Active Solute Transport Research Articles

Source of net water and electrolyte loss following intestinal ischaemia.

Ischaemia of the dog intestine lasting 1 h causes desquamation of the epithelium at the villus tips and congestion in the villus capillaries. The crypt cells are relatively undamaged. These changes are associated with a loss of active transport of organic solutes, determined in vitro, a reduction in mucosal sucrase activity and an abolition of glucose absorption in vivo. A profuse net loss of water and electrolytes into the lumen in vivo develops. The net sodium loss is due primarily to an inhibition of the lumen-blood flux of this ion, the blood-lumen flux being relatively unchanged. In uraemic dogs, the loss of urea into the lumen is the same in control and ischaemic loops, testifying to the lack of change in the unidirectional water flow from blood to lumen. Perfusion of the dog intestine with 1% Triton X-100 leads to morphological changes that have certain similarities with those provoked by ischaemia. Damage was restricted to the villus tips, protection from further alterations apparently being provided by a mucus layer that forms on the mucosal surface; the crypt region remained unchanged. After 10 min exposure, organic solute transport in vitro and glucose absorption in vivo were both reduced by not abolished; sodium and water absorption in vivo were suppressed, but no net secretion occurred. To account for these observations, we have suggested that the normal crypt cell is a secretory element with respect to sodium and water. During maturation, its absorptive properties develop such that the mature enterocyte, possessing both absorptive and secretory mechanisms, is capable of net absorption of sodium. After destruction of the villus tips, net secretion continues in the crypts; if there are insufficient villus cells remaining to ensure reabsorption, a net secretory capacity is observed.

Effect of cell-substratum interaction on hemicyst formation by MDCK cells.

On impermeable substrata MDCK cells, a cell line derived from normal dog kidney, forms a confluent monolayer that is studded with numerous hemicysts. Previous studies with this cell line suggest that thes hemicysts develop as a result of active fluid accumulation between cell sheet and substratum. However, the formation of hemicysts as a multifocal phenomenon is still unexplained. The results presented here show that the hemicysts are not only expressions of active transport of solutes and water, but also of cell-substratum interaction. The increase in number and size of the hemicyst produced by dbcAMP may be explained by a decrease in the adhesive strength to substrata produced by this compound. Moreover, when the strength of the cell-substratum adhesion was increased the number of hemicysts was reduced or abolished. On the contrary, when this strength was reduced, larger hemicysts occurred, covering practically all the area available for growth. Results from cinematographic time lapse studies, showing that 90% of the area of the monolayer is able to produce hemicysts, also suggest that hemicyst formation as a multifocal phenomenon is more an expression of local variations in cell-substratum interaction than of regional changes in transepithelial active transport.

Polarity of the blood-brain barrier: Distribution of enzymes between the luminal and antiluminal membranes of brain capillary endothelial cells

The subcellular distribution in brain capillaries of alkaline phosphatase and Na+, K+-ATPase was investigated by two methods. Cytochemical studies using whole brain perfusion and electron microscopic examination indicated that alkaline phosphatase activity was located in both the luminal and antiluminal cytoplasmic membranes of the brain capillary endothelial cells. By contrast, the K+-dependent phosphatase activity associated with Na+, K+-ATPase was located in only the antiluminal membrane. Biochemical studies using membranes prepared by homogenization of isolated brain capillaries and density gradient centrifugation resulted in identification of two plasma membrane fractions. The light fraction contained alkaline phosphatase but very little Na+, K+-ATPase while the heavier fraction contained both enzyme activities. In addition, gamma-glutamyl transpeptidase showed a distribution similar to alkaline phosphatase while 5'-nucleotidase activity was distributed with the Na+, K+-ATPase activity. We conclude that the luminal and antiluminal membranes of brain capillaries are biochemically and functionally different. This polarity should permit active solute transport across brain capillary endothelial cells which are the cells responsible for the blood-brain barrier.

PH gradient-stimulated transport of urate and p-aminohippurate in dog renal microvillus membrane vesicles.

