Transepithelial Na+ transport and the intracellular fluids: A computer study

Summary Computer simulations of tight epithelia under three experimental conditions have been carried out, using the rheogenic nonlinear model of Lew, Ferreira and Moura (Proc. Roy. Soc. London. B 206:53–83, 1979) based largely on the formulation of Koefoed-Johnsen and Ussing (Acta Physiol. Scand.42:298–308, 1958). First, analysis of the transition between the short-circuited and open-circuited states has indicated that (i) apical Cl^– permeability is a critical parameter requiring experimental definition in order to analyze cell volume regulation, and (ii) contrary to certain experimental reports, intracellular Na⁺ concentration (c _Na ^c ) is expected to be a strong function of transepithelial clamping voltage. Second, analysis of the effects of lowering serosal K⁺ concentration (c _K ^s ) indicates that the basic model cannot simulate several well-documented observations; these defects can be overcome, at least qualitatively, by modifying the model to take account of the negative feedback interaction likely to exist between the apical Na⁺ permeability andc _Na ^c . Third, analysis of the effects induced by lowering mucosal Na⁺ concentration (c _Na ^m ) strongly supports the concept that osmotically induced permeability changes in the apical intercellular junctions play a physiological role in conserving the body's stores of NaCl. The analyses also demonstrate that the importance of Na⁺ entry across the basolateral membrane is strongly dependent upon transepithelial potential,c _Na ^m andc _K ^s ; under certain conditions, net Na⁺ entry could be appreciably greater across the basolateral than across the apical membrane.     

Membrane transport models with fast and slow reactions: General analytical solution for a single relaxation

Summary Membrane transport models are usually expressed on the basis of chemical kinetics. The states of a transporter are related by rate constants, and the time-dependent changes of these states are given by linear differential equations of first order. To calculate the time-dependent transport equation, it is necessary to solve a system of differential equations which does not have a general analytical solution if there are more than five states. Since transport measurements in a complex system rarely provide all the time constants because some of them are too rapid, it is more appropriate to obtain approximate analytical solutions, assuming that there are fast and slow reaction steps. The states of the fast steps are related by equilibrium constants, thus permitting the elimination of their differential equations and leaving only those for the slow steps. With a system having only two slow steps, a single differential equation is obtained and the state equations have a single relaxation. Initial conditions for the slow reactions are determined after the perturbation which redistribute the states related by fast reactions. Current and zero-trans uptake equations are calculated. Curve fitting programs can be used to implement the general procedure and obtain the model parameters.     

A General Model for the Dynamics of the Cell Volume

The conservation of the cell volume within values compatible with the overall cell functions represents an ubiquitous property, shared by cells comprising the whole biological world. Water transport across membranes constitutes the main process associated to the dynamics of the cell volume, its chronic and acute regulations therefore represent crucial aspects of cell homeostasis. In spite of the biological diversity, the dynamics of the cell volume exhibits common basic features in the diverse types of cells. The purpose of this study is to show that there is a general model capable to describe the basic aspects of the dynamics of the cell volume. It is demonstrated here that the steady states of this model represent asymptotically stable configurations. As illustrations, several cases of non-polarized (i.e., symmetrical) and polarized (e.g., epithelial) cells performing water transport are shown here to represent particular cases of the general model. From a biological perspective, the existence of a general model for the dynamics of the cell volume reveals that, in spite of physiological and morphological peculiarities, there is a basic common design of the membrane transport processes. In view of its stability properties, this basic design may represent an ancestral property that has proven to be successful regarding the overall homeostatic properties of cells.     

