Vacuolar Symplast as a Regulated Pathway for Water Flows in Plants

Indirect immunofluorescent microscopy and a tonoplast-specific marker enzyme were used to demonstrate the occurrence of pyrophosphatase within the plasmodesmata in the elongation zone of maize root segments. The pulsed field gradient NMR method (PFG NMR) was applied to study restricted self-diffusion of water molecules in the root segments under normal conditions and after the inhibition of respiration with sodium azide (10 mM NaN₃, 30 min). The results led to the conclusion that vacuoles in the root segments examined are interconnected into a unified intercellular continuum and that intervacuolar connections are formed by desmotubules within the plasmodesmata. The water permeability of the vacuolar symplast appears to be controlled by an ATP-dependent process. The experimental data can provide a methodological approach to studying water permeability of the vacuolar symplast with the PFG NMR technique.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 372–377.Original Russian Text Copyright © 2005 by Velikanov, Volobueva, Belova, Gaponenko.     

Regulation of Water Permeability of Vacuolar Symplast

Effect of exogenous ABA and an inhibitor of energy metabolism NaN₃ on water permeability of the desmotubules and tonoplast as the structural elements of vacuolar symplast ensuring water permeability of this transport system was investigated. The methodological approach based on the use of NMR with magnetic field pulse gradient is described in detail. It was shown that ABA affects water permeability of the vacuolar symplast in the root cells of maize (Zea mays L.) seedlings by temporary increase in water permeability of its membrane (tonoplast) and does not modify water permeability of desmotubules. At the same time, the effect of sodium azide is related to the disturbance of water permeability in the latter, and this evidence is corroborated by the additivity in the effects of the two above-mentioned agents on diffusion decay of spin echo produced by vacuolar symplast water molecules. ABA effect was detected only at high exogenous concentrations (10^?4 M). The effect of ABA on water permeability of the tonoplast did not depend on or was weakly related to intracellular concentration of ATP, whereas the open state of desmotubules was ATP-dependent. Observations were made on the role of aquaporins in the ABA influence on tonoplast water permeability and the physiological role of high ABA concentrations.     

Nonexponential Diffusion Extinction of Spin Echo from Water Protons in Wheat Root

The method of NMR spin echo with the magnetic field pulse gradient was used for studying self-diffusion of water molecules in the radial root direction. It was shown on the basis of physiologopharmacological methods of modification of the object that the coefficients of water self-diffusion resulting from computer decomposition of nonexponential diffusion extinction of the relative echo amplitude in the root to components are related to the subcellular and supercellular organization (structure) of the root and reflect changes in water permeability of the two transport channels of plasmodesmas.     

ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT

Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow.     

Studies on water transport through the sweet cherry fruit surface: IX. Comparing permeability in water uptake and transpiration

Water uptake and transpiration were studied through the surface of intact sweet cherry (Prunus avium L.) fruit, exocarp segments (ES) and cuticular membranes (CM) excised from the cheek of sweet cherry fruit and astomatous CM isolated from Schefflera arboricola (Hayata) Hayata, Citrus aurantium L., and Stephanotis floribunda Brongn. leaves or from Lycopersicon esculentum Mill. and Capsicum annuum L. var. annuum Fasciculatum Group fruit. ES and CM were mounted in diffusion cells. Water (deionized) uptake into intact sweet cherry fruit, through ES or CM interfacing water as a donor and a polyethyleneglycol (PEG 6000, osmotic pressure 2.83 MPa)-containing receiver was determined gravimetrically. Transpiration was quantified by monitoring weight loss of a PEG 6000-containing donor (2.83 MPa) against dry silica as a receiver. The permeability coefficients for osmotic water uptake and transpiration were calculated from the amount of water taken up or transpired per unit surface area and time, and the driving force for transport. Permeability during osmotic water uptake was markedly higher than during transpiration in intact sweet cherry fruit (40.2-fold), excised ES of sweet cherry fruit (12.5- to 53.7-fold) and isolated astomatous fruit and leaf CM of a range of species (on average 23.0-fold). Partitioning water transport into stomatal and cuticular components revealed that permeability of the sweet cherry fruit cuticle for water uptake was 11.9-fold higher and that of stomata 56.8-fold higher than the respective permeability during transpiration. Increasing water vapor activity in the receiver from 0 to 1 increased permeability during transpiration across isolated sweet cherry fruit CM about 2.1-fold. Permeability for vapor uptake from saturated water vapor into a PEG 6000 receiver solution was markedly lower than from liquid water, but of similar magnitude to the permeability during self-diffusion of ³H₂O in the absence of osmotica. The energy of activation for self-diffusion of water across ES or CM was higher than for osmotic water uptake and decreased with increasing stomatal density. The data indicate that viscous flow along an aqueous continuum across the sweet cherry fruit exocarp and across the astomatous CM of selected species accounted for the higher permeability during water uptake as compared to self-diffusion or transpiration.     

