An ATP-dependent iron transport system in isolated rat liver nuclei

A concerted translational control is responsible for maintaining an iron level in the cytosol that is both adequate for the synthesis of iron-containing proteins and does not represent a danger to the cell. However, little is known about how iron level is controlled in the nucleus. Nuclei of rat liver take up iron from ferric citrate by a process that is dependent on ATP. This system shares several properties with known P-type ATPases, suggesting that a P-type ATPase in the nuclear membrane is responsible for iron transport. (i) Adenosine 5'-(beta,gamma-iminodiphosphate), a non-hydrolyzable ATP analogue, does not support iron uptake; (ii) the uptake is strongly inhibited by vanadate; (iii) there is an absolute requirement for Mg2+; and (iv) reagents that oxidize SH groups inhibit uptake, and this inhibition can be prevented by dithiothreitol. The energy of activation for the uptake (11.5 kcal/mol) and the Km for ATP (0.4 mM) are similar to values for other known cation transport ATPases. Inhibitors of Na+,K+-ATPase, sarcoplasmic reticulum Ca2+-ATPase, proton V-ATPase, and nuclear Ca2+-ATPase have no effect on uptake. Ferric citrate can be replaced by Fe-ATP as a source of iron for the transport system; however, two other stronger iron chelators, Tiron and desferrioxamine, completely inhibit the uptake. Taken together, these data strongly suggest that an Fe-ATPase, distinct from other known P-type ATPases, is responsible for iron transport in the nucleus.     

Active transport properties of porcine choroid plexus cells in culture

We have investigated the transport properties of cultured porcine choroid plexus cells grown on permeable membranes and in serum-free medium. Withdrawal of serum yielded cell cultures with permeabilities low enough to establish and maintain a pH-gradient between the two compartments of the filter system and to allow apical fluid secretion. This became possible because of ten-fold increased electrical resistance of 1700 Omega cm2 in the absence of serum. These plexus epithelial cells transported phenol red, fluorescein, riboflavin and penicillin G from the apical to the basolateral side. KM values and vmax were determined and come close to in vivo values. Competitive inhibition with probenicid showed that the organic anion transporter is involved. Riboflavin transport however was not completely inhibited and did not respond quantitatively to the stilben derivate SITS that blocks the Cl-/HCO3--exchanger. We assume that an additional transport system exists for riboflavin. Ascorbic acid and myo-inositol were transported from the basolateral to the apical side in vitro which strongly resembles the in vivo transport from the blood to the cerebrospinal fluid. Again the experimental in vitro KM values come close to the in vivo values. The established epithelial cell culture model thus closely mimics the blood-CSF-barrier and may be a useful tool to further elucidate transport to and from the brain.     

Culture and characterization of sinusoidal endothelial cells isolated from human liver

BACKGROUND: Epidemiologic data concerning skin diseases in many rural areas in sub-Saharan Africa are not available. Little is known about the effect of regular treatment schedules by paramedical staff (especially community health workers) in the primary healthcare system on the severity and prevalence of dermatoses. METHODS: 5780 school and pre-school children from 13 primary schools in four sublocations in rural western Kenya (Kisumu District) were examined for dermatoses by the author, together with community health workers in 1993. On-the-spot training and weekend seminars about important and common dermatoses were also given. In 1994 a dermatology program was started within the primary healthcare system. Twelve trained community health workers carried out regular school visits once a week and diagnosed and treated pupils with dermatoses. Treatment was performed with gentian violet 1% solution for bacterial skin infections, Whitfield's ointment for dermatophytoses, benzylbenzoate emulsion 25% for scabies, and hydrocortisone acetate 1% cream for eczemas. All schools were visited again in 1995 to evaluate the long-term effects of the program. RESULTS: In 1993, the prevalence rate for dermatoses was 32.4%. Most of the skin diseases found were of infective origin (27.1% were caused by bacteria, 21.6% by fungi, and 17.6% by arthropods, mainly scabies mites). Dermatitis accounted for 3.5%. In 1995, the prevalence of dermatoses declined to 29.6% (p<0.05), and this reduction was most strongly observed for tropical ulcers and tinea capitis. Additionally, there was an improvement in the extent and severity of skin diseases. CONCLUSIONS: This study defines, for the first time, the number and extent of skin diseases in children in rural Kisumu District; most dermatoses were of infective origin. The study demonstrates that community health workers in the primary healthcare system are capable of dealing successfully with the most common dermatoses in children following a short training period.     