Claudins and ki-67: potential markers to differentiate low- and high-grade transitional cell carcinomas of the urinary bladder

Updated classification of urothelial cell cancer differentiates low-grade and high-grade cancers, which determines potential clinical outcome. Substantial interobserver variability necessitates new biomarkers to ensure classification. Claudins' specific expression pattern characterizes normal tissues, different tumor types, and defined grades of tumor differentiation. The aim of this study was to examine the expression pattern of claudins and proliferation marker Ki-67 in low-grade and high-grade urothelial cell cancers compared with independent control samples of non-tumorous urothelium, as well as to reveal the predictive usefulness of claudins. The expression of claudins-1, -2, -3, -4, -5, -7, and -10 and Ki-67 was studied with quantitative immunohistochemistry and real-time RT-PCR with relative quantification in 103 samples: 86 urothelial cell cancers (27 low grade, 59 high grade) and 17 non-tumorous urothelia. Results were analyzed regarding overall survival and recurrence-free period as well. High-grade tumors overall showed significantly higher claudin-4 and Ki-67 and significantly lower claudin-7 expression when compared with low-grade ones. High-grade tumors revealed significantly shorter overall survival in Kaplan-Meier analysis. Claudin-4, claudin-7, and Ki-67 might be used as potential markers to differentiate low-grade and high-grade urothelial cell cancers, thereby possibly enhancing accuracy of pathological diagnosis and adding further information to clinical outcome.     

A network thermodynamic method for numerical solution of the Nernst-Planck and Poisson equation system with application to ionic transport through membranes

Simple techniques of network thermodynamics are used to obtain the numerical solution of the Nernst-Planck and Poisson equation system. A network model for a particular physical situation, namely ionic transport through a thin membrane with simultaneous diffusion, convection and electric current, is proposed. Concentration and electric field profiles across the membrane, as well as diffusion potential, have been simulated using the electric circuit simulation program, SPICE. The method is quite general and extremely efficient, permitting treatments of multi-ion systems whatever the boundary and experimental conditions may be.     

Surface hydrophobicity and water transport of the toad urinary bladder: Effects of vasopressin

Summary The present study investigated whether the hydrophobic properties (wettability) of the luminal surface of the toad urinary bladder might play a role in modulating water transport across this epithelium. In the absence of vasopressin (ADH), water transport across the tissue was low, while luminal surface hydrophobicity (water contact angle) was relatively high. Following stimulation by ADH, water transport increased and surface hydrophobicity decreased. The addition of indomethacin to inhibit ADH-induced prostaglandin synthesis did not reduce these actions of ADH. In an attempt to alter water transport in this tissue, a liposomal suspension of surface-active phospholipids was administered to the luminal surface. This addition had no detectable influence on the low basal rates of water transport, but blocked the ADH-induced stimulation of water transport. We suggest that surface-active phospholipids on the toad bladder luminal membrane may contribute to the hydrophobic characteristics of this tissue. ADH may act to decrease surface hydrophobicity, facilitating the movement of water molecules across an otherwise impermeable epithelium. This surface alteration may be associated with the appearance of water channels in the apical membrane.     

A method for the enumeration of bacterial adhesion to epithelial cells using image analysis

Abstract Computerised image analysis was utilised to enumerate the attachment of Staphylococcus epidermidis to HEp2 cell monolayers. A differential staining technique was employed such that individual staphylococcal cells stood out in sharp contrast against the uneven cell surface and granular contents of the epithelial cells. The primary image analysis operation involved subtracting an out-of-focus image from an in-focus image of the bacteria on the monolayer, thereby accentuating the bacterial image. Enumeration, using a particle counting routine, was rapid and reproducible, facilitating counting in excess of 700 bacteria per field at ×500 magnification. The computerised programme compared favourably with manual counting and would provide a rapid, objective and morphologically discriminatory method for evaluating bacterial attachment to various tissues.     

Differential effects of aldosterone and ADH on intracellular electrolytes in the toad urinary bladder epithelium