State of water and its diffusion across lipid bilayers: effect of hydration degree

The state of adsorbed water (estimated from the dependence of the shape of the 1H NMR spectrum on the angle between the normal to the bilayers and the direction of the magnetic field) and the diffusion of water molecules in the direction of the normal to the bilayers (estimated by 1H NMR spectroscopy with the impulse gradient of magnetic field) in microscopically oriented dioleoylphosphatidylcholine bilayers have been studied depending on hydration. The dependences of the shape of the NMR spectrum on angle differ qualitatively only at concentrations of water greater and less than the concentration that is achieved upon hydration from saturated vapors chi(eq) (about 23 weight %). At concentrations below chi(eq), all water present in samples enters the hydrate shells of polar "heads" of lipids or is in the state of "rapid exchange" with the water of hydrate shells, with the result that the signal of spin echo for water is observed only in a narrow range of angles close to the "magic angle", 54 degrees C. At concentrations above xhi(eq), the signal of spin echo for water is retained at all orientations, indicating probably that part of water between the bilayers ("quasi-free water") is in the state of a "slow exchange" with water "bound" to polar "heads". It was found that the coefficient of self-diffusion of water across the system of bilayers inversely depends on the degree of hydration, which is described in the Tanner model with consideration of the self-diffusion of water molecules in the hydrophobic moiety of the bilayer. The permeability of the bilayer, the coefficient of distribution of molecules between the water and lipid phases, and the coefficient of self-diffusion of water in the hydrophobic moiety of the bilayer were estimated.     

Water state and diffusion through lipid bilayers: Effect of hydration degree

The dependences of adsorbed water state (obtained from the variations in ¹H NMR spectra with the angle between the bilayer normal and magnetic field direction) and water diffusion along the bilayer normal (measured using pulsed field gradient ¹H NMR) on hydration degree have been studied in macroscopically oriented bilayers of dioleoylphosphatidylcholine. The angle dependences of the shape of NMR spectrum are qualitatively different only for water concentrations higher and lower than that achieved by hydration from saturated vapors (χ_eq, about 23%). At concentrations lower than χ_eq, all water in the sample either makes the hydration shells of the lipid polar heads or is in fast exchange with the shell water, so the spin-echo signal from water is detected only within a narrow range of angles close to the magic angle, 54.7°. At concentration exceeding χ_eq, the spin-echo signal from water is retained at all orientations, suggesting that a portion of water between bilayers (quasi-free water) slowly exchanges with water bound to the polar heads. There is an inverse dependence of the coefficient of water self-diffusion through the bilayer system on the hydration degree, which is described in the Tanner model with account of water self-diffusion in the hydrophobic part of the bilayer. Bilayer permeability, distribution coefficient of molecules between aqueous and lipid phases, and water self-diffusion coefficient in the hydrophobic region of the bilayer are estimated.     

Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion.