The transport mechanism of urate and p-aminohippurate (PAH) was evaluated in microvillus membrane vesicles isolated from the renal cortex of the mongrel dog. Imposition of a transmembrane pH gradient (pHo less than pH1) markedly accelerated the uptake of [14C]urate and [3H]PAH and caused the transient accumulation ("overshoot") of each anion above its final level of uptake. The transport of urate and PAH under both stimulated (pHo less than pHi) and basal (pHo = pHi) conditions was insensitive to valinomycin-induced K+ diffusion potentials. The pH gradient-stimulated uptake of 25 microM [14C]urate and 1.0 microM [3H]PAH was significantly inhibited by 1.2 mM PAH, urate, furosemide, salicylate, or probenecid. The effect of each inhibitor on [14C]urate transport was identical to the effect of the same inhibitor on [3H]PAH flux. We conclude that the transport of urate and PAH in dog renal microvillus membrane vesicles occurs via a pH gradient-stimulated electroneutral carrier-mediated process such as 1:1 H+-anion cotransport or OH-anion exchange. Such a transport mechanism may possibly play a role in effecting uphill urate reabsorption in the proximal tubule. Moreover, this study demonstrates that secondary active solute transport in epithelial membranes may be coupled to the electrochemical gradient of an ion other than Na+.

Restoration of active transport of solutes and oxidative phosphorylation by naphthoquinones in irradiated membrane vesicles from Mycobacterium phlei.

Irradiation of the inverted membrane vesicles of Mycobacterium phlei with light at 360 nm inactivated the natural menaquinone [MK(9)(II-H)] and resulted in a loss of substrate oxidation, pH gradient, membrane potential, active transport of proline or calcium ions, and oxidative phosphorylation. Restoration of the protonmotive force and active transport occurred on addition of naphthoquinones such as vitamin K(1), menadione, or lapachol to the irradiated membrane vesicles. However, coupled phosphorylation was restored only by vitamin K(1). Menadione and lapachol did not act as uncoupling agents. The magnitude of the pH gradient and membrane potential in the quinone-restored system was a reflection of the rate of oxidation and was correlated with the rate of uptake of proline or Ca(2+). These results are consistent with the chemosmotic hypothesis proposed for the energy transducing mechanism for active transport and further demonstrate that the complete respiratory chain is not required to drive active transport. In contrast, the data suggest that in addition to the driving force (protonmotive force) necessary to establish oxidative phosphorylation, a specific spatial orientation of the respiratory components, such as the naphthaquinones, is essential for the utilization of the proton gradient or membrane potential or both. Bypass of electrons from the respiratory chain with menadione may explain the inability of this quinone to restore oxidative phosphorylation; however, lapachol restores oxidation by the same electron transport pathway as the natural menaquinone but fails to restore phosphorylation. Because all three quinones restore the protonmotive force, other factors that are discussed must be considered in understanding the mechanism of oxidative phosphorylation.

Mise en évidence d'activités enzymatiques au niveau de l'haustorium d'une phanérogame parasite : Striga hermonthica (Scrophulariaceae)

Acid phosphatase, ATPase, peroxidase, succinate dehydrogenase, and cytochrome oxidase activities have been demonstrated in the haustorium of Striga hermonthica, a parasitic phanerogam on millet (Pennisetum typhoideum var. Sonna 3). Their localization has been specified and their possible role discussed. These enzymatic activities seem to be related to the host penetration mechanism and also with the process of active transport of solutes between host and parasite. A tracheid radial system has also been discovered in the mature haustorium.

Determination of the membrane potential in bacterial membrane vesicles from the accumulation of N-methyldeptropine

Active transport of solutes by cytoplasmic membrane vesicles from bacteria can be driven by electron transfer in respiratory chains, anaerobic electron transfer systems or by cyclic electron transfer systems (reviewed [ 1,2]). Electron transfer results in the generation of an electrochemical proton gradient across the membrane which is composed of an electrical gradient (A$) and a chemical proton gradient (ApH) according to the equation A&+ = A$-2.3 RT/F log ApH. The generation of a membrane potential and a pH gradient, induced by electron transfer, has been demonstrated in membrane vesicles from several bacteria [3,4]. The transmembrane pH gradient can be determined from the accumulation of radioactively labeled weak acids or bases as measured by means of flow dialysis [5]. For the determination of the membrane potential several methods have been employed [3]. A very reliable method for small cells and membrane vesicles has been introduced [6]. These investigators demonstrated that lipophilic organic cations or anions distribute across the membrane in response to the membrane potential. The membrane potential can be calculated from the concentration gradient reached at steady state level of accumulation with the Nernst equation:

Solute transport process in intestinal epithelial cells.

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na+ and K+ concentrations, significant gains of Na+ and K+ occurred with glucose transport. The quantitative relationships among net gains of Na+, K+ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na+ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na+-efflux and K+-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.

