Modulation of the epithelial sodium channel by serine proteases

Abstract The epithelial sodium channel (ENaC) is composed of three homologous subunits α, ß, and γ. This channel is involved in the regulation of sodium balance, which influences the periciliary liquid level in the lung, and blood pressure via the kidney. ENaC expressed in Xenopus laevis oocytes is preferentially and rapidly assembled into heteromeric αßγ complexes. Expression of homomeric α or heteromeric αß and αγ complexes lead to channel expression at the cell surface wíth low activities. Recent studies have demonstrated that α and γ (but not ß) ENaC subunits undergo proteolytic cleavage by endogenous proteases (i.e. furin) correlating with increased channel activity. We therefore assayed the full-length subunits and their cleavage products at the cell surface, as well as in the intracellular pool for all homo- and heteromeric combínations (α, ß, γ, ßγ, αß, αγ, ßγ and αßγ) and measured the corresponding channel activities as amiloride-sensitive sodíum transport (INa). We showed that upon assembly, cleavage of the y ENaC subunit ís responsible for increasing INa. We further demonstrated that in disease states such as cystic fibrosis (CF) where there is disequilibrium in the proteaseprotease inhibitor balance, ENaC is over-activated by the serine protease elastase (NE). We demonstrated that elevated NE concentrations can cleave cell surface expressed γ ENaC (but not α, or ß ENaC), suggesting a causal relationship between γ ENaC cleavage and ENaC activation, taking place at the plasma membrane. In addition, we demonstrated that the serine protease inhibitor (serpin) serpinH1, which is co-expressed with ENaC in the distal nephron is capable of inhibiting the channel by preventing cleavage of the γ ENaC subunit. Aldosterone mediated increases in INa aze known to be inhibted by TGFß. TGFß is also known to increase serpinHl expression. The demonstrated inhibition of γ ENaC cleavage and channel activation by serpinH1 may be responsible for the effect of TGFß on aldosterone stimulation in the distal nephron. In summary, we show that cleavage of the γ subunit, but not the α or ß subunit is linked to channel activation in three seperate contexts. Résumé Le canal épithélial à sodium (ENaC) est constitué de trois sous-unités homologues α, ß, and γ. Ce canal est impliqué dans le maintien de la balance sodique qui influence le niveau du liquide périciliaire du poumon et la pression sanguine via le rein. Dans les ovocytes de Xenopus laevis ENaC est préférentiellement et rapidement exprimé en formant un complexe hétéromérique αßγ. En revanche, l'expression homomérique de α ou hétéromérique des complexes αß et αγ conduit à une expression à la surface cellulaire d'un canal ENaC ne possédant qu'une faible activité. Des études récentes ont mis en évidence que les sous-unités α et γ d'ENaC (mais pas ß) sont coupées par des protéases endogènes (les farines) et que ces clivages augmentent l'activité du canal. Nous avons donc analysé, aussi bien à la surface cellulaire que dans le cytoplasme, les produits des clivages de combinaison homo- et hétéromérique des sous-unités d'ENaC (α, ß, γ, ßγ, αß, αγ, ßγ et αßγ). En parallèle, nous avons étudié l'activité correspondante à ces canaux par la mesure du transport de sodium sensible à l'amiloride (INa). Nous avons montré que lors de l'assemblage des sous-unités d'ENaC, le clivage de γ correspond à l'augmentation de INa. Nous avons également mis en évidence que dans une maladie telle que la fibrose cystique (CF) caractérisée par un déséquilibre de la balance protéase-inhibiteur de protéase, ENaC est suractivé par une sérine protéase nommée élastase (NE). L'augmentation de la concentration de NE clive γ ENaC exprimé à la surface cellulaire (mais pas α, ni ß ENaC) suggérant une causalité entre le clivage d'ENaC et son activation à la membrane plasmique. De plus, nous avons démontré que l'inhibiteur de sérine protéase (serpin) serpinH1, qui est co-exprimé avec ENaC dans le néphron distal, inhibe l'activité du canal en empêchant le clivage de la sous-unité γ ENaC. Il est connu que le INa induit par l'aldostérone peut être inhibé par TGFß. Or TGFß augmente l'expression de serpinH1. L'inhibition du clivage de γ ENaC et de l'activation du canal par la serpinH1 que nous avons mis en évidence pourrait ainsi être responsable de l'effet de TGFß sur la stimulation du courant par l'aldostérone dans le néphron distal. En résumé, nous avons montré que le clivage de la sous-unité γ, mais pas des sous-unités α et ß, est lié à l'activation du canal dans trois contextes distincts. Résumé tout public Le corps humain est composé d'environ 10 000 milliards de cellules et d'approximativement 60% d'eau. Les cellules du corps sont les unités fondamentales de la vie et elles sont dépendantes de certains nutriments et molécules. Ces nutriments et molécules sont dissous dans l'eau qui est présente dans et hors des cellules. Le maintien d'une concentration adéquate - de ces nutriments et de ces molécules dans l'eau à l'intérieur et à l'extérieur des cellules est -..essentiel pour leur survie. L'eau hors des cellules est nommée le fluide extracellulaire et peut être subdivisée en fluide interstitiel, qui se trouve autour des cellules, et en plasma, qui est le fluide des vaisseaux sanguins. Les fluides, les nutriments et les molécules sont constamment échangés entre les cellules, le fluide interstitiel, et le plasma. Le plasma circule dans le système circulatoire afin de distribuer les nutriments et molécules dans tout le corps et afin d'enlever les déchets cellulaires. Le rein joue un rôle essentiel dans la régulation du volume et de la concentration du plasma en éliminant sélectivement les nutriments et les molécules via la formation de l'urine. L'être humain possède deux reins, constitués chacun d'environ 1 million de néphrons. Ces derniers sont responsables de réabsorber et de sécréter sélectivement les nutriments et les molécules. Le canal épithélial à sodium (ENaC) est localisé à la surface cellulaire des néphrons et est responsable de la réabsorption du sodium (Na+). Le Na+ est présent dans quasiment toute la nourriture que nous mangeons et représente, en terme de molécule, 50% du sel de cuisine. Si trop de sodium est consommé, ENaC est inactif, si bien que le Na+ n'est pas réabsorbé et quitte le corps par l'urine. Ce mécanisme permet d'éviter que la concentration plasmatique de Na+ ne devienne trop grande, ce qui résulterait en une augmentation de la pression sanguine. Si trop peu de Na+ est consommé, ENaC réabsorbe le Na+ de l'urine primaire ce qui permet de conserver la concentration de Na+ et de prévenir une diminution de la pression sanguine par une perte de Na+. ENaC est aussi présent dans les cellules des poumons qui sont les organes permettant la respiration. La respiration est aussi essentielle pour la survie des cellules. Les poumons ne doivent pas contenir trop de liquide afin de permettre la respiration, mais en même temps ils ne doivent pas non plus être trop secs. En effet, ceci tuerait les cellules et empêcherait aussi la respiration. ENaC permet de maintenir un niveau d'humidité approprié dans les poumons en absorbant du Na+ ce qui entraîne un mouvement osmotique d'eau. L'absorption de sodium par ENaC ~ est augmentée par les protéases (in vitro et ex vivo). Les protéases sont des molécules qui peuvent couper d'autres molécules à des endroits précis. Nous avons démonté que certaines protéases augmentent l'absorption de Na+ en coupant ENaC à des endroits spécifiques. L'inhibition de ces protéases diminue le transport de Na+ et empêche le clivage d'ENaC. Dans certaines maladies telle que la mucoviscidose, des protéases sont suractivées et augmentent l'activité d'ENaC de manière inappropriée conduisant à une trop forte absorption de Na+ et à un déséquilibre de la muqueuse des poumons. Cette étude est donc particulièrement importante dans le cadre de la recherche thérapeutique de ce genre de maladie.

