Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase : an integrated, animated model

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na+ and K+ translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P-2c-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E-1/E-2-ATPase as it undergoes conformational changes between the E-1 and E-2 forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na+ and K+ translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the "core engine" of the pump, with respect to ATP binding, cation transport, and ADP and P-i release.

Receptor occupancy vs. induction of Na+-K+-ATPase and Na+ transport by aldosterone.

In the urinary bladder of the toad Bufo marinus aldosterone (between 0.8 and 100 nM) stimulates Na+ transport [half-maximal induction concentration (K1/2) = 6.5 nM]. At low hormone concentrations (0.8-8 nM), the increase of Na+ transport between 0.75 and 2.5 h is accompanied by a fall in transepithelial resistance (R). Higher hormone concentrations (30-800 nM) induce an additional resistance-independent fraction of Na+ transport within 2.5-8 h. From 6 h on, aldosterone (between 0.2 and 20 nM) stimulates in the same tissue the biosynthesis rate of the alpha- and beta-subunits of Na+-K+-ATPase (K1/2 = 3 and 1.5 nM, respectively). New pump synthesis is thus not a prerequisite for the early mineralocorticoid response but might be linked to the late transport event. The mineralocorticoid response is usually ascribed to interaction with the higher affinity type 1 receptor. In the present study we show, however, that at least 55% of the overall Na+ transport response is linked to nuclear occupation of the lower affinity type 2 receptors [dissociation constant (Kd) = 50 nM, maximum number of binding sites (Nmax) = 315 fmol/mg protein]. Distinct aldosterone effects, such as the fall in R and the increase in Na+-K+-ATPase synthesis, are more closely related to occupation of type 1 receptors (Kd = 0.3 nM, Nmax = 23 fmol/mg protein). At maximal induction of these latter parameters, only about 20% of type 2 receptors are occupied. These results suggest that both types of aldosterone receptors are involved in the mediation of the full mineralocorticoid response: type 1 in the early and late and type 2 particularly in the late tissue response.

Harvesting and transport of root biomass from fast-growing poplar plantations

Abstract

Effects of erythropoietin on muscle O2 transport during exercise in patients with chronic renal failure.

Erythropoietin (rHuEPO) has proven to be effective in the treatment of anemia of chronic renal failure (CRF). Despite improving the quality of life, peak oxygen uptake after rHuEPO therapy is not improved as much as the increase in hemoglobin concentration ([Hb)] would predict. We hypothesized that this discrepancy is due to failure of O2 transport rates to rise in a manner proportional to [Hb]. To test this, eight patients with CRF undergoing regular hemodialysis were studied pre- and post-rHuEPO ([Hb] = 7.5 +/- 1.0 vs. 12.5 +/- 1.0 g x dl-1) using a standard incremental cycle exercise protocol. A group of 12 healthy sedentary subjects of similar age and anthropometric characteristics served as controls. Arterial and femoral venous blood gas data were obtained and coupled with simultaneous measurements of femoral venous blood flow (Qleg) by thermodilution to obtain O2 delivery and oxygen uptake (VO2). Despite a 68% increase in [Hb], peak VO2 increased by only 33%. This could be explained largely by reduced peak leg blood flow, limiting the gain in O2 delivery to 37%. At peak VO2, after rHuEPO, O2 supply limitation of maximal VO2 was found to occur, permitting the calculation of a value for muscle O2 conductance from capillary to mitochondria (DO2). While DO2 was slightly improved after rHuEPO, it was only 67% of that of sedentary control subjects. This kept maximal oxygen extraction at only 70%. Two important conclusions can be reached from this study. First, the increase in [Hb] produced by rHuEPO is accompanied by a significant reduction in peak blood flow to exercising muscle, which limits the gain in oxygen transport. Second, even after restoration of [Hb], O2 conductance from the muscle capillary to the mitochondria remains considerably below normal.

