Intestinal glutathione transport system: a possible detoxication role

The epithelium of the small intestine act by the formation of GSH-S-conjugation, as a first line of defence against various ingested toxic chemicals. GSH and GSH-dependent enzymes are present in the gastrointestinal wall. We and others have characterized the GSH-specific transport systems in intestinal brush-border and in basolateral membrane vesicles, in which gamma-glutamyltranspeptidase (gamma-GT) activity was inactivated by AT-125. In the present study we use inhibition experiments, kinetic studies, trans-stimulation of GSH uptake and HPLC determination to demonstrate (for the first time) that GSH and two GSH-S-conjugates (chosen as model compounds) share a common transport system. Plasma GSH-S-conjugates that may enter the intestinal cells via basolateral membrane, and GSH-S-conjugates that form in intestinal cells, may be eliminated directly by this GSH transporter across brush-border membranes or transported into lumen to the active site of gamma-GT; they are then further metabolized and excreted by various routes. This transport system may thus contribute to the intestinal detoxication role.     

Intestinal uptake and transport of proteins in the adult rat.

The transport of immunoglobulin and ferritin across the intestinal mucosa of adult rats provides an excellent model for transcellular protein transport study. Intestinal uptake and transcellular transport have been extensively studied in the neonatal rat, but not to such an extent in the adult rat. The transport of 125I labelled bovine immunoglobulin G and ferritin was studied in 100 days old rats using intestinally administered proteins. Antigen was estimated in the tissues by reacting extracts against specific immune antiserum prepared in rats, and visualization studies were carried out by fluorescence microscopy and direct deposition autoradiography at electron microscopic level. From these studies, it can be seen that these proteins are taken up by the intestinal cells and transported, antigenically intact, across the barriers to the body organs.     

Kinetic analysis of two component systems in transmembrane transport (multiple forms of transport systems)

Summary The uptake of substrates often involves two or more transport systems. The characterization of single components of multiple transport systems requires setting up certain criteria for their determination. Some experimental data were used to demonstrate the necessity of such criteria. Ideal constellations ofK andV values of the different transport systems for a single substrate to separate the various components by graphical analysis are discussed. The discussion is based on the slopes and the intercepts of the two extreme tangent equations.

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Zusammenfassung Die Aufnahme von Substraten in die Zelle erfolgt oft über zwei oder mehr Transportsysteme. Die Charakterisierung einer einzelnen Komponente solcher multipler Transportsysteme erfordert die Aufstellung gewisser Kriterien. Einige experimentelle Daten wurden herangezogen, um die Notwendigkeit solcher Kriterien zu zeigen. Ideale Konstellationen derK- undV- Werte der verschiedenen Transportsysteme, die eine Trennung der verschiedenen Komponenten durch graphische Analyse ermöglichen, werden diskutiert. Es werden drei graphische Auftragungen erörtert. Die Diskussion beruht auf den Verhältnissen der Steigungen und Achsenabschnitte der zwei extremen Tangenten der Kurven in diesen Auftragungsarten.

     

High- and low-affinity zinc transport systems and their possible role in zinc efficiency in bread wheat

There is considerable variability among wheat (Triticum aestivum L.) cultivars in their ability to grow and yield well in soils that contain very low levels of available Zn. The physiological basis for this tolerance, termed Zn efficiency, is unknown. We investigated the possible role of Zn(2+) influx across the root cell plasma membrane in conferring Zn efficiency by measuring short-term (65)Zn(2+) uptake in two contrasting wheat cultivars, Zn-efficient cv Dagdas and Zn-inefficient cv BDME-10. Plants were grown hydroponically under sufficient and deficient Zn levels, and uptake of (65)Zn(2+) was measured over a wide range of Zn activities (0.1 nM-80 microM). Under low-Zn conditions, cv BDME-10 displayed more severe Zn deficiency symptoms than cv Dagdas. Uptake experiments revealed the presence of two separate Zn transport systems mediating high- and low-affinity Zn influx. The low-affinity system showed apparent K(m) values similar to those previously reported for wheat (2-5 microM). Using chelate buffered solutions to quantify Zn(2+) influx in the nanomolar activity range, we uncovered the existence of a second, high-affinity Zn transport system with apparent K(m) values in the range of 0.6 to 2 nM. Because it functions in the range of the low available Zn levels found in most soils, this novel high-affinity uptake system is likely to be the predominant Zn(2+) uptake system. Zn(2+) uptake was similar for cv Dagdas and cv BDME-10 over both the high- and low-affinity Zn(2+) activity ranges, indicating that root Zn(2+) influx does not play a significant role in Zn efficiency.     