Spatially discrete models of counter-current mass transport for application to the kidney

Within the kidney the transport of many solute species and water is accomplished by the parallel operation of several counter-current transport systems. The simulation of these processes requires the solution of a non-linear, two-point boundary value problem with many simultaneous equations. Standard formulations of the problem involve the solution of a set of one-dimensional, plug-transport equations. Rather than seek an accurate solution of this spatially continuous model, which is only an approximation to the real system, we investigated spatially discrete analogs. The motivation for use of spatially discrete models is to simulate with much more efficiency the detailed simultaneous transport processes of many solutes and water. The membrane flux equations of these models describe solvent drag as well as passive and active transport of solutes. In this study, we investigated a perfectly mixed compartment model and an improved Euler (semi-discretized) model. The solutions of these models were compared to the numerical solution of a standard spatially continuous model. For efficient numerical solution of the continuous model, the equations were arranged to yield a band matrix structure and a triangular decomposition procedure was applied. The model solutions were compared for convergence, efficiency, and accuracy for a variety of parameter values. With membrane parameter values typical of tubule segments of the rat kidney, a spatial step size as large as 1 mm (roughly one-seventh the length of the renal medulla of the rat) was found to be suitable for both discrete models.

Effect of ouabain and colloid osmotic pressure on renal tubule cell volume

Proximal renal tubule cell volume increases in ouabain but cell swelling is limited by the tubule basement membrane (TBM) and the colloid osmotic pressure from the bath protein. We compared the effect of ouabain, external protein concentration, and TBM on cell volume of proximal convoluted (PCT), proximal straight (PST), and cortical collecting tubules (CCT). We blocked active solute transport with ouabain and evaluated cell size by measuring the outer diameter of nonperfused tubules. Proximal tubules in ouabain swelled 35-40% in isoncotic medium and 20-25% further in hyponcotic medium (0.3 g/100 ml albumin), but PCT swelled faster than PST. The CCT swelled minimally in similar mediums, indicating pronounced heterogeneity in the response of cortical nephron segments to ouabain. In the presence of ouabain, all tubules swelled extensively when we removed the TBM with collagenase. In the hyponcotic medium fluid flux across the peritubular membrane was 0.081, 0.049, and 0.030 nl/min per mm tubule length for PCT, PST, and CCT, respectively. The rates of fluid flux in PCT and PST were proportional to estimates of the respective basolateral surface areas. We suggest that differences in swelling rates between proximal segments reflect variations in surface area rather than intrinsic peritubular membrane permeability to solute and water.

Electrical properties and active solute transport in rat small intestine. II. Conductive properties of transepithelial routes.

The transepithelial resistance, the cell membrane resistance and the ratio of resistances of the serosal (baso-lateral) to the mucosal (brush border) cell membrane were measured in rat duodenum, jejunum and ileum by means of microelectrode techniques. These measured values were not affected in the presence of actively transported solutes in the mucosal bathing fluid. Contribution of an electrical conductance through the extracellular shunt pathway to the total transepithelial conductance was quantitatively estimated using an electrically equivalent circuit analysis. These values estimated in respective tissues of small intestine were approx. 95% of the total transepithelial conductance, remaining unaffected by an active solute transport. From these data, the changes in emf's of the mucosal and serosal membrane induced by D-glucose or glycine were separately evaluated.

A model of the human renal medulla

A comprehensive mathematical model of the renal medulla is presented and validated. It is used here to simulate the human kidney. The model formulation is based upon mass-balance equations describing flow of water, electrolyte, and urea through the various structures of the medulla. These structures include representations of both the cortical and juxtamedullary nephrons plus collecting ducts, vasa recta, and interstitium. The simulation produces a concentration increase in the inner zone without active transport of solutes from any tubule within that zone. Analysis of this behaviour indicates that passive mechanisms are adequate for the human kidney but may be inadequate for other mammalian species. The simulation also reproduces observed urea excretion rates over the normal range of diuresis to hydropenia and demonstrates a route for urea recirculation via the thin ascending limb of Henle.

Electrical properties and active solute transport in rat small intestine. I. Potential profile changes associated with sugar and amino acid transports.

Addition of D-glucose to the mucosal fluid resulted in a significant depolarization of the mucosal membrane potential (V-m) in rat duodenum, jejunum, and ileum accompanied by an increase in the transepithelial potential difference (PDt). On the other hand, L-glucose did not induce PDt and Vm changes. Glycine applied from the mucosal side also induced Vm-depolarization and PDt-increment in the ileum. Phlorizin added to the mucosal fluid or ouabain added to the serosal fluid inhibited the sugar-dependent changes in PDt and Vm. According to the analysis with an equivalent circuit model for the epithelium, it was concluded that an actively transported solute induced not only a depolarization of the mucosal (brush border) membrane but also a hyperpolarization of the serosal (baso-lateral) membrane of an epithelial cell, so that the origin of solute-induced PDt changes should be attributed to changes in emf's at both membranes. The hyperpolarization of the serosal membrane in the presence of an actively transported solute was attributed to a mechanism of serosal electrogenic sodium pump stimulated by the increase in the extrusion rate of Na+ co-transported into the cell with sugar or amino acid.