Entropic transport: kinetics, scaling, and control mechanisms

We show that transport in the presence of entropic barriers exhibits peculiar characteristics which makes it distinctly different from that occurring through energy barriers. The constrained dynamics yields a scaling regime for the particle current and the diffusion coefficient in terms of the ratio between the work done to the particles and available thermal energy. This interesting property, genuine to the entropic nature of the barriers, can be utilized to effectively control transport through quasi-one-dimensional structures in which irregularities or tortuosity of the boundaries cause entropic effects. The accuracy of the kinetic description has been corroborated by simulations. Applications to different dynamic situations involving entropic barriers are outlined.

Characterization of cerebral glucose dynamics in vivo with a four-state conformational model of transport at the blood-brain barrier.

Determination of brain glucose transport kinetics in vivo at steady-state typically does not allow distinguishing apparent maximum transport rate (T(max)) from cerebral consumption rate. Using a four-state conformational model of glucose transport, we show that simultaneous dynamic measurement of brain and plasma glucose concentrations provide enough information for independent and reliable determination of the two rates. In addition, although dynamic glucose homeostasis can be described with a reversible Michaelis-Menten model, which is implicit to the large iso-inhibition constant (K(ii)) relative to physiological brain glucose content, we found that the apparent affinity constant (K(t)) was better determined with the four-state conformational model of glucose transport than with any of the other models tested. Furthermore, we confirmed the utility of the present method to determine glucose transport and consumption by analysing the modulation of both glucose transport and consumption by anaesthesia conditions that modify cerebral activity. In particular, deep thiopental anaesthesia caused a significant reduction of both T(max) and cerebral metabolic rate for glucose consumption. In conclusion, dynamic measurement of brain glucose in vivo in function of plasma glucose allows robust determination of both glucose uptake and consumption kinetics.

Regulation of sodium transport in the cortical collecting duct : physiological role of GILZ in vitro and in vivo : characterisation of Na+ transport in the mCCDcl1 cell line