Gene expression of syndecans and betaglycan in isolated rat liver cells

Competence as communication skills and as skilled practice of asepsis were studied by observing four nurses while interacting with patients and performing intravenous procedures. Nurses were observed using sterile equipment for methods of intravenous therapy. Asepsis is performed frequently, but through misunderstanding in the learning of asepsis or improper model learning the nurses may establish incorrect routines. When performing procedures, unexpected factors can distract both the expert and the inexperienced, resulting in a failure to apply basic aseptic techniques. The nurses showed an interest in the patient by listening and giving responses. Nurses may control interactions with the patient by using undesirable communication skills which include incomplete sentences, incomplete explanations and closed questions. Asked to evaluate their own behavior, the nurses did not estimate whether or not the appropriate skills were applied in observed situations. To improve the quality of nursing care performance it is recommended to further develop and apply skill training programs.     

The diffusional transport of water and small solutes in isolated endothelial cells and erythrocytes

The diffusional permeability coefficients, PD, for tritiated water (3HHO) 14C-antipyrine (AP) and 14C-iodoantipyrine (IAP) in isolated calf pulmonary artery endothelial cells and dog erythrocytes are measured with the linear diffusion technique at 11.5, 15, 20 and 37 degrees C. The PD values for both cell populations follow the sequence 3HHO > IAP > AP at each of the temperatures. PD for water is higher in the erythrocyte compared to the endothelial cells. The differences in PD for AP and IAP in the erythrocytes and endothelial cells are not dramatic and are similar to the differences seen in comparing permeation of the same solute through bilayers of different composition. A comparison of the values of PD calculated for the endothelial cells with those for isolated capillaries and the structured endothelium in whole lungs validates the use of the isolated cells as models for the endothelial cells in situ. Incubation of the endothelial cells with cis-vaccenic acid or cholesterol produces a reduction in PD for water and antipyrine. These data are analyzed in terms of Stokesian and non-Stokesian diffusion. The interpretation which best accommodates the data is that the phospholipid area of the membrane, rather than the hydrocarbon core, provides the greatest resistance to permeation for these solutes.     

Adenine dinucleotide-mediated activation of glycogen phosphorylase in isolated liver cells

PTH is incriminated as an uraemic toxin involved in the pathogenesis of anaemia in chronic renal failure. This fact was the background of our present studies performed in 14 patients with noninflammatory acute renal failure (NARF). Plasma levels of erythropoietin (EPO) and parathyroid hormone (PTH) were estimated in the anuric/oliguric (a/o) and polyuric (p) phase of NARF. In the a/o phase plasma EPO levels were predominantly normal, although inappropriately low to the degree of anaemia. In 50% of patients with NARF episodic short-term increases of plasma EPO levels were noticed which were not caused by worsening of anaemia. In the p phase plasma EPO concentrations were in the normal range (17.9 +/- 3.3 mU/ml) in spite of the same degree of anaemia as in the a/o phase. Plasma PTH levels were significantly elevated during the a/o phase (1.14 +/- 0.1 ng/ml), with a tendency to decline in the p phase (0.87 +/- 0.2 ng/ml). No correlation was found between plasma EPO and PTH concentrations. Results presented in this study suggest presence of relative EPO deficiency both during the a/o and p phases of NARF. As plasma PTH levels were not significantly correlated with serum EPO concentrations, its role in the pathogenesis of suppressed EPO levels seems unproven. Results presented in this study suggest deterioration of the physiological feedback between EPO secretion and the magnitude of erythropoiesis in NARF.     

A compartmental model for hepatic transport of taurocholic acid in isolated perfused rat liver

In order to characterize the transport of bile acids through the liver and to study the influence of drugs on these processes, a kinetic model for hepatobiliary transport of taurocholic acid (TC) using the isolated perfused liver was developed. After the system was brought to a steady state by infusing TC at a constant rate, a tracer dose of 14C-TC was injected into the medium. The medium disappearance of 14C-TC followed a first-order kinetic with a single rate constant. The plot of the biliary secretion rate of radioactivity versus time revealed a curve composed of at least three exponential components. From the described results and the present knowledge of hepatobiliary transport of bile acids we proposed a three compartment model, composed of a perfusion medium compartment and two liver compartments. Parameters calculated from the model constants agreed well with model-independent estimations. The influence of bromosulfophthalein (BSP) on the kinetic parameters was studied to compare the result with the known effect of BSP on hepatic transport of taurocholic acid. BSP decreased the constant describing the fractional transfer of taurocholic acid from medium into the liver, which is in agreement with the inhibition of hepatic uptake of taurocholic acid by BSP. Thus a three compartment model may adequately define the hepatobiliary transport of taurocholic acid in the isolated perfused rat liver.     