A specific effect of Cu2+ eliciting selective changes in the permeability of intact Saccharomyces cerevisiae cells is described. When 100 microM CuCl2 was added to a cell suspension in a buffer of low ionic strength, the permeability barrier of the plasma membranes of the cells was lost within 2 min at 25 degrees C. The release of amino acids was partial, and the composition of the amino acids released was different from that of those retained in the cells. Mostly glutamate was released, but arginine was mainly retained in the cells. Cellular K+ was released rapidly after CuCl2 addition, but 30% of the total K+ was retained in the cells. These and other observations suggested that Cu2+ caused selective lesions of the permeability barrier of the plasma membrane but did not affect the permeability of the vacuolar membrane. These selective changes were not induced by the other divalent cations tested. A novel and simple method for differential extraction of vacuolar and cytosolic amino acid pools by Cu2+ treatment was established. When Ca2+ was added to Cu2+-treated cells, a large amount of Ca2+ was sequestered into vacuoles, with formation of an inclusion of a Ca2+-polyphosphate complex in the vacuoles. Cu2+-treated cells also showed enhanced uptake of basic amino acids and S-adenosylmethionine. The transport of these substrates showed saturable kinetics with low affinities, reflecting the vacuolar transport process in situ. With Cu2+ treatment, selective leakage of K+ from the cytosolic compartment appears to create a large concentration gradient of K+ across the vacuolar membrane and generates an inside-negative membrane potential, which may provide a driving force of uptake of positively charged substances into vacuoles. Cu2+ treatment provides a useful in situ method for investigating the mechanisms of differential solute pool formation and specific transport phenomena across the vacuolar membrane.     

Water self-diffusion behavior in yeast cells studied by pulsed field gradient NMR

The water self-diffusion behavior in yeast cell water suspension was investigated by pulsed field gradient NMR techniques. Three types of water were detected, which differ according to the self-diffusion coefficients: bulk water, extracellular and intracellular water. Intracellular and extracellular water self-diffusion was restricted; the sizes of restriction regions were approximately 3 and 15-20 microm, respectively. The smallest restriction size was determined as inner cell size. This size and also cell permeability varied with the growth phase of yeast cell. Cell size increased, but permeability decreased with increasing growth time. The values of cell permeabilities P(1)(d) obtained from time dependence of water self-diffusion coefficient were in good agreement with the permeabilities obtained from the exchange rate constants P(1)(eff). The values of P(1)(eff) were 7 x 10(-6), 1.2 x 10(-6) and 1.6 x 10(-6) m/s, and P(1)(d) were 6.3 x 10(-6), 8.4 x 10(-7), 1.5 x 10(-6) m/s for yeast cells incubated for 9 h (exponential growth phase), 24 h (end of exponential growth phase), and 48 h (stationary growth phase), respectively.     

A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.     

Transport of potassium ions across planar lipid membranes by the antibiotic,grisorixin: I. The equilibrium state and self-diffusion K+ fluxes

Summary ⁴²K⁺ tracer flux and steady-state conductance measurements were carried out with bilayer lipid membranes containing grisorixin, a carboxylic polyether antibiotic. When the membranes are placed between two bulk aqueous solutions of identical composition, the exchange or self-diffusion transmembrane flux of potassium is measured by a method which allows the characterization of the bilayer K⁺ permeability at the equilibrium state. The K⁺ self-diffusion flux increases with the pH in the range pH 6 to pH 9 and reaches a constant value for values above 9. This can be directly related to the increase of the surface concentration of the 11 complex formed by K⁺ and the deprotonated polyether at both bilayer membrane interfaces. The transport model initially proposed by Pressman and coworkers (Proc. Natl. Acad. Sci. USA 58:1949–1956, 1967) is again taken into consideration in the quantitative analysis of the flux data. The transmembrane transport of K⁺ results from the translocation of its neutral complex with grisorixin and the association-dissociation of the antibiotic with either potassium or conditions by a translocation process of the acidic grisorixin. Using the data of some previous studies for mixed ionophorelipid monolayers at the air/water interface and the present results for the self-diffusion flux measurements, it was possible to propose an evaluation of the more important parameters characterizing the transport; namely, the total surface concentration of grisorixin, the interfacial pK and the translocation rate constant of its potassium neutral complex. The method proposed could be extended easily to other carboxylic polyethers, which would lead to an interesting comparison of their ionophoric properties using model membrane systems.     

The effect of temperature on membrane hydraulic conductivity

The objective of this study was to use the temperature dependence of water permeability to suggest the physical mechanisms of water transport across membranes of osmotically slowly responding cells and to demonstrate that insight into water transport mechanisms in these cells may be gained from easily performed experiments using an electronic particle counter. Osmotic responses of V-79W Chinese hamster fibroblast cells were measured in hypertonic solutions at various temperatures and the membrane hydraulic conductivity was determined. The results were fit with the general Arrhenius equation with two free parameters, and also fit with two specific membrane models each having only one free parameter. Data from the literature including that for human bone marrow stem cells, hamster pancreatic islets, and bovine articular cartilage chondrocytes were also examined. The results indicated that the membrane models could be used in conjunction with measured permeability data at different temperatures to investigate the method of water movement across various cell membranes. This approach for slower responding cells challenges the current concept that the presence of aqueous pores is always accompanied by an osmotic water permeability value, P(f)>0.01 cm/s. The possibility of water transport through aqueous pores in lower-permeability cells is proposed.     