SUMMARY Regulation of sodium excretion by the kidney is a key mechanism in the long term regulation of blood pressure, and when altered it constitutes a risk factor for the appearance of arterial hypertension. Aldosterone, which secretion depends upon salt intake in the diet, is a steroid hormone that regulates sodium reabsorption in the distal part of the nephron (functional unit of the kidney) by modulating gene transcription. It has been shown that it can act synergistically with the peptidic hormone insulin through the interaction of their signalisation pathways. Our work consisted of two distinct parts: 1) the in vitro and in vivo characterisation of Glucocorticoid-Induced Leucine Zipper (GILZ) (an aldosterone-induced gene) mechanism of action; 2) the in vitro characterisation of insulin mechanism of action and its interaction with aldosterone. GILZ mRNA, coded by the TSC22D3 gene, is strongly induced by aldosterone in the cell line of principal cells of the cortical collecting duct (CCD) mpkCCDc14, suggesting that GILZ is a mediator of aldosterone response. Co-expression of GILZ and the amiloride-sensitive epithelial sodium channel ENaC in vitro in the Xenopus oocyte expression system showed that GILZ has no direct effect on the ENaC-mediated Na+ current in basal conditions. To define the role of GILZ in the kidney and in other organs (colon, heart, skin, etc.), a conditional knock-out mouse is being produced and will allow the in vivo study of its role. Previous data showed that insulin induced a transepithelial sodium transport at supraphysiological concentrations. Insulin and the insulin-like growth factor 1 (IGF-1) are able to bind to each other receptor with an affinity 50 to 100 times lower than to their cognate receptor. Our starting hypothesis was that the insulin effect observed at these supraphysiological concentrations is actually mediated by the IGF receptor type 1 (IGF-1R). In a new cell line that presents all the characteristics of the principal cells of the CCD (mCCDc11) we have shown that both insulin and IGF-1 induce a physiologically significant increase of Na+ transport through the activation of IGF-1R. Aldosterone and insulin/IGF-1 have an additive effect on Na+ transport, through the activation of the PI3-kinase (PI3-K) pathway and the phosphorylation of the serum- and glucocorticoid-induced kinase 1 (Sgk1) by the IGF-1R, and the induction of Sgk1 expression by aldosterone. Thus, Sgk1 integrates IGF-1/insulin and aldosterone effects. We suggest that IGF-1 is physiologically relevant in the modulation of sodium balance, while insulin can only regulate Na+ transport at supraphysiological conditions. Both hormones would bind to the IGF-1R and induce Na+ transport by activating the PI3-K PDK1/2 - Sgk1 pathway. We have shown for the first time that Sgk1 is expressed and phosphorylated in principal cells of the CCD in basal conditions, although the mechanism that maintains Sgk1 phosphorylation is not known. This new role for IGF-1 suggests that it could be a salt susceptibility gene. In effect, IGF-1 stimulates Na+ and water transport in the kidney in vivo. Moreover, 35 % of the acromegalic patients (overproduction of growth hormone and IGF-1) are hypertensives (higher proportion than in normal population), and genetic analysis suggest a link between the IGF-1 gene locus and blood pressure. RÉSUMÉ La régulation de l'excrétion rénale de sodium (Na+) joue un rôle principal dans le contrôle à long terme de la pression sanguine, et ses altérations constituent un facteur de risque de l'apparition d'une hypertension artérielle. L'aldosterone, dont la sécrétion dépend de l'apport en sel dans la diète, est une hormone stéroïdienne qui régule la réabsorption de Na+ dans la partie distale du nephron (unité fonctionnelle du rein) en contrôlant la transcription de gènes. Elle peut agir de façon synergistique avec l'hormone peptidique insuline, probablement via l'interaction de leurs voies de signalisation cellulaire. Le but de notre travail comportait deux volets: 1) caractériser in vitro et in vivo le mécanisme d'action du Glucocorticoid Induced Leucine Zipper (GILZ) (un gène induit par l'aldosterone); 2) caractériser in vitro le mécanisme d'action de l'insuline et son interaction avec l'aldosterone. L'ARNm de GILZ, codé par le gène TSC22D3, est induit par l'aldosterone dans la lignée cellulaire de cellules principales du tubule collecteur cortical (CCD) mpkCCDc14, suggérant que GILZ est un médiateur potentiel de la réponse à l'aldosterone. La co-expression in vitro de GILZ et du canal à Na+ sensible à l'amiloride ENaC dans le système d'expression de l'oocyte de Xénope a montré que GILZ n'a pas d'effet sur les courants sodiques véhiculées par ENaC en conditions basales. Une souris knock-out conditionnelle de GILZ est en train d'être produite et permettra l'étude in vivo de son rôle dans le rein et d'autres organes. Des expériences préliminaires ont montré que l'insuline induit un transport transépithelial de Na+ à des concentrations supraphysiologiques. L'insuline et l'insulin-like growth factor 1 (IGF-1) peuvent se lier à leurs récepteurs réciproques avec une affinité 50 à 100 fois moindre qu'à leur propre récepteur. Nous avons donc proposé que l'effet de