A stimulus-activated conductance in isolated taste epithelial membranes.

Membrane vesicles isolated from the cutaneous taste epithelium of the catfish were incorporated into phospholipid bilayers on the tips of patch pipettes. Voltage-dependent conductances were observed in approximately 50% of the bilayers and single-channel currents having conductances from 8 to greater than 250 pS were recorded. In 40% of the bilayers displaying no voltage-dependent conductances, micromolar concentrations of L-arginine, a potent stimulus for one class of catfish amino acid taste receptors, activated a nonselective cation conductance. The L-arginine-gated conductance was concentration-dependent, showing half-maximal activation in response to approximately 15 microM L-arginine. L-Arginine-activated channels had unitary conductances of 40-50 pS and reversed between -6 and +18 mV with pseudointracellular solution in the pipette and Ringer in the bath. L-Alanine, a potent stimulus for the other major class of catfish amino acid taste receptors, did not alter bilayer conductance. D-Arginine, which is a relatively ineffective taste stimulus for catfish but a good cross-adapter of the L-arginine-induced neural response, had no effect on bilayer conductance at concentrations below 200 microM. However, increasing concentrations of D-arginine from 1 to 100 microM progressively suppressed the L-arginine-activated conductance, suggesting that D-arginine competed for the L-arginine receptor, but did not activate the associated cation channel. This interpretation is consonant with recent biochemical binding studies in this system. These results suggest that L-arginine taste receptor proteins in the catfish are part of or closely coupled to cation-selective channels which are opened by L-arginine binding.     

A rapid method for the evaluation of the ionic permeabilities across epithelial cell membranes

This short note presents a recipe for the calculation of the ionic permeabilities across epithelial cell membranes. The method requires the Goldman-Hodgkin-Katz formalism as well as the consideration of the equivalent electrical circuit for an epithelial cell. The equivalent electrical circuit is solved in terms of the equivalent electromotive forces coupled in series with the ionic resistances of both cell membranes (apical and basolateral). The present procedure is feasible for any leaky epithelial cell membrane with the condition that this membrane (apical or basolateral) does not contain primary or secondary mechanisms for active transport.     

Electrogenic proton transport in epithelial membranes

Summary Certain polar epithelial cells have strong transport capacities for protons and can be examinedin vitro as part of an intact epithelial preparation. Recent studies in the isolated turtle bladder and other tight urinary epithelia indicate that the apical membranes of the carbonic anhydrase-containing cell population of these tissues contain an electrogenic proton pump which has the characteristics of a proton-translocating ATPase. The translocation of protons is tightly coupled to the energy of ATP hydrolysis. Since the pump translocates protons without coupling to the movement of other ions, it may be regarded as an ideal electrogenic pump. The apparent simplicity of the functional properties has led to extensive studies of the characteristics of this pump and of the cellular organization of the secondary acid-base flows in the turtle bladder. Over a rather wide range of electrochemical potential gradients for protons ( ) across the epithelium, the rate of H⁺ transport is nearly linear with . The formalisms of equivalent circuit analysis and nonequilibrium thermodynamics have been useful in describing the behavior of the pump, but these approaches have obvious limitations. We have attempted to overcome some of these limitations by developing a more detailed set of assumptions about each of the transport steps across the pump complex and to formulate a working model for proton transport in the turtle bladder that can account for several otherwise unexplained experimental results. The model suggests that the real pump is neither a simple electromotive force nor a constant current source. Depending on the conditions, it may behave as one or the other.     