Effect of basement membrane and colloid osmotic pressure on renal tubule cell volume.

Renal tubule cell volume is thought to be kept constant by a cation pump. When active transport is blocked, intracellular impermeant solutes cause cells to swell. Cell size is then determined by transmembrane hydrostatic and colloid osmotic forces. We studied the importance of passive transmembrane forces in determining cell size in isolated rabbit proximal straight tubules (PST). We blocked active solute transport with ouabain and evaluated subsequent changes in cell size by measuring outer diameter of nonperfused tubules. Tubules in a ouabain and 6 g/100 ml protein bath swelled only 40% above control. However, removal of the tubule basement membrane with collagenase dissipated a transmembrane hydrostatic pressure and caused more swelling. Final cell volume was determined largely by bath protein concentration. Tubules in ouabain and collagenase swelled enormously in hyponcotic protein, moderately in isoncotic protein, and could be shrunk below control in hyperoncotic protein. Intracellular colloid osmotic pressure was estimated to exceed 38 cmH20. We conclude that hydrostatic and colloid osmotic forces are major determinants of cell size in isolated PST treated with ouabain.

Fluid transport by rabbit preimplantation blastocysts in vitro.

The large expansion of the rabbit blastocyst between the 3rd and 7th day post coitum(p.c.) is due to the accumulation of fluid in the blastocoele cavity (Daniel, 1964). Na+, Cl− and HCO3− appear to be actively transported across the trophectoderm into the blastocoele (Cross, 1973, 1974) and water moves passively secondary to NaCl accumulation (Borland, Biggers & Lechene, 1976; see also review by Borland, 1977). The trophoblast cell is physiologically polarized in that Na is transported into the blastocoele by Na/K pumps (Na+–K+ ATPase, E.C. 3.6.1.3) located on the juxtacoelic plasma membrane of the trophoblast cells (Biggers, Borland & Lechene, 1977). This functional polarity is also reflected by the anatomical polarity of the trophectoderm. Membrane junctional complexes are located at the apices of the trophoblast cell and are adjacent to fluid-filled intercellular spaces that face the blastocoele cavity (Gamow & Daniel, 1970; Enders, 1971; Hastings & Enders, 1975; Ducibella, Albertini, Anderson & Biggers, 1975). Diamond & Bossert (1967) have proposed that such closed-end channels may be the site of active solute transport that creates local standing osmotic gradients which cause water flow across epithelia.

On the influence of a leaky tight junction on water and solute transport in epithelia

The role of a leaky tight junction in epithelia is examined by considering the flow of water and solute through a channel consisting of two sections representing the intercellular space and tight junction. Two cases are considered, flow through a channel with a circular cross-section and flow between parallel planes. Analytical solutions are obtained using the isotonic convection approximation. The flow is driven by active transport of solute and imposed concentration and pressure differences. Particular attention is paid to the flux of solute through the tight junction. It is shown that the shape of the channel cross-section is important. The theory is applied to the rat proximal tube epithelium. It is deduced that the emergent osmolarity is close to that predicted for a closed tight junction, but that transepithelial hydrostatic pressure differences are potentially important. The influence of transepithelial concentration differences appears to be unimportant in this model.

The relationship between the electrochemical proton gradient and active transport in Escherichia coli membrane vesicles.