l'insuline soit médié par le récepteur à l'IGF type 1 (IGF-1R). Dans une nouvelle lignée cellulaire qui présente toutes les caractéristiques des cellules principales du CCD (mCCDc11) nous avons montré que les deux hormones induisent une augmentation physiologiquement significative du transport du Na+ par l'activation des IGF-1 R. Aldosterone et insuline/IGF-1 ont un effet additif sur le transport de Na+, via l'activation de la voie de la PI3-kinase et la phosphorylation de la serum- and glucocorticoid-induced kinase 1 (Sgk1) par l'IGF-1R, dont l'expression est induite par l'aldosterone. Sgk1 intègre les effets de l'insuline et l'aldosterone. Nous proposons que l'IGF-1 joue un rôle dans la modulation physiologique de la balance sodique, tandis que l'insuline régule le transport de Na+ à des concentrations supraphysiologiques. Les deux hormones agissent en se liant à l'IGF-1R et induisent le transport de Na+ en activant la cascade de signalisation PI3-K - PDK1/2 - Sgk1. Nous avons montré pour la première fois que Sgk1 est exprimée et phosphorylée dans des conditions basales dans les cellules principales du CCD, mais le mécanisme qui maintient sa phosphorylation n'est pas connu. Ce nouveau rôle pour l'IGF-1 suggère qu'il pourrait être un gène impliqué de susceptibilité au sel. Aussi, l'IGF-1 stimule le transport rénal de Na+ in vivo. De plus, 35 % des patients atteints d'acromégalie (surproduction d'hormone de croissance et d'IGF-1) sont hypertensifs (prévalence plus élevée que la population normale), et des analyses génétiques suggèrent un lien entre le locus du gène de l'IGF-1 et la pression sanguine. RÉSUMÉ GRAND PUBLIC Nos ancêtres se sont génétiquement adaptés pendant des centaines de millénaires à un environnement pauvre en sel (chlorure de sodium) dans la savane équatoriale, où ils consommaient moins de 0,1 gramme de sel par jour. On a commencé à ajouter du sel aux aliments avec l'apparition de l'agriculture (il y a 5000 à 10000 années), et aujourd'hui une diète omnivore, qui inclut des plats préparés, contient plusieurs fois la quantité de sodium nécessaire pour notre fonction physiologique normale (environ 10 grammes par jour). Le corps garde sa concentration constante dans le sang en s'adaptant à une consommation très variable de sel. Pour ceci, il module son excrétion soit directement, soit en sécrétant des hormones régulatrices. Le rein joue un rôle principal dans cette régulation puisque l'excrétion urinaire de sel change selon la diète et peut aller d'une quantité dérisoire à plus de 36 grammes par jour. L'attention qu'on prête au sel est liée à sa relation avec l'hypertension essentielle. Ainsi, le contrôle rénal de l'excrétion de sodium et d'eau est le principal mécanisme dans la régulation de la pression sanguine, et une ingestion excessive de sel pourrait être l'un des facteurs-clé déclenchant l'apparition d'un phénotype hypertensif. L'hormone aldosterone diminue l'excrétion de sodium par le rein en modulant l'expression de gènes qui pourraient être impliqués dans la sensibilité au sel. Dans une lignée cellulaire de rein l'expression du gène TSC22D3, qui se traduit en la protéine Glucocorticoid Induced Leucine Zipper (GILZ), est fortement induite par l'aldosterone. Ceci suggère que GILZ est un médiateur potentiel de l'effet de l'aldosterone, et pourrait être impliqué dans la sensibilité au sel. Pour analyser la fonction de GILZ dans le rein plusieurs approches ont été utilisées. Par exemple, une souris dans laquelle GILZ est spécifiquement inactivé dans le rein est en train d'être produite et permettra l'étude du rôle de GILZ dans l'organisme. De plus, on a montré que GILZ, en conditions basales, n'a pas d'effet direct sur la protéine transportant le sodium à travers la membrane des cellules, le canal sodique épithélial ENaC. On a aussi essayé de trouver des protéines qui interagissent directement avec GILZ utilisant une technique appelée du « double-hybride dans la levure », mais aucun candidat n'a émergé. Des études ont montré que, à de hautes concentrations, l'insuline peut aussi diminuer l'excrétion de sodium. A ces concentrations, elle peut activer son récepteur spécifique, mais aussi le récepteur d'une autre hormone, l'Insulin-Like Growth Factor 1 (IGF-1). En plus, l'infusion d'IGF-1 augmente la rétention rénale de sodium et d'eau, et des mutations du gène codant pour l'IGF-1 sont liées aux différents niveaux de pression sanguine. On a utilisé une nouvelle lignée cellulaire de rein développée dans notre laboratoire, appelée mCCDc11, pour analyser l'importance relative des deux hormones dans l'induction du transport de sodium. On a montré que les deux hormones induisent une augmentation significative du transport de sodium par l'activation de récepteurs à l'IGF-1 et non du récepteur à l'insuline. On a montré qu'à l'intérieur de la cellule leur activation induit une augmentation du transport sodique par le biais du canal ENaC en modifiant la quantité de phosphates fixés sur la protéine Serumand Glucocorticoid-induced Kinase 1 (Sgk1). On a finalement montré que l'IGF-1 et l'aldosterone ont un effet additif sur le transport de sodium en agissant toutes les deux sur Sgk1, qui intègre leurs effets dans le contrôle du transport de sodium dans le rein.