Ion-exchange resins as stationary phase in reactive chromatography

Dynamic behavior of bothisothermal and non-isothermal single-column chromatographic reactors with an ion-exchange resin as the stationary phase was investigated. The reactor performance was interpreted by using results obtained when studying the effect of the resin properties on the equilibrium and kinetic phenomena occurring simultaneously in the reactor. Mathematical models were derived for each phenomenon and combined to simulate the chromatographic reactor. The phenomena studied includes phase equilibria in multicomponent liquid mixture¿ion-exchange resin systems, chemicalequilibrium in the presence of a resin catalyst, diffusion of liquids in gel-type and macroporous resins, and chemical reaction kinetics. Above all, attention was paid to the swelling behavior of the resins and how it affects the kinetic phenomena. Several poly(styrene-co-divinylbenzene) resins with different cross-link densities and internal porosities were used. Esterification of acetic acid with ethanol to produce ethyl acetate and water was used as a model reaction system. Choosing an ion-exchange resin with a low cross-link density is beneficial inthe case of the present reaction system: the amount of ethyl acetate as well the ethyl acetate to water mole ratio in the effluent stream increase with decreasing cross-link density. The enhanced performance of the reactor is mainly attributed to increasing reaction rate, which in turn originates from the phase equilibrium behavior of the system. Also mass transfer considerations favor the use ofresins with low cross-link density. The diffusion coefficients of liquids in the gel-type ion-exchange resins were found to fall rapidly when the extent of swelling became low. Glass transition of the polymer was not found to significantlyretard the diffusion in sulfonated PS¿DVB ion-exchange resins. It was also shown that non-isothermal operation of a chromatographic reactor could be used to significantly enhance the reactor performance. In the case of the exothermic modelreaction system and a near-adiabatic column, a positive thermal wave (higher temperature than in the initial state) was found to travel together with the reactive front. This further increased the conversion of the reactants. Diffusion-induced volume changes of the ion-exchange resins were studied in a flow-through cell. It was shown that describing the swelling and shrinking kinetics of the particles calls for a mass transfer model that explicitly includes the limited expansibility of the polymer network. A good description of the process was obtained by combining the generalized Maxwell-Stefan approach and an activity model that was derived from the thermodynamics of polymer solutions and gels. The swelling pressure in the resin phase was evaluated by using a non-Gaussian expression forthe polymer chain length distribution. Dimensional changes of the resin particles necessitate the use of non-standard mathematical tools for dynamic simulations. A transformed coordinate system, where the mass of the polymer was used as a spatial variable, was applied when simulating the chromatographic reactor columns as well as the swelling and shrinking kinetics of the resin particles. Shrinking of the particles in a column leads to formation of dead volume on top of the resin bed. In ordinary Eulerian coordinates, this results in a moving discontinuity that in turn causes numerical difficulties in the solution of the PDE system. The motion of the discontinuity was eliminated by spanning two calculation grids in the column that overlapped at the top of the resin bed. The reactive and non-reactive phase equilibrium data were correlated with a model derived from thethermodynamics of polymer solution and gels. The thermodynamic approach used inthis work is best suited at high degrees of swelling because the polymer matrixmay be in the glassy state when the extent of swelling is low.

