Calcium uptake by isolated rat intestinal cells

Intestinal cells were isolated by a combination of mechanical and enzymatic means, and their calcium uptake was assayed by a rapid filtration procedure. Calcium uptake was a time- and concentration-dependent process that was markedly elevated at 25 and 37°C, as compared to 0°C. Cells isolated from rat duodenum exhibited higher uptakes than cells from jejunum, which in turn took up more calcium than cells from the ileurn. Duodenal cells from vitamin D-deficient animals took up less calcium than cells from vitamin D-replete cells. In vivo vitamin D repletion with 1,25-dihydroxyvitamin D₃ raised calcium uptake by duodenal cells from treated animals toward that of cells from replete rats. Furthermore, calcium uptake by duodenal cells from vitamin D-deficient animals approximated that of ileal cells from replete rats. These findings with isolated cells parallel prior findings of tissue calcium transport and suggest that cellular calcium uptake may be related to the saturable component of intestinal calcium absorption. Isolated intestinal cells may therefore constitute one experimental model for the study of transcellular calcium transport.     

Calcium uptake by enzyme-treated permeabilized myometrial cells

Isolated myometrial cells prepared by collagenase and pronase treatment of the tissue were rendered permeable to charged molecules and Ca ions. ATP dependent Ca uptake studied in these cells showed that Ca uptake by the intracellular organelles was well preserved. Inhibitors of mitrochondrial metabolism caused about 90% inhibition of Ca uptake by the cells. Whereas D-600 had no effect on Ca uptake, diethylstilbestrol caused a signficant inhibition. This preparation which requires no homogenization should prove useful for study of Ca uptake and possibly other processes requiring exogenous ATP.     

Free fatty acid uptake is increased in doxorubicin-resistant glioblastoma cells

We have investigated the mechanism by which the rat glioblastoma C6 cell line resistant to doxorubicin presented an increase of free fatty acid utilization from the nutrient source as compared to the wild sensitive strain. We have shown that this was not due to an accelerated turnover of the phospholipid hydrophobic or hydrophilic moieties, but to an important increase of the uptake of the free fatty acids. This was demonstrated by studies performed with linoleic and linolenic acids during very short incubations, at low temperatures, and in the absence of albumin in the medium. This enhancement of free fatty acid uptake may explain the differences which exist in the acyl group composition of membrane lipids between doxorubicin-sensitive and -resistant C6 cells, because of the suppression of the essential fatty acid-deficient status of the cells.     

Calcium homeostasis of epithelial cells

1. Cytosolic free Ca2+ is an important regulator of ion transport processes in epithelial cells. 2. Free Ca2+ concentration is regulated by a concerted action of Ca2+ transport systems in plasma membranes and intracellular organelles. 3. These transport systems were studied in intestinal and renal cortical cells with emphasis on the transport capacities and Ca2+ affinities. 4. Ca2+ accumulation by permeabilized cells was compared to Ca2+ uptake by isolated organelles and membrane fractions. 5. Effects induced by cell or organelle isolation methods and the influence of temperature and pH on Ca2+ transport capacities were studied.     

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.

     

Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. We found that Mertk protein is absent from RCS, but not wild-type, tissues and cultured RPE cells. Delivery of rat Mertk to cultured RCS RPE cells by means of a recombinant adenovirus restored the cells to complete phagocytic competency. Infected RCS RPE cells ingested exogenous outer segments to the same extent as wild-type RPE cells, but outer segment binding was unaffected. Mertk protein progressively co-localized with outer segment material during phagocytosis by primary RPE cells, and activated Mertk accumulated during the early stages of phagocytosis by RPE-J cells. We conclude that Mertk likely functions directly in the RPE phagocytic process as a signaling molecule triggering outer segment ingestion.     

Choline uptake is increased by Ca++ and liposomes+ Ca++ in ascites tumor cells

Steady-state uptake of choline by Lettre-Ehrlich tumor cells in vitro, resulting in cell-to-medium ratios of 10 or more, is significantly increased by 0.2-1.0 mM Ca++ as well as by dipalmitoyl phosphatidyl choline multilamellar liposomes + Ca++. The increases occur in spite of a decrease in carrier affinity, as indicated by the Km, and therefore result either from increased carrier velocity or utilization of new carriers. About half of the labelled choline which is taken up is firmly bound to cells. That label which freely leaves cells is phosphocholine, thus, these cells utilize choline mainly in phospholipid synthesis. Choline and nitrogen mustard (HN2) share a plasma membrane carrier but the intracellular distribution of HN2 into DNA, RNA and protein, contrasts with that of choline, into phospholipid.     