In the previous paper [ramos, S., and Kaback, H.R. (1977), Biochemistry 16 (preceding paper in this issue)], it was demonstrated that Escherichia coli membrane vesicles generate a large electrochemical proton gradient (delta-muH+) under appropriate conditions, and some of the properties of delta-muH+ and its component forces [i.e., the membrane potential (delta psi) and the chemical gradient of protons (deltapH)] were described. In this paper, the relationship between delta-muH+, delta psi, and deltapH and the active transport of specific solutes is examined. Addition of lactose or glucose 6-phosphate to membrane vesicles containing the appropriate transport systems results in partial collapse of deltapH, providing direct evidence for the suggestion that respiratory energy can drive active transport via the pH gradient across the membrane. Titration studies with valinomycin and nigericin lead to the conclusion that, at pH 5.5, there are two general classes of transport systems: those that are driven primarily by delta-muH+ (lactose, proline, serine, glycine, tyrosine, glutamate, leucine, lysine, cysteine, and succinate) and those that are driven primarily by deltapH (glucose 6-phosphate, D-lactate, glucuronate, and gluconate). Importantly, however, it is also demonstrated that at pH 7.5, all of these transport systems are driven by delta psi which comprises the only component of delta-muH+ at this external pH. In addition, the effect of external pH on the steady-state levels of accumulation of different solutes is examined, and it is shown that none of the pH profiles correspond to those observed for delta-muH+, delta psi, or deltapH. Moreover, at external pH values above 6.0-6.5, delta-muH+ is insufficient to account for the concentration gradients established for each substrate unless the stoichiometry between protons and accumulated solutes is greater than unity. The results confirm many facets of the chemiosmotic hypothesis, but they also extend the concept in certain important respects and allow explanations for some earlier observations which seemed to preclude the involvement of chemiosmotic phenomena in active transport.

Active Transport of Solutes in Bacterial Membrane Vesicles

The metabolic energy-dependent processes are thought to be the major bacterial mechanisms involved in the accumulation of solute in the cytoplasm. The two processes differ in an important aspect: in group translocation, the transported molecule is changed covalently during passage through the membrane, while in active transport the solute is accumulated in an unmodified form in the interior of the cell. It has been demonstrated that energy released by the electron transfer systems is not coupled to active transport via ATP. It has not yet been thoroughly established whether ATP can serve as the major source of energy for active transport in bacteria grown under glycolytic conditions it might be possible that electron-transfer systems which do not contain cytochromes supply the energy for active transport under these conditions. To obtain a complete understanding of the mechanism of active transport, a number of features remain to be elucidated. Among them are: involvement of electron-transfer intermediates in the translocation of protons; the role of the electrical potential, and the pH-gradient, in the energy coupling to active transport of different solutes; the molecular properties of the transport carriers and the mechanism of solute translocation.

Micropuncture study of the sodium and potassium content in the nephridial and coelomic fluids in a marine invertebrate Sabella pavonina savigny (Polychaeta annelida)

1. 1. In the thoracic coelomic fluid of Sabella, the potassium concentration is much higher than in the external marine medium whereas sodium concentration is identical in both. 2. 2. The antero-posterior decreasing osmotic gradient in the coelomic fluid is partly explained by the drop in sodium concentration. 3. 3. Along the proximal nephridial limb the sodium concentration increases but the potassium concentration does not change. There is an inverse relationship between the concentration of the ion in both nephridial and coelomic fluids; the concentration declines longitudinally in the coelomic compartment and rises in the nephridial one. This observation suggests that sodium movements might occur from the coelomic to the nephridial compartment. The ultrastructural organisation of the proximal nephridial wall indicate that active transport of solutes might occur at this site. 4. 4. The K + and Na + concentration in the fluid increase along the distal nephridial limb and the terminal duct. In the final urine, the K + concentration is three times higher and the Na concentration 10% higher than in the external medium: urine hypertonicity is explained by the sodium and potassium concentrations.

Energetics and molecular biology of active transport in bacterial membrane vesicles

Bacterial membrane vesicles retain the same sidedness as the membrane in the intact cell and catalyze active transport of many solutes by a respiration-dependent mechanism that does not involve the generation of utilization of ATP or other high-energy phosphate compounds. In E. coli vesicles, most of these transport systems are coupled to an electrochemical gradient of protons (deltamuH+, interior negative and alkaline) generated primarily by the oxidation of D-lactate or reduced phenazine methosulfate via a membrane-bound respiratory chain. Oxygen or, under appropriate conditions, fumarate or nitrate can function as terminal electron acceptors, and the site at which deltamuH+ is generated is located before cytochrome b1 in the respiratory chain. Certain (N-dansyl)aminoalkyl-beta-D-galactopyranosides (Dns-gal) and N(2-nitro-4-azidophenyl)aminoalkyl 1-thio-beta-D-galactopyranosides (APG) are competitive inhibitors of lactose transport but are not transported themselves. Various fluorescence techniques, direct binding assays, and photoinactivation studies demonstrate that the great bulk of the lac carrier protein (ca. 95%) does not bind ligand in the absence of energy-coupling. Upon generation of a deltamuH+ (interior negative and alkaline), binding of Dns-gal and APG-dependent photoinactivation are observed. The data indicate that energy is coupled to the initial step in the transport process, and suggest that the lac carrier protein may be negatively charged.