Summary Quantitative electron microprobe analysis was employed to compare the effects of aldosterone and ADH on the intracellular electrolyte concentrations in the toad urinary bladder epithelium. The measurements were performed on thin freeze-dried cryosections utilizing energy dispersive x-ray microanalysis. After aldosterone, a statistically significant increase in the intracellular Na concentration was detectable in 8 out of 9 experiments. The mean Na concentration of granular cells increased from 8.9±1.3 to 13.2±2.2 mmol/kg wet wt. A significantly larger Na increase was observed after an equivalent stimulation of transepithelial Na transport by ADH. On average, the Na concentration in granular cells increased from 12.0±2.3 to 31.4±9.3 mmol/kg wet wt (5 experiments). We conclude from these results that aldosterone, in addition to its stimulatory effect on the apical Na influx, also exerts a stimulatory effect on the Na pump. Based on a significant reduction in the Cl concentration of granular cells, we discuss the possibility that the stimulation of the pump is mediated by an aldosterone-induced alkalinization.Similar though less pronounced concentration changes were observed in basal cells, suggesting that this cell type also participates in transepithelial Na transport. Measurements in mitochondria-rich cells provided no consistent results.     

Amiloride stimulation of sodium transport in the presence of calcium and a divalent cation chelator

Amiloride in nM to μM concentrations stimulates the short circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) of the toad urinary bladder by as much as 120% when applied in conjunction with apical Ca²⁺ and a divalent cation chelator. A significant decrease in transepithelial resistance ($\text{R}{}_{\mathrm{<mtext>t</mtext>}}$) is observed simultaneously. This response is spontaneously reversible and its amplitude is dependent upon apical sodium concentrations. The stimulated $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ persisted when acetazolamide (1 mM) was introduced, HPO₄^2? substituted for HCO₃^? or SO₄^2? replaced Cl^?. Consequently, the increase in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ is not due to the change of Cl^?, H⁺ or HCO₃^? flux. This behavior in a ‘tight’ epithelium may be related to the mechanism controlling apical sodium permeability.     

A rapid method for generating sufficient amounts of uniform genotype-specific material from the woody perennial grapevine for ion transport studies

Experimentation with woody perennials may be difficult due to slow plant growth and a lack of sufficient amounts of uniform plant material. In this study, we sought to determine whether rooted leaves could be used as a substitute for whole plants in ion accumulation studies. Grapevine leaves are particularly amenable for rooting since their petioles are of sufficient length for dipping in rooting hormone and for holding the leaf above the soil surface. To determine whether rooted leaves would be useful for salinity experiments, we investigated the ion uptake characteristics of rooted leaves derived from a backcross population that differed in Cl^– accumulation. Long-term ion accumulation studies conducted over several weeks and short-term radioactive uptake studies conducted over several hours were performed. The data showed that the Cl^– content of rooted grapevine leaves from different genotypes grown at 25 and 50 mM NaCl was similar to data reported by others. Short-term radioactive uptake assays did not always reveal differences in uptake between the genotypes. Therefore, we suggest that rooted leaves under certain conditions may provide a space-efficient method for generating sufficient amounts of plant material. This material could be used for studying whole plant, molecular and electrophysiological aspects of ion transport and for conducting experiments where root material from specific genotypes is required.     

Serial estimates of body water content and distribution during the first postnatal month in baboons

Water content of the various body compartments was estimated serially during the first postnatal month in six term baboon neonates. In absolute volumes (mL), total body water, intracellular water, and plasma volume all increased linearly with body weight and postnatal age, although the effect of weight was predominant. In proportion to body weight (mL/kg), total body water, intracellular water, and plasma volume increased linearly with postnatal age whereas extracellular water and red cell volume decreased. There was no linear relation between the proportions of any of the water volumes and body weight.     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.     

An improved numerical method for strong coupling of excitation and contraction models in the heart

Quantifying the interactions between excitation and contraction is fundamental to furthering our understanding of cardiac physiology. To date simulating these effects in strongly coupled excitation and contraction tissue models has proved computationally challenging. This is in part due to the numerical methods implemented to maintain numerical stability in previous simulations, which produced computationally intensive problems. In this study, we analytically identify and quantify the velocity and length dependent sources of instability in the current established coupling method and propose a new method which addresses these issues. Specifically, we account for the length and velocity dependence of active tension within the finite deformation equations, such that the active tension is updated at each intermediate Newton iteration, within the mechanics solution step. We then demonstrate that the model is stable and converges in a three-dimensional rod under isometric contraction. Subsequently, we show that the coupling method can produce stable solutions in a cube with many of the attributes present in the heart, including asymmetrical activation, an inhomogeneous fibre field and a nonlinear constitutive law. The results show no instabilities and quantify the error introduced by discrete length updates. This validates our proposed coupling framework, demonstrating significant improvement in the stability of excitation and contraction simulations.     