Pathways of glutathione metabolism and transport in isolated proximal tubular cells from rat kidney

Cellular uptake and metabolism of exogenous glutathione (GSH) in freshly isolated proximal tubular (PT) cells from rat kidney were examined in the absence and presence of inhibitors of GSH turnover [acivicin, L-buthionine-S,R-sulfoximine (BSO)] to quantify and assess the role of different pathways in the handling of GSH in this renal cell population. Incubation of PT cells with 2 or 5 mM GSH in the presence of acivicin/BSO produced 3- to 4-fold increases in intracellular GSH within 10-15 min. These significantly higher intracellular concentrations were maintained for up to 60 min. At lower concentrations of extracellular GSH, an initial increase in intracellular GSH concentrations was observed, but this was not maintained for the 60-min time course. In the absence of inhibitors, intracellular concentrations of GSH increased to levels that were 2- to 3-fold higher than initial values in the first 10-15 min, but these dropped below initial levels thereafter. In both the absence and presence of acivicin/BSO, PT cells catalyzed oxidation of GSH to glutathione disulfide (GSSG) and degradation of GSH to glutamate and cyst(e)ine. Exogenous tert-butyl hydroperoxide oxidized intracellular GSH to GSSG in a concentration-dependent manner and extracellular GSSG was transported into PT cells, but limited intracellular reduction of GSSG to GSH occurred. Furthermore, incubation of cells with precursor amino acids produced little intracellular synthesis of GSH, suggesting that PT cells have limited biosynthetic capacity for GSH under these conditions. Hence, direct uptake of GSH, rather than reduction of GSSG or resynthesis from precursors, may be the primary mechanism to maintain intracellular thiol redox status under toxicological conditions. Since PT cells are a primary target for toxicants, the ability of these cells to rapidly take up and metabolize GSH may serve as a defensive mechanism to protect against chemical injury.     

Gene-dose effect on carnitine transport activity in embryonic fibroblasts of JVS mice as a model of human carnitine transporter deficiency

Recently, the marked decline in renal carnitine reabsorption has been thought to account fotr the systemic carnitine deficiency in juvenile visceral steatosis (JVS) mice. We have conducted a kinetic analysis using embryonic fibroblasts derived from normal, heterozygous, and homozygous jvs mice and found that the high-affinity carnitine transporter (Km = 5.5 microM), which shows Na+ and temperature dependency and stereospecificity, is defective in homozygous jvs mice. Moreover, a gene dose-dependent decrease of carnitine transport activity, which was due to a decrease in the number of the transporter molecules, was found in heterozygous jvs mice. Similar phenomena have been observed in human primary carnitine deficiency. Therefore, JVS mice may be useful for understanding this extremely rare human hereditary disorder.     

Multiformity of elongation factor eEF-2 isolated from rat liver cells

Two fractions of eEF-2 (M(r) approx. 100,000 and M(r) approx. 65,000) were isolated from post-ribosomal supernatant of the rat liver cells. Only eEF-2, with mol. weight of about 100,000 Da, can be phosphorylated, but only eEF-2, with mol. weight of about 65,000 Da, was isolated from the active polyribosomes. The existence of two eEF-2 forms with different properties in the rat liver cells is striking and uncovers new aspects for the cellular function of this protein.     

Chloride-dependent transport of NH4+ into bee retinal glial cells

Mammalian astrocytes convert glutamate to glutamine and bee retinal glial cells convert pyruvate to alanine. To maintain such amination reactions these glial cells may take up NH4+/NH3. We have studied the entry of NH4+/NH3 into bundles of glial cells isolated from bee retina by using the fluorescent dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium concentration of about 5 mM. This acidifying response to ammonium was abolished by the loop diuretic bumetanide (100 microM) and by removal of extracellular Cl-. These results strongly suggest that ammonium enters the cell by contransport of NH4+ with Cl-. Removal of extracellular Na+ did not abolish the NH(4+)-induced acidification. The NH(4+)-induced pH change was unaffected when nearly all K+ conductance was blocked with 5 mM Ba2+ showing that NH4+ did not enter through Ba(2+)-sensitive ion channels. Application of 2 mM NH4+ led to a large increase in total intracellular proton concentration estimated to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH3, we suggest that when NH4+ entered the cells, NH3 left, so that protons were shuttled into the cell. This shuttle, which was strongly dependent on internal and external pH, was quantitatively modelled. In retinal slices, 2 mM NH4+ alkalinized the extracellular space: this alkalinization was reduced in the absence of bath Cl-. We conclude that NH4+ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH4+(K+)-Cl- cotransporter described in kidney cells.     