Aquaporins of plasma membranes of epithelial cells

The early 90s have brought us a discovery of a new class of membrane proteins--aquaporins with a function of transmembrane water channels. Being genetically closed proteins aquaporins are members of a large family of channel-forming proteins called MIPs (major intrinsic proteins). All aquaporins, except AQP4, are mercury-sensitive. Many aquaporins have been cloned and identified. Polyclonal antibodies grown against some of them promoted numerous studies of aquaporin localization and distribution in animal and plant tissues. Up to the present, 10 and 2 aquaporins have been described in mammalian and amphibian epithelial tissues, respectively. One of described aquaporins, AQP2, whose localization is confined to kidney collecting duct principal cells, has been found to be a hormone-depending water channel. The insertion of apical vesicles bearing AQP2 was shown to be regulated by vasopressin, meanwhile all other aquaporins are inserted into the plasma membrane constitutively. There is a vast evidence showing that the integrity of microtubules is necessary for both pathways of aquaporin insertion. AQP2 is important for normal kidney functioning and AQP2 mutations cause water-balance disorders. On the contrary, the AQP1 mutations are not accompanied by any evident clinical pathology. This review is focused on a discussion of the data so far available on aquaporin distribution in different animal tissues.     

Performance optimization of polysulfone ultrafiltration membranes for riboflavin separation using design experiments

Polysulfone membranes containing different concentrations of polyethylene glycol (M.Wt: 600 Da) as additive was prepared using N,N-dimethyl formamide as solvent. The parameters chosen for the study of separation of vitamin B₂ were concentrations of additive and feed and solvent evaporation time. Box-Behnken design methods and analysis of variance have been applied to the experimental separation of vitamin B₂ by polysulfone membranes. Box-Behnken design with three levels of additive concentration (2.5, 6.25 and 10 wt%), feed concentration (0.002, 0.005 and 0.008%) and solvent evaporation time (10, 20 and 30 s) is used for the identification of significant effects and interaction for the batch studied. Second order polynomial regression model which was used for analysis of the experiment made significant effect. The experimental values are in good correlation with predicted values, and the correlation coefficient was found to be 0.9194.     

Voltage-clamp experiments on oxidized cholesterol membranes modified with excitability-inducing material and comparison with a model

The conductance of oxidized cholesterol membranes modified with excitability-inducing material was observed in membranes containing either single conductance channels or 100–1000 channels. Membranes containing single channels have several conductance states for each voltage polarity, and the current through membranes containing many channels decays with at least two, and probably three, time constants following a step change in voltage (voltage-clamp). The time constants differ by about an order of magnitude. The multi-state behavior seems more pronounced in membranes made from highly oxidized cholesterol. Although a given conductance state could occur at either positive or negative voltages, each state was much more frequent at one polarity or the other. The most frequently observed single-channel conductance states in 0.1 M NaCl are about 0.3, 0.1, 0.03, 0.0 nΩ^-1 for negative voltages and 0.25, 0.05, 0.03, and 0.0 nΩ^-1 for positive voltages. The current following a voltage clamp decays to a quasi-steady state within 1 min for positive voltages and 1–15 min for negative voltages. When the holding voltage is −20 mV, the decay constants and quasi-steady state conductances as functions of clamping voltage are reasonably well described by either a three-state model of the conductance or a two-state model applied independently at negative and positive voltages. However, for high voltages, the quasi-steady state does not appear to approach a state in which all the channels are in a low conductance state.     

Fluorescent and dispersion experiments on biological membranes under micro-gravity

Almost all biological processes, especially those involved in signal reception and signal transduction, depend on the physical and physiological properties of biological membranes. It has been shown, that neuronal tissue and the speed of the action potential (AP) which is the basic neuronal unit of all nervous activity, is sensitive to changes in gravity as well as to other weak external forces. We strongly suppose the membrane to be the most important factor in gravitational responses although it is very difficult to observe the effects of gravity changes on these fragile thermodynamic systems. Therefore we developed two different experiments to measure the structural changes and the lateral membrane tension of spheroid cells under microgravity.     

Studies on Box-Behnken design experiments: cellulose acetate-polyurethane ultrafiltration membranes for BSA separation

Ultrafiltration membranes were prepared from mixtures of cellulose acetate-polyurethane blend membranes. During the last 1 or 2 decades, the concentration purification and separation of Albumin by ultrafiltration through semipermeable membranes have been put into practice and hence membrane separation is considered as the unit operation. The blend solution was prepared from cellulose acetate and polyurethane in polar solvent in presence of polyvinylpyrrolidone as additive