Influence of glutathione (GSH) on net uptake of sulphate and sulphate transport in tobacco plants

Net uptake of sulphate by the roots, and its transport to theshoot was inhibited in intact tobacco plants by 1 h exposureof its roots to 0.1 or 1.0 mM GSH. The relative amount of sulphatetaken up that was transported to the shoot was not affectedby this treatment. Apparently, sulphate uptake, but not xylemloading of sulphate, was inhibited by GSH. Similar results wereobtained when L-cysteine was used instead of GSH. As L-cysteine,mediated inhibition of sulphate, net uptake was counteractedby exposure of the roots to BSO, GSH synthesis seems to be requiredfor inhibition of sulphate uptake by L-cysteine. However, exposureto 0.1 or 1.0mM GSH did not result In an enhanced GSH levelin roots or shoots. Also the cysteine contents remained unchanged.Small changes in metabolically active pools of GSH or cysteineseem to be responsible for the inhibition of sulphate uptakeand its transport to the shoot. Flap-feeding of GSH to a matureleaf resulted in an inhibition of net uptake of sulphate bythe roots and its transport to the shoot. These findings supportthe hypothesis that GSH produced in the leaves acts as a signalto control sulphur nutrition of plants. Key words: Sulphate transport, sulphate uptake, xylem loading, Nicotiana, Solanaceae, glutathione, cysteine, buthionine sulphoximine     

On the formation and possible biological role of 25-hydroxycholesterol

The oxysterol 25-hydroxycholesterol is a widely used compound displaying an array of pharmacological actions in in vitro systems and cell based experimental systems. In spite of the frequent use of this compound over the last few decades and a large number of studies in vitro and in vivo, its mechanism of formation in vivo is still not well understood.     

Urate transport in erythrocytes: possible role of a transport membrane]

Urate transport seems dependant of ATP. So, we studied urate behaviour in relation with the isolated membrane: we observed a linkage between urate and a membrane protein of which molecular weight was estimated to twenty thousand. The exact role of this protein remains to be clarify.     

Sustained oscillations of transmembrane Ca2+ fluxes in mitochondria and their possible biological significance

Sustained oscillations of transmembrane fluxes of Ca2+ and other ions in isolated mitochondria are described. The data are presented that the major cause of the oscillations is the Ca2+-induced Ca2+ efflux from the mitochondrial matrix and spontaneous opening/closing of the permeability transition pore in the inner mitochondrial membrane. Conditions favourable for the generation of oscillations are considered. The role of phospholipid peroxidation and hydrolysis in the generation of [Ca2+] oscillations is emphasized. Literature data concerning [Ca2+] changes in the mitochondrial matrix in intact cells and the data on the participation of mitochondria in intracellular Ca2+ oscillation and in the Ca2+ wave propagation are reviewed. The hypothesis that mitochondria are able to generate [Ca2+] oscillations in intact cells is put forward. It is assumed that Ca2+ oscillations can protect mitochondria of resting cells from osmotic shock and oxidative stress.     

Bisoliton mechanism of electron transport in biological systems

A nonlinear mechanism describing the transport of paired electrons in protein molecules is suggested. It is shown that electron-phonon interaction leads to the pairing of two electrons with local chain deformation in a state called a bisoliton. The parameters of the bisoliton and the stability conditions with respect to its decay into two-soliton state are estimated.I dedicate this paper to the memory of my teacher Professor A.S. Davydov, who attracted me to nonlinear science and was my colleague.     