Differential expression and regulation of nucleoside transport systems in rat liver parenchymal and hepatoma cells

Primary cultures of rat-liver parenchymal cells show carrier-mediated nucleoside uptake by a mechanism that mainly involves concentrative, Na+-dependent transport activity. In contrast, the hepatoma cell line FAO shows high nucleoside transport activity, although it is mostly accounted for by Na+-independent transport processes. This is associated with a low amount of sodium purine nucleoside transporter (SPNT) mRNA. SPNT encodes a purine-preferring transporter expressed in liver parenchymal cells. To analyze whether SPNT expression is modulated during cell proliferation, SPNT mRNA levels were determined in the early phase of liver growth after partial hepatectomy and in synchronized FAO cells that had been induced to proliferate. SPNT mRNA amounts increased as early as 2 hours after partial hepatectomy. FAO cells induced to proliferate after serum refeeding show an increase in SPNT mRNA levels, which is followed by an increase in Na+-dependent nucleoside uptake and occurs before the peak of 3H-thymidine incorporation into DNA. FAO cells also express significant equilibrative nucleoside transport activity, which may be accounted for by the expression of the nitrobenzylthioinosine (NBTI)-sensitive and -insensitive isoforms, rat equilibrative nucleoside transporter 1 (rENT1) and rENT2, respectively. Interestingly, rENT2 mRNA levels follow a similar pattern to that described for SPNT when FAO cells are induced to proliferate, whereas rENT1 appears to be constitutively expressed. Liver parenchymal cells show low and negligible mRNA levels for rENT1 and rENT2 transporters, respectively, although most of the equilibrative transport activity found in hepatocytes is NBTI-resistant. It is concluded that: 1) SPNT expression is regulated both in vivo and in vitro in a way that appears to be dependent on cell cycle progression; 2) SPNT expression may be a feature of differentiated hepatocytes; and 3) equilibrative transporters are differentially regulated, rENT2 expression being cell cycle-dependent. This is consistent with its putative role as a growth factor-induced delayed early response gene.     

Purification and partial sequence of proteins involved in the cholic acid transport into rat liver hepatocytes

Two proteins, in previous work labeled by affinity markers derived from taurocholic acid, were purified and partially sequenced. Antibodies were raised against purified proteins, and cross-reactions were carefully checked. The influence of these antibodies upon taurocholic acid import into vesicles from rat liver plasma membranes was measured, and showed a distinct inhibition of transport in the case of the 54 kD protein.     

The N-terminal domain of rat liver carnitine palmitoyltransferase 1 mediates import into the outer mitochondrial membrane and is essential for activity and malonyl-CoA sensitivity

The rat liver carnitine palmitoyltransferase 1 (L-CPT1), an integral outer mitochondrial membrane (OMM) protein, is the key regulatory enzyme of fatty acid oxidation and is inhibited by malonyl-CoA. In vitro import of L-CPT1 into the OMM requires the presence of mitochondrial receptors and is stimulated by ATP but is membrane potential-independent. Its N-terminal domain (residues 1-150), which contains two transmembrane segments, possesses all of the information for mitochondrial targeting and OMM insertion. Deletion of this domain abrogates protein targeting, whereas its fusion to non-OMM-related proteins results in their mitochondrial targeting and OMM insertion in a manner similar to L-CPT1. Functional analysis of chimeric CPTs expressed in Saccharomyces cerevisiae shows that this domain also mediates in vivo protein insertion into the OMM. When the malonyl-CoA-insensitive CPT2 was anchored at the OMM either by a specific OMM signal anchor sequence (pOM29) or by the N-terminal domain of L-CPT1, its activity remains insensitive to malonyl-CoA inhibition. This indicates that malonyl-CoA sensitivity is an intrinsic property of L-CPT1 and that its N-terminal domain cannot confer malonyl-CoA sensitivity to CPT2. Replacement of the N-terminal domain by pOM29 results in a less folded and less active protein, which is also malonyl-CoA-insensitive. Thus, in addition to its role in mitochondrial targeting and OMM insertion, the N-terminal domain of L-CPT1 is essential to maintain an optimal conformation for both catalytic function and malonyl-CoA sensitivity.