Modeling the Individual Growth of Tetrahymena Sp. and its Population Consequences

ABSTRACT. Three types of mathematical growth models are presented to describe the individual growth of the ciliate Tetrahymena sp. feeding on the bacterium Pseudomonas fluorescens . Both organisms were isolated from a domestic waste-water treatment plant. Growth of individual ciliates and the consequences for the whole population are considered. Experimental data, obtained by following the individual ciliate during its lifespan from cell division to cell division, are used for parameter estimations. Differences between growth models for individuals turn out to have little effect on the specific population growth rate and the mean cell volume. In case of exponential growth of individuals the unstructured and structured population models are equivalent, even in time-variant environments. This knowledge can be applied in the stability analysis of food chains or forced systems. The results obtained facilitates quantification of protozoa biomass as a function of bacterial biomass in chemostats. More specifically, it highlights the dynamic behaviour of bacteria and protozoa in waste-water treatment plants.     

Characteristics of the volume- and chloride-dependent K transport in human erythrocytes homozygous for hemoglobin C

Summary In human red cells homozygous for hemoglobin C (CC), cell swelling and acid pH increase K efflux and net K loss in the presence of ouabain (0.1mm) and bumetanide. We report herein, that K influx is also dependent on cell volume in CC cells: cell swelling induces a marked increase in the maximal rate (from 6 to 18 mmol/liter cell × hr) and in the affinity for external K (from 77±16mm to 28±3mm) of K influx. When the external K concentration is varied from 0 to 140mm, K efflux from CC and normal control cells is unaffected. Thus, K/K exchange is not a major component of this K movement. K transport through the pathway of CC cells is dependent on the presence of chloride or bromide; substitution with nitrate, acetate or thiocyanate inhibits the volume- and pH-dependent K efflux. When CC cells are separated according to density, a sizable volume-dependent component of K efflux can be identified in all the fractions and is the most active in the least dense fraction. N-ethylmaleimide (NEM) markedly stimulates K efflux from CC cells in chloride but not in nitrate media, and this effect is present in all the fractions of CC cells separated according to density. The persistence of this transport system in denser CC cells suggests that not only cell age, but also the presence of the positively charged C hemoglobin is an important determinant of the activity of this system. These data also indicate that the K transport pathway of CC cells is not an electrodiffusional process and is coupled to chloride.     

Atrial natriuretic peptide and cGMP activate sodium transport through PKA-dependent pathway in the urinary bladder of the Japanese tree frog

The effects of atrial natriuretic peptide (ANP) and cGMP on transepithelial ion transport were examined in the urinary bladder of the Japanese tree frog, Hyla japonica, using Ussing chamber voltage-clamp and whole-cell patch-clamp techniques. When the bladders were exposed to 4.4×10⁻¹¹ to 10⁻⁶ M ANP or 10⁻⁷ to 3×10⁻⁴ M 8-Br-cGMP, both the transepithelial potential difference (PD) and the short-circuit current (Isc) were significantly increased in a concentration-response manner. The cGMP-dependent responses were inhibited in a Na⁺-free bath solution and in the presence of amiloride. The cGMP-dependent increases in Isc were significantly inhibited by specific PKA inhibitors (5×10⁻⁷ M KT-5720 and >10⁻⁵ M H-89), but not by a specific PKG inhibitor (5×10⁻⁷ M KT-5823). ANP-dependent increases in Isc were also significantly inhibited by KT-5720. In the patch-clamp study, ANP and cGMP significantly increased in inward currents involving Na⁺ uptake. These results suggest that a cross-talk mechanism exists between cAMP and cGMP signaling pathways, which leads to Na⁺ transport in the frog urinary bladder. In addition, the cGMP-dependent increases in Isc were partially inhibited by 10⁻⁴ M l-cis-diltiazem, a specific inhibitor of cyclic nucleotide-gated (CNG) channels. These results also suggest a relation between CNG channels and the cGMP-dependent increases in Na⁺ absorption of the frog urinary bladder.     