Rod structures of bound water: a possible role in self-organization of biological systems and nondissipative energy transmission

Cluster–rod structure were designed, which are comprised of tetrahedral atoms with a typical torsion angle of ~38° at interatomic bonds. These structures correspond to a muscle tissue and clathrin lattice by their metrics and topology and can be formed by bound water in these systems. It is shown that the considered rod structures, which are fragments of bound water structures, can also be involved in nondissipative energy transmission as elastic energy storing structures. The estimated length of the bound water rod structure required to absorb the energy of decomposition of an ATP molecule into ADP and a phosphate group is comparable with myosin head sizes and its step along an actin filament. A mechanism of cooperative transition of the rod structure to a fragment of the ice Ih structure was demonstrated. This transition is accompanied by nondissipative release of stored energy.     

Receptor-mediated uptake and 'retroendocytosis' of high-density lipoproteins by cholesterol-loaded human monocyte-derived macrophages: possible role in enhancing reverse cholesterol transport

Human monocyte-derived macrophages (MDM) are cholesterol-loaded, and the rates of uptake, degradation and resecretion of high-density lipoproteins are measured and compared to the rates in control cells. Results show the binding activity of these lipoproteins is upregulated in cholesterol-loaded cells; the bound and internalized lipoproteins are not degraded to any appreciable extent but primarily resecreted as a larger particle. The enhancement of binding activity for high-density lipoproteins is arrested when cycloheximide is added to the medium, suggesting that protein synthesis is involved. Preliminary evidence also indicates that HDL3 (without apoE) after internalisation is converted intracellularly to a larger apoE-containing HDL2-like particles. Thus, MDM appears to possess specific receptors for HDL3 without apoE that may function to facilitate HDL-mediated removal of excess cholesterol from cells.     

Contribution of mitochondrial GSH transport to matrix GSH status and colonic epithelial cell apoptosis

Previously, we showed that cellular glutathione/glutathione disulfide (GSH/GSSG) play an important role in apoptotic signaling, and early studies linked mitochondrial GSH (mtGSH) loss to enhanced cytotoxicity. The current study focuses on the contribution of mitochondrial GSH transport and mitochondrial GSH/GSSG status to apoptosis initiation in a nontransformed colonic epithelial cell line, NCM460, using menadione (MQ), a quinone with redox cycling bioreactivity, as a model of oxidative challenge. Our results implicate the semiquinone radical in MQ-mediated apoptosis, which was associated with marked oxidation of the mitochondrial soluble GSH and protein-bound thiol pools, mitochondria-to-cytosol translocation of cytochrome c, and activation of caspase-9. MQ-induced apoptosis was potentiated by inhibition of mtGSH uptake in accordance with exacerbated mitochondrial GSSG (mtGSSG) and protein-SSG and compromised mitochondrial respiratory activity. Moreover, cell apoptosis was prevented by N-acetyl-L-cysteine (NAC) pretreatment, which restored cellular redox homeostasis. Importantly, mtGSH transport inhibition effectively blocked NAC-mediated protection in accordance with its failure to attenuate mtGSSG. These results support the importance of mitochondrial GSH transport and the mtGSH status in oxidative cell killing.     

The possible role of intimal convective transport in atherogenesis

The possibility of fluid flux within the thickened subendothelial intima is considered. Both the media and the endothelium were already shown to be major hydraulic barriers. It is hypothesized that if the hydraulic conductivity of the inbetween layer of the subendothelial intima is considerably higher, then fluid flux in the downstream (axial) direction is likely to occur within the intima as a result of the luminal blood pressure wave. Macromolecular species (as lipoproteins) would then be transported axially by the fluid. This convective transport may give rise to the formation of early atheromas. The proposed mechanism is in accord with several clinical and experimental observations.     