The self-diffusion of water in Artemia cysts

The self-diffusion coefficient of water molecules has been measured by nuclear magnetic resonance in cysts of Artemia over a wide range of hydration. Compared to the value for bulk water, the diffusion coefficient is reduced by a factor of 7 at the highest hydration and by approximately 100 at the lowest hydration. The results are used to evaluate various models that have been proposed to account for the reduction of water self-diffusion coefficients in complex systems.     

The blood-brain barrier in a reptile, Anolis carolinensis

An electron microscopic study was made of the ultrastructure and permeability of the capillaries in the cerebral hemispheres of the lizard, Anolis carolinensis. The brain of Anolis is vascularized by a loop-type pattern consisting exclusively of arteriovenous capillary loops. The ultrastructure of the endothelium and the arrangement of the various layers from the capillary lumen to the central nervous tissue is similar to that of mammals. The endothelial cells form a continuous layer around the lumen and are joined by tight interendothelial junctions. The basal lamina of the endothelium is also continuous and encloses pericyte processes. The cells of the nervous tissue rest directly on the basal lamina of the capillary and are separated from each other by a 200 Å space. Intravenously injected horseradish peroxidase (MW 40,000) and ferritin (MW 500,000) were used to study the permeability of the capillaries. The entry of horseradish peroxidase and ferritin into the intercellular spaces of the brain is restricted by the tightness of the interendothelial junctions. No vesicular transport of either tracer occurs; however, ferritin does enter the endothelial cells in vacuoles. No tracer molecules are present in the basal lamina, pericytes, or nervous tissue. The different responses of the endothelial cell to the tracers used in this study suggest that endocytotic activities of endothelial cells involve different processes. Vacuoles formed by marginal folds, vacuoles formed by endothelial surface projections or deep invaginations of the plasma membrane, 600–800 Å vesicles, and coated vesicles all seem to differ in the nature of the substances which they endocytose.     

GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi‐enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non‐exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter‐mediated pathway and the vesicle trafficking‐mediated pathway. No molecules involved in the vesicle trafficking‐mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan‐colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis‐sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome‐like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9‐mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.     

Mechanical stabilization of desiccated vegetative tissues of the resurrection grass Eragrostis nindensis: does a TIP 3;1 and/or compartmentalization of subcellular components and metabolites play a role?

During dehydration, numerous metabolites accumulate in vegetative desiccation-tolerant tissues. This is thought to be important in mechanically stabilizing the cells and membranes in the desiccated state. Non-aqueous fractionation of desiccated leaf tissues of the resurrection grass Eragrostis nindensis (Ficalho and Hiern) provided an insight into the subcellular localization of the metabolites (because of the assumptions necessary in the calculations the data must be treated with some caution). During dehydration of the desiccant-tolerant leaves, abundant small vacuoles are formed in the bundle sheath cells, while cell wall folding occurs in the thin-walled mesophyll and epidermal cells, leading to a considerable reduction in the cross-sectional area of these cells. During dehydration, proline, protein, and sucrose accumulate in similar proportions in the small vacuoles in the bundle sheath cells. In the mesophyll cells high amounts of sucrose accumulate in the cytoplasm, with proline and proteins being present in both the cytoplasm and the large central vacuole. In addition to the replacement of water by compatible solutes, high permeability of membranes to water may be critical to reduce the mechanical strain associated with the influx of water on rehydration. The immunolocalization of a possible TIP 3;1 to the small vacuoles in the bundle sheath cells may be important in both increased water permeability as well as in the mobilization of solutes from the small vacuoles on rehydration. This is the first report of a possible TIP 3;1 in vegetative tissues (previously only reported in orthodox seeds).     

Roles of Aquaporin-3 Water Channels in Volume-Regulatory Water Flow in a Human Epithelial Cell Line

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl₂) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.     