Entropic transport in confined media: a challenge for computational studies in biological and soft-matter systems

Transport in small-scale biological and soft-matter systems typically occurs under confinement conditions in which particles proceed through obstacles and irregularities of the boundaries that may significantly alter their trajectories. A transport model that assimilates the confinement to the presence of entropic barriers provides an efficient approach to quantify its effect on the particle current and the diffusion coefficient. We review the main peculiarities of entropic transport and treat two cases in which confinement effects play a crucial role, with the appearance of emergent properties. The presence of entropic barriers modifies the mean first-passage time distribution and therefore plays a very important role in ion transport through micro- and nano-channels. The functionality of molecular motors, modeled as Brownian ratchets, is strongly affected when the motor proceeds in a confined medium that may constitute another source of rectification. The interplay between ratchet and entropic rectification gives rise to a wide variety of dynamical behaviors, not observed when the Brownian motor proceeds in an unbounded medium. Entropic transport offers new venues of transport control and particle manipulation and new ways to engineer more efficient devices for transport at the nanoscale.

The competitive position of the Nordic wood industry in Germany : intangible quality dimensions

Abstract

ERK1/2 controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell of the cortical collecting duct of the mouse kidney.

The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 pathway as shown in vivo by immunostaining of phosphorylated active ERK1/2 (pERK1/2). The MEK1/2-ERK1/2 pathway controls many different ion transports both in proximal and distal nephron, raising the question of whether this pathway is involved in the basal and/or hormone-dependent transepithelial sodium reabsorption in the principal cell of the cortical collecting duct (CCD), a process mediated by the apical epithelial sodium channel and the basolateral sodium pump (Na,K-ATPase). To answer this question we used ex vivo microdissected CCDs from normal mouse kidney or in vitro cultured mpkCCDcl4 principal cells. Significant basal levels of pERK1/2 were observed ex vivo and in vitro. Aldosterone and vasopressin, known to up-regulate sodium reabsorption in CCDs, did not change ERK1/2 activity either ex vivo or in vitro. Basal and aldosterone- or vasopressin-stimulated sodium transport was down-regulated by the MEK1/2 inhibitor PD98059, in parallel with a decrease in pERK1/2 in vitro. The activity of Na,K-ATPase but not that of epithelial sodium channel was inhibited by MEK1/2 inhibitors in both unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface biotinylation showed that intrinsic activity rather than cell surface expression of Na,K-ATPase was controlled by pERK1/2. PD98059 also significantly inhibited the activity of Na,K-ATPase ex vivo. Our data demonstrate that the ERK1/2 pathway controls Na,K-ATPase activity and transepithelial sodium transport in the principal cell and indicate that basal constitutive activity of the ERK1/2 pathway is a critical component of this control.

Ville, tourisme et transport : la Compagnie du chemin de fer Lausanne-Ouchy (1869-1914)

Inauguré le 16 mars 1877, le chemin de fer entre Lausanne et le petit port touristique d'Ouchy constitue une entreprise aux dimensions techniques et financières pharaoniques. Premier funiculaire à câble de Suisse, le Lausanne-Ouchy est aussi le premier au monde à être équipé d'un système de traction hydraulique. L'objectif de cette contribution n'est toutefois pas d'analyser les conditions de réalisation de cet exploit technologique, mais la naissance et l'évolution d'une compagnie de transport étroitement liée à l'établissement du système touristique lausannois. La demande de mobilité créée par l'afflux d'étrangers et l'apport des milieux touristiques au capital sont en effet indispensables à la création et à la survie de l'entreprise, dont la santé financière reste longtemps problématique. En retour, le funiculaire modifie le système de transport régional et national, donnant d'importantes impulsions au développement touristique.