Integrin-linked kinase is an essential link between integrins and uptake of bacterial pathogens by epithelial cells

Entry of Streptococcus pyogenes or group A streptococcus (GAS) into host cells is mediated by fibronectin bound to surface proteins, M1 or PrtF1, forming a bridge to alpha5beta1 integrins. This interaction leads to cytoskeletal rearrangement and uptake of streptococci. We postulated that integrin-linked kinase (ILK), which directly associates with integrins, is the universal link between integrins and several bacterial pathogens. We showed that inhibition of ILK expression by siRNA silencing, or ILK kinase activity by chemical inhibitors or expression of a dominant negative form of ILK reduced M1-mediated invasion of epithelial cells up to 80%. To evaluate the ILK requirement for PrtF1-mediated GAS invasion, a M1-PrtF1+ recombinant strain within the M1 background was constructed. Inhibition of ILK kinase activity also significantly reduced invasion of epithelial cells by this recombinant and wild-type strain JRS4 that expresses PrtF1. In addition, impaired ILK kinase activity results in significant reduction of integrin-dependent invasion mediated by invasins of two other important pathogens, Staphylococcus aureus and Yersinia spp. This study suggests that bacterial pathogens evolved different molecules and strategies to exploit the host integrin signalling pathway for their survival.     

Restituting intestinal epithelial cells exhibit increased transducibility by adenoviral vectors

BACKGROUND AND AIMS: While mature enterocytes are resistant to transduction by adenovirus type 5 (Ad5) vectors, undifferentiated cells are transduced much more efficiently. Our purpose was to study enterocyte transduction in models of intestinal wound healing. METHODS: Transduction was studied ex vivo using cultures of endoscopic biopsies and in vitro utilizing Caco-2 cells in models of mucosal wound healing. Vectors carried either the LacZ or the luciferase gene. CAR (coxsackievirus and adenovirus receptor) and integrins were studied with transduction inhibition and immunofluorescent staining. RESULTS: Increased transduction efficiency was observed for a subset of enterocytes with a flattened de-differentiated phenotype present at the edge of cultured biopsies. In the in vitro systems, expanding Caco-2 cell monolayers exhibited increased transducibility that was time- and dose-dependent, reaching virtually 100% in cells along the leading edge at high viral load. Bioluminescence activity of transduced expanding monolayers was up to 3-fold greater than that of non-expanding monolayers ('fence' system, 48 h, MOI 1000, p < 0.05). Mitomycin C pre-treatment did not affect levels of transduction in expanding monolayers. At the highest viral load tested, CAR or integrin blocking prior to virus application resulted in 39.4% and 45.4% reduction in transduction levels (p < 0.05). Immunofluorescence revealed altered expression of CAR on the migrating edge of the Caco-2 cultures and the expression of CAR on the apical membrane of biopsy enterocytes. CONCLUSIONS: Increased CAR and integrin accessibility in migrating enterocytes mediates increased transduction by Ad5 vectors. This subset of enterocytes provides a target for the delivery of genes of interest for both research and gene therapy applications.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent K_m value of 46 μM and a V_max of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na⁺ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na⁺ was related to the transmembraneous Na⁺-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent Km value of 46 microM and a Vmax of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na+ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na+ was related to the transmembraneous Na+-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Phosphate uptake by kidney epithelial (LLC-PK1) cells

Phosphate uptake by the cultured kidney epithelial cell (LLC-PK1) was studied. The uptake was Na+ dependent, saturable with respect to phosphate and Na+, and energy dependent. The characteristics of the cell uptake system resembled the properties of phosphate transport in the kidney. Parathyroid hormone, dibutyryl cyclic AMP, and forskolin decreased Na+-dependent phosphate uptake. These agonists did not affect Na+-dependent alpha-methylglucoside uptake. Vasopressin and isoproterenol, which do not affect renal phosphate transport, did not inhibit phosphate uptake by the cell. These findings suggest that the cultured cell system may be a useful experimental model for studies of renal phosphate transport and its regulation.     

Cytoplasmic organization and quantitation of microtubules in bovine mammary epithelial cells during lactation and involution

Summary Ultrastructural examination of milk secretory cells from lactating bovine mammary gland revealed presence of numerous microtubules in the apical and paranuclear cytoplasm, particularly in the vicinity of Golgi components. Most microtubules were oriented perpendicular to the apical plasma membrane and appeared to form a framework around Golgi dictyosomal elements and secretory vesicles. In comparison, non-secretory cells obtained from involuting glands displayed few microtubules and these were randomly located throughout the cytoplasm with no particular orientation.     