N.m.r. imaging of intact biological systems

Internal images of structured objects may be obtained with n.m.r. by labelling component parts with different magnetic field strengths and therefore recognizably different n.m.r. frequencies. A linear field gradient generates a one-dimensional projection of nuclear density and a variety of techniques are employed to manipulate this one-dimensional probe to yield internal images in two and three dimensions. In the past few years, n.m.r. imaging, sometimes also called zeugmatography or spin mapping, has been applied progressively to provide proton images of small phantoms, fruit, vegetables and small animals, and finally to in vivo imaging of the human body; it promises to provide a valuable means of interior investigation of intact biological systems generally. For medical imaging the method is non-invasive, does not use ionizing radiations, appears to be without hazard and penetrates bony cavities without attenuation. Furthermore, other n.m.r. parameters, for example, relaxation times and fluid flow, may also be mapped; there is evidence that the relaxation times from tumours are significantly longer than those from corresponding normal tissue. Effort to date has mostly been concentrated on proton n.m.r., but some work has been done with other nuclei. Three examples are shown of n.m.r. images of intact biological systems: a fruit, an animal and a human system. The discussion includes the quantitative nature of the images, tissue discrimination, the relation between the resolution in the image and image acquisition time, attenuation and phase shift of the r.f. field in the biological tissue, and magnets suitable for n.m.r. imaging. In principle, all conventional n.m.r. techniques can be combined with n.m.r. methods in order to investigate heterogeneous systems. Overhauser imaging is briefly discussed.     

ATP-dependent GSH and glutathione S-conjugate transport in skate liver: role of an Mrp functional homologue

Multidrug resistance-associated proteins 1 and 2 (Mrp1 and Mrp2) are thought to mediate low-affinity ATP-dependent transport of reduced glutathione (GSH), but there is as yet no direct evidence for this hypothesis. The present study examined whether livers from the little skate (Raja erinacea) express an Mrp2 homologue and whether skate liver membrane vesicles exhibit ATP-dependent GSH transport activity. Antibodies directed against mammalian Mrp2-specific epitopes labeled a 180-kDa protein band in skate liver plasma membranes and stained canaliculi by immunofluorescence, indicating that skate livers express a homologous protein. Functional assays of Mrp transport activity were carried out using (3)H-labeled S-dinitrophenyl-glutathione (DNP-SG). DNP-SG was accumulated in skate liver membrane vesicles by both ATP-dependent and ATP-independent mechanisms. ATP-dependent DNP-SG uptake was of relatively high affinity [Michaelis-Menten constant (K(m)) = 32 +/- 9 microM] and was cis-inhibited by known substrates of Mrp2 and by GSH. Interestingly, ATP-dependent transport of (3)H-labeled S-ethylglutathione and (3)H-labeled GSH was also detected in the vesicles. ATP-dependent GSH transport was mediated by a low-affinity pathway (K(m) = 12 +/- 2 mM) that was cis-inhibited by substrates of the Mrp2 transporter but was not affected by membrane potential or pH gradient uncouplers. These results provide the first direct evidence for ATP-dependent transport of GSH in liver membrane vesicles and support the hypothesis that GSH efflux from mammalian cells is mediated by members of the Mrp family of proteins.     

The uptake and the transport of14C-labeled epibrassinolide in intact seedlings of cucumber and wheat

The uptake and the transport of exogenously applied epibrassinolide (EBR) in seedlings of cucumber and wheat were examined by autoradiography using¹⁴C-EBR.¹⁴C-EBR was applied to roots, young and mature leaves, and the shoot apex. When applied to roots,¹⁴C-EBR was readily taken up and was swiftly transported throughout both plant species. When¹⁴C-EBR was applied to the adaxial surface of a young cucumber leaf, it was readily taken up, but was very slowly transported. In cucumber leaves,¹⁴C-EBR was transported throughout the treated leaf after 3 days of treatment, and then it was transported to upper leaves from the treated leaf after 7 days. Some 6.3% of applied¹⁴C-EBR was transported to the newly expanded leaves. In wheat leaves,¹⁴C-EBR was transported only in the apical direction from the treated spot after 3 days of treatment, but it was not transported from the treated leaf to the other leaves or organs even after 7 days. Some 1.3% of applied¹⁴C-EBR was transported to the tip area of the treated leaf. These results indicate that exogenous EBR applied to intact plants is acropetally transported.