Proliferation of anchorage-dependent contact-inhibited cells. II: experimental results and validation of the theoretical models

This study establishes that the cellular automata models developed in an earlier article capture the essential features of the proliferation process for anchorage-dependent contact-inhibited cells. Model predictions are in excellent agreement with experimental data obtained with bovine pulmonary artery endothelial cells. The models are particularly suitable for predictive purposes since they have no adjustable parameters. All model parameters can be easily obtained from a priori measurements. Our studies also show that proliferation rates are very sensitive to the spatial distributions of seed cells. The adverse effects of seeding heterogeneities become more pronounced as a cell population approaches confluency and they should be accounted for in experimental studies attempting to assess the response of cells to external stimuli.     

A new technique for estimating rates of carboxylation and electron transport in leaves of C3 plants for use in dynamic global vegetation models

The possible responses of the terrestrial biosphere to future CO₂ increases and associated climatic change are being investigated using dynamic global vegetation models (DG VMs) which include the Farquhar et al. (1980) biochemical model of leaf assimilation as the primary means of carbon capture. This model requires representative values of the maximum rates of Rubisco activity, V_max, and electron transport, J_max, for different vegetation types when applied at the global scale. Here, we describe an approach for calculating these values based on measurements of the maximum rate of leaf photosynthesis (A_max) ¹³C discrimination. The approach is tested and validated by comparison with measurements of Rubisco activity assayed directly on wild-type and transgenic Nicotiana tabacum (tobacco) plants with altered Rubisco activity grown under ambient and elevated CO₂ mole fractions with high and low N-supply. V_max and J_max values are reported for 18 different vegetation types with global coverage. Both variables were linearly related reinforcing the idea of optimal allocation of resources to photosynthesis (light harvesting vs. Rubisco) at the global scale. The reported figures should be of value to the further development of vegetation and ecosystem models employing mechanistic DGVMs.     

General rate equations and rejection criteria for the rapid equilibrium carrier model of cotransport

It is demonstrated that under fixed activator conditions the general flux equation for the rapid equilibrium carrier model of cotransport can be written entirely in terms of five independent kinetic constants. Thus the kinetic parameters from any experiment carried out under the same activator conditions must necessarily be expressible in terms of these five constants. These predicted relationships between experimental kinetic parameters provide rejection criteria for the model, a number of which are derived here. Generalization of the treatment to the case where a competitive substrate is present on both sides of the membrane is also given.     

Coupling satellite data with in situ measurements and numerical modeling to study fine suspended-sediment transport: a study for the lagoon of New Caledonia

This paper investigates the potential of remotely sensed data to map turbidity in a coral reef lagoon and to calibrate a numerical model of fine suspended-sediment transport. Simultaneous measurements of turbidity depth-profile and above-water spectral reflectance integrated according Landsat 7 ETM+ band 2 spectral sensitivity provide a linear regression relationship for the southwest lagoon of New Caledonia (r²=0.95, n=40). This relationship is applied to an empirically atmospherically corrected Landsat ETM+ image of the lagoon acquired on October 23, 2002. A comparison between Landsat estimates of turbidity and concurrent measurements at 14 stations indicates that the mean standard error in the satellite-estimated turbidity is 17.5%. The numerical model introduced in Douillet et al. (2001) is used to simulate the transport of fine suspended sediments in the lagoon in October 2002. A calibration of the erosion rate coefficient required by the model is proposed using in situ turbidity profiles and the remotely sensed turbidity field. In situ data are used to tune locally the erosion rate coefficient, while satellite data are used to determine its spatial zonation. We discuss necessary improvements in coupled studies of fine-sediment transport in coastal zones, namely relationships between turbidity and sediment concentration, integration of wave influence in the model, and correction of bottom reflection in satellite data processing.