Gas exchange of the umbu tree under semi-arid conditions

A study was carried out at Embrapa Semi-Árido, Petrolina-PE, Brazil, aiming to understand the gas exchange process of the umbu tree (Spondias tuberosa Arr. Cam.) in the dry and rainy seasons. Stomatal conductance, transpiration, photosynthesis and internal CO2 concentration were obtained with a portable infrared gas analyzer (IRGA). During the dry season the umbu tree showed a much lower stomatal conductance early in the morning, as soon as the vapor pressure deficit increased, apparently affecting CO2 assimilation more than transpiration. The highest values were detected around 6:00 am but decreased to the lowest points between 10:00 am and 2:00 pm. During the rainy season, however, stomatal conductance, transpiration and photosynthesis were significantly higher, reaching the highest values between 8:00 and 10:00 am and the lowest around 2:00 pm. It was also observed at 4:00 pm, mainly during the rainy season, an increase on these variables indicating that the umbu tree exhibits a two-picked daily course of gas exchange.

Reduction of conjugated dienoic carboxylic acids and esters with sodium dithionite

Reduction of 2,4-alkadienoic acids and esters with sodium dithionite was carried out in aqueous solution or under PTC conditions to give, almost exclusively, Z:E isomeric mixtures of the corresponding 3-alkenoic acids and esters, respectively, in good yields.

Water status and gas exchange of umbu plants (Spondias tuberosa Arr. Cam.) propagated by seeds and stem cuttings

The experiment was carried out at the Embrapa Semi-Árido, Petrolina-PE, Brazil, in order to study the physiological responses of umbu plants propagated by seeds and by stem cuttings under water stress conditions, based on leaf water potential and gas exchange measurements. Data were collected in one-year plants established in pots containing 30 kg of a sandy soil and submitted to twenty-day progressive soil water deficit. The evaluations were based on leaf water potential and gas exchange data collection using psychrometric chambers and a portable infra-red gas analyzer, respectively. Plants propagated by seeds maintained a significantly higher water potential, stomatal conductance, transpiration and photosynthesis under decreasing soil water availability. However, plants propagated by stem cuttings were unable to maintain a favorable internal water balance, reflecting negatively on stomatal conductance and leaf gas exchange. This fact is probably because umbu plants propagated by stem cuttings are not prone to formation of root tubers which are reservoirs for water and solutes. Thus, the establishing of umbu plants propagated by stem cuttings must be avoided in areas subjected to soil water deficit.

Epithelial sodium transport and its control by aldosterone: the story of our internal environment revisited.

Transcription and translation require a high concentration of potassium across the entire tree of life. The conservation of a high intracellular potassium was an absolute requirement for the evolution of life on Earth. This was achieved by the interplay of P- and V-ATPases that can set up electrochemical gradients across the cell membrane, an energetically costly process requiring the synthesis of ATP by F-ATPases. In animals, the control of an extracellular compartment was achieved by the emergence of multicellular organisms able to produce tight epithelial barriers creating a stable extracellular milieu. Finally, the adaptation to a terrestrian environment was achieved by the evolution of distinct regulatory pathways allowing salt and water conservation. In this review we emphasize the critical and dual role of Na(+)-K(+)-ATPase in the control of the ionic composition of the extracellular fluid and the renin-angiotensin-aldosterone system (RAAS) in salt and water conservation in vertebrates. The action of aldosterone on transepithelial sodium transport by activation of the epithelial sodium channel (ENaC) at the apical membrane and that of Na(+)-K(+)-ATPase at the basolateral membrane may have evolved in lungfish before the emergence of tetrapods. Finally, we discuss the implication of RAAS in the origin of the present pandemia of hypertension and its associated cardiovascular diseases.

Preparation of alpha-labeled aldehydes by base-catalyzed exchange reactions

A simple, efficient protocol for the preparation of α-labeled aldehydes based on H/D exchange catalyzed by 4-(N,N-dimethylamino)pyridine or Et3N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.

Preparation of alpha-labeled aldehydes by base-catalyzed exchange reactions

A simple, efficient protocol for the preparation of α-labeled aldehydes based on H/D exchange catalyzed by 4-(N,N-dimethylamino)pyridine or Et3N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.