A mathematical model of solute coupled water transport in toad intestine incorporating recirculation of the actively transported solute

A mathematical model of an absorbing leaky epithelium is developed for analysis of solute coupled water transport. The non-charged driving solute diffuses into cells and is pumped from cells into the lateral intercellular space (lis). All membranes contain water channels with the solute passing those of tight junction and interspace basement membrane by convection-diffusion. With solute permeability of paracellular pathway large relative to paracellular water flow, the paracellular flux ratio of the solute (influx/outflux) is small (2-4) in agreement with experiments. The virtual solute concentration of fluid emerging from lis is then significantly larger than the concentration in lis. Thus, in absence of external driving forces the model generates isotonic transport provided a component of the solute flux emerging downstream lis is taken up by cells through the serosal membrane and pumped back into lis, i.e., the solute would have to be recirculated. With input variables from toad intestine (Nedergaard, S., E.H. Larsen, and H.H. Ussing, J. Membr. Biol. 168:241-251), computations predict that 60-80% of the pumped flux stems from serosal bath in agreement with the experimental estimate of the recirculation flux. Robust solutions are obtained with realistic concentrations and pressures of lis, and with the following features. Rate of fluid absorption is governed by the solute permeability of mucosal membrane. Maximum fluid flow is governed by density of pumps on lis-membranes. Energetic efficiency increases with hydraulic conductance of the pathway carrying water from mucosal solution into lis. Uphill water transport is accomplished, but with high hydraulic conductance of cell membranes strength of transport is obscured by water flow through cells. Anomalous solvent drag occurs when back flux of water through cells exceeds inward water flux between cells. Molecules moving along the paracellular pathway are driven by a translateral flow of water, i.e., the model generates pseudo-solvent drag. The associated flux-ratio equation is derived.     

Water transport in aquaporins: osmotic permeability matrix analysis of molecular dynamics simulations

Single-channel osmotic water permeability (p(f)) is a key quantity for investigating the transport capability of the water channel protein, aquaporin. However, the direct connection between the single scalar quantity p(f) and the channel structure remains unclear. In this study, based on molecular dynamics simulations, we propose a p(f)-matrix method, in which p(f) is decomposed into contributions from each local region of the channel. Diagonal elements of the p(f) matrix are equivalent to the local permeability at each region of the channel, and off-diagonal elements represent correlated motions of water molecules in different regions. Averaging both diagonal and off-diagonal elements of the p(f) matrix recovers p(f) for the entire channel; this implies that correlated motions between distantly-separated water molecules, as well as adjacent water molecules, influence the osmotic permeability. The p(f) matrices from molecular dynamics simulations of five aquaporins (AQP0, AQP1, AQP4, AqpZ, and GlpF) indicated that the reduction in the water correlation across the Asn-Pro-Ala region, and the small local permeability around the ar/R region, characterize the transport efficiency of water. These structural determinants in water permeation were confirmed in molecular dynamics simulations of three mutants of AqpZ, which mimic AQP1.     

Specific vacuolation of frog urinary bladder granular cell after ADH stimulation of water transport

The present study deals with an analysis of specific traits of cell vacuolation induced by water flow and ADH. During incubation of frog urinary bladders in Ringer's solution diluted 2-fold, the water content of the bladder wall increased by an average of 19%. In case of ADH-stimulated water flow the water content increased by an average of 15.7%. Cell swelling induced by hypotonic conditions on the serosal side resulted in a drastic decrease of the response to the hydroosmotic action of ADH. Electron microscopy revealed significant differences between cells hydrated in the above conditions. Two-fold hypotonicity of the serosal solution caused a slight swelling of all types of cells accompanied by a narrowing of intercellular spaces. With ADH stimulation of water transport (at maximal water movement) granular cells were characterized by the presence of irregularly shaped giant vacuoles with processes. The limiting membranes of the vacuoles were closely connected with microtubules and microfilaments. The electron microscopic study of these cells by the freeze-substitution method revealed, in addition to giant vacuoles, a highly complex system of microtubules 35-40 nm in diameter. A morphological similarity was observed between the vacuolar systems of these granular cells and the contractile vacuole complex of protozoans. Possible mechanisms for the participation of giant vacuoles, electron-dense canaliculi, microtubules and microfilaments in transcellular water flow across epithelium are discussed.