Calcium ion uptake in isolated pancreas cells induced by secretagogues.

1. Secretagogues of pancreatic enzyme secretion: pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, caerulein and the Ca2+ ionophore A 23187 stimulate 45Ca uptake into isolated rat pancreatic cells, whereas adrenaline, isoproterenol, secretin, dibutyrylic cyclic adenosine 3',5'-monophosphate and dibutyrylic cyclic guanosine 3',5'-monophosphate have no effect on 45Ca uptake. 2. A graphical analysis of the Ca2+ uptake curves reveals at least two phases: a fast phase, probably due to binding of Ca2+ to the membrane and a slow phase representing Ca2+ transport into cells. Both phases are stimulated by pancreozymin and carbamylcholine. 3. The 45Ca-exchangeable pool size is increased by both carbamylcholine and pancreozymin, whereas a significant increase of total content of cell calcium was too small to be detected. 4. Atropine blocks the stimulatory effect of carbamylcholine completely but not that of pancreozymin. The Ca2+ antagonist D600 blocks the stimulatory effects of both carbamylcholine and pancreozymin only partially. 5. The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca2+ transfer into the cell most probably through an increase of the cell membrane permeability for Ca2+.     

Glucose uptake is increased in trained vs. untrained muscle during heavy exercise.

Endurance training increases muscle content of glucose transporter proteins (GLUT-4) but decreases glucose utilization during exercise at a given absolute submaximal intensity. We hypothesized that glucose uptake might be higher in trained vs. untrained muscle during heavy exercise in the glycogen-depleted state. Eight untrained subjects endurance trained one thigh for 3 wk using a knee-extensor ergometer. The subjects then performed two-legged glycogen-depleting exercise and consumed a carbohydrate-free meal thereafter to keep muscle glycogen concentration low. The next morning, subjects performed dynamic knee extensions with both thighs simultaneously at 60, 80, and until exhaustion at 100% of each thigh's peak workload. Glucose uptake was similar in both thighs during exercise at 60% of thigh peak workload. At the end of 80 and at 100% of peak workload, glucose uptake was on average 33 and 22% higher, respectively, in trained compared with untrained muscle (P < 0.05). Training increased the muscle content of GLUT-4 by 66% (P < 0. 05). At exhaustion, glucose extraction correlated significantly (r = 0.61) with total muscle GLUT-4 protein. Thus, when working at a high load with low glycogen concentrations, muscle glucose uptake is significantly higher in trained than in untrained muscle. This may be due to the higher GLUT-4 protein concentration in trained muscle.     

MIF production by dendritic cells is differentially regulated by Toll-like receptors and increased during rheumatoid arthritis

Macrophage migration inhibitory factor (MIF) is clearly associated with rheumatoid arthritis (RA) disease severity. However, the regulation of MIF during the course of RA has not been subjected to similar scientific scrutiny. The aim of our study was to investigate the role of various Toll-like receptors (TLRs) and inflammatory mediators on MIF production by dendritic cells (DCs) in healthy controls and RA patients. DCs were cultured from 12 healthy donors and 12 RA patients. Triggering via TLR mediated pathways was achieved using various TLR specific ligands alone or in combination: Pam3Cys for TLR2, LPS and recombinant extra domain A containing fibronectin for TLR4 and Poly(I:C) and R848 for TLR3 and TLR7, respectively. In addition, iDCs from healthy controls were incubated with various cytokines, RANKL and CD40L for 48 h. MIF levels were measured using an ELISA assay. Stimulation of DCs by TLR4 ligands resulted in higher MIF production compared to immature DCs from healthy controls (p<0.002) and RA patients (p<0.002). DCs from RA patients produced higher MIF levels than healthy controls both at the immature stage (p<0.04) as well after full maturation via TLR2 (p<0.04) and TLR4 (p<0.001) triggering. Incubation with TLR3 and TLR7 ligands resulted in a significantly decreased secretion of MIF in RA patients and controls. Simultaneous incubation of TLR4 with either TLR3 or TLR7 ligands resulted in a decrease of MIF secretion when compared to TLR4 stimulation alone. The secretion of MIF increased when DCs were stimulated with TNF-alpha, RANKL and CD40L. The secretion of MIF by dendritic cells is differentially regulated by TLRs. In addition, TNF-alpha, RANKL, and CD40L augment MIF production by DCs and thus play a potential role in the amplification of the inflammatory loop in RA.