the results may suggestthe involvement of cell- or tissue-specific posttranslationalmodification(s) and/or the existence of an auxiliary proteinthat may account for the differences in the characteristics of theintestinal RFC when expressed inXenopus oocytes compared with whenexpressed in intestinal epithelial cells.

     

Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4

The cellular mechanisms regulating intestinal proliferation anddifferentiation remain largely undefined. Previously, we showed anearly induction of the cyclin-dependent kinase (CDK) inhibitor p21^Waf1/Cip1 in Caco-2 cells, ahuman colon cancer line that spontaneously differentiates into a smallbowel phenotype. The purpose of our present study was to assess thetiming of cell cycle arrest in relation to differentiation in Caco-2cells and to examine the mechanisms responsible for CDK inactivation.Caco-2 cells undergo a relativeG₁/S block and cease toproliferate at day3 postconfluency; an increase in theactivity of terminally differentiated brush-border enzymes (sucrase andalkaline phosphatase) was noted at day6 postconfluency. Cell cycle block wasassociated with suppression of both CDK2 and CDK4 activities, which areimportant for G₁/S progression.Treatment of the CDK immune complexes with the detergent deoxycholate(DOC) resulted in restoration of CDK2, but not CDK4, activity atday 3 postconfluency, suggesting the presence of inhibitory protein(s)binding to the cyclin/CDK2 complex at this time point. An increasedbinding of p21^Waf1/Cip1 to CDK2complexes at day3 postconfluency was noted, suggesting a potential role for p21^Waf1/Cip1in CDK2 inactivation; however, immunodepletion ofp21^Waf1/Cip1 from Caco-2 proteinextracts demonstrated thatp21^Waf1/Cip1 is only partiallyresponsible for CDK2 suppression atday 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears tocontribute to the CDK2 inactivation noted atdays6 and12 postconfluency. Taken together, ourresults suggest that multiple mechanisms contribute to CDK suppressionduring Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leadsto G₁ arrest and inhibition ofproliferation that precede Caco-2 cell differentiation.

     

Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely (P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.     

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

The general phosphate need in mammalian cells is accommodated by members of the P_i transport (PiT) family (SLC20), which use either Na⁺ or H⁺ to mediate inorganic phosphate (P_i) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na⁺-dependent P_i (NaP_i) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with ³²P_i as a traceable P_i source. For PiT1, the Michaelis-Menten constant for P_i was determined as 322.5 ± 124.5 µM. PiT2 was analyzed for the first time and showed positive cooperativity in P_i uptake with a half-maximal activity constant for P_i of 163.5 ± 39.8 µM. PiT1- and PiT2-mediated Na⁺-dependent P_i uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na⁺ dependency patterns. However, only PiT2 was capable of Na⁺-independent P_i transport at acidic pH. Study of the impact of divalent cations Ca²⁺ and Mg²⁺ revealed that Ca²⁺ was important, but not critical, for NaP_i transport function of PiT proteins. To gain insight into the NaP_i cotransport function, we analyzed PiT2 and a PiT2 P_i transport knockout mutant using ²²Na⁺ as a traceable Na⁺ source. Na⁺ was transported by PiT2 even without P_i in the uptake medium and also when P_i transport function was knocked out. This is the first time decoupling of P_i from Na⁺ transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E⁵⁵ and E⁵⁷⁵ are responsible for linking P_i import to Na⁺ transport in PiT2. inorganic phosphate transport; retroviral receptor; SLC20     

Characterization of inorganic phosphate transport in osteoclast-like cells

Osteoclasts possess inorganic phosphate (P_i) transport systems to take up external P_i during bone resorption. In the present study, we characterized P_i transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-B ligand (RANKL). In undifferentiated RAW264.7 cells, P_i transport into the cells was Na⁺ dependent, but after treatment with RANKL, Na⁺-independent P_i transport was significantly increased. In addition, compared with neutral pH, the activity of the Na⁺-independent P_i transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na⁺-independent system consisted of two components with K_m of 0.35 mM and 7.5 mM. The inhibitors of P_i transport, phosphonoformic acid, and arsenate substantially decreased P_i transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K⁺ ionophore, valinomycin, significantly suppressed P_i transport activity. Analysis of BCECF fluorescence indicated that P_i transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K⁺ ion stimulated P_i transport, suggesting that membrane voltage is involved in the regulation of P_i transport activity. Finally, bone particles significantly increased Na⁺-independent P_i transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a P_i transport system with characteristics that are different from those of other Na⁺-dependent P_i transporters. We conclude that stimulation of P_i transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment. Na⁺-dependent phosphate cotransporter; RAW264.7; phosphate uptake     

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration

Polyamines are essential for cell migrationduring early mucosal restitution after wounding in the gastrointestinaltract. Activity of voltage-gated K⁺ channels (Kv) controlsmembrane potential (E_m) that regulates cytoplasmicfree Ca²⁺ concentration([Ca²⁺]_cyt) by governing thedriving force for Ca²⁺ influx. This study determinedwhether polyamines are required for the stimulation of cell migrationby altering K⁺ channel gene expression,E_m, and[Ca²⁺]_cyt in intestinal epithelialcells (IEC-6). The specific inhibitor of polyamine synthesis,-difluoromethylornithine (DFMO, 5 mM), depleted cellularpolyamines (putrescine, spermidine, and spermine), selectivelyinhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression,and resulted in membrane depolarization. Because IEC-6 cells did notexpress voltage-gated Ca²⁺ channels, the depolarizedE_m in DFMO-treated cells decreased [Ca²⁺]_cyt as a result of reduceddriving force for Ca²⁺ influx through capacitativeCa²⁺ entry. Migration was reduced by 80% in thepolyamine-deficient cells. Exogenous spermidine not only reversed theeffects of DFMO on Kv1.1 channel expression, E_m,and [Ca²⁺]_cyt but also restoredcell migration to normal. Removal of extracellular Ca²⁺ orblockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cellmigration by exogenous spermidine in polyamine-deficient cells. Theseresults suggest that polyamine-dependent intestinal epithelial cellmigration may be due partially to an increase of Kv1.1 channelexpression. The subsequent membrane hyperpolarization raises[Ca²⁺]_cyt by increasing the drivingforce (the electrochemical gradient) for Ca²⁺ influx andthus stimulates cell migration.

     

Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine

The aim of this study was to investigate expression and relative contribution of human thiamin transporter (hTHTR)-2 toward overall carrier-mediated thiamin uptake by human intestinal epithelial cells. Northern blot analysis showed that the message of the hTHTR-2 is expressed along the native human gastrointestinal tract with highest expression being in the proximal part of small intestine. hTHTR-2 protein was found, by Western blot analysis, to be expressed at the brush-border membrane (BBM), but not at the basolateral membrane, of native human enterocytes. This pattern of expression was confirmed in studies using a fusion protein of hTHTR-2 with the enhanced green fluorescent protein (hTHTR2-EGFP) expressed in living Caco-2 cells grown on filter. Pretreating Caco-2 cells (which also express the hTHTR-2 at RNA and protein levels) with hTHTR-2 gene-specific small interfering RNA (siRNA) led to a significant (P < 0.01) and specific inhibition (48%) in carrier-mediated thiamin uptake. Similarly, pretreating Caco-2 cells with siRNA that specifically target hTHTR-1 (which is expressed in Caco-2 cells) also significantly (P < 0.01) and specifically inhibited (by 56%) carrier-mediated thiamin uptake. When Caco-2 cells were pretreated with siRNAs against both hTHTR-2 and hTHTR-1 genes, an almost complete inhibition in carrier-mediated thiamin uptake was observed. These results show that the message of hTHTR-2 is expressed along the human gastrointestinal tract and that expression of its protein in intestinal epithelia is mainly localized to the apical BBM domain. In addition, results show that this transporter plays a significant role in carrier-mediated thiamin uptake in human intestine.     

Active Uptake of CO2 by the Diatom Navicula pelliculosa

Mass spectrometry has been used to investigate the transportof CO₂ in the freshwater diatom Navicula pelliculosa. The timecourseof CO₂ formation in the dark after addition of 100 mmol m^–3dissolved inorganic carbon (DIC) to cell suspensions showedthat no external carbonic anhydrase (CA) was present in thesecells. Upon illumination, cells pre-incubated at pH 75 with100 mmol m^–3 DIC, removed almost all free CO₂ from themedium at an initial rate of 285 µmol CO₂ mg^–1Chl h^–1. Equilibrium between HCO₃^– and CO₂ in themedium occurred rapidly upon addition of bovine CA, showingthat CO₂ depletion resulted from a selective uptake of CO₂ ratherthan an uptake of all inorganic carbon species. However, photosyntheticO₂ evolution rate remained constant after CO₂ had been depletedfrom the medium indicating that photosynthesis is sustainedprimarily by active HCO₃^– uptake. Treatment of cells with2-iodoacetamide (83 mol m^–3) completely inhibited CO₂fixation but had little effect on CO₂ transport since initialrates of CO₂ depletion were about 81% that of untreated cells.Transfer of iodoacetamide-treated cells to the dark caused arapid increase in the CO₂ concentration in the medium largelydue to the efflux of the unfixed intracellular DIC pool whichwas found to be about 194 times the concentration of that inthe external medium. These results indicate that Navicula pelliculosaactively takes up molecular CO₂ against a concentration gradientby a process distinct from HCO₃^– transport. Key words: Dissolved inorganic carbon, carbonic anhydrase, bicarbonate transport, CO₂ transport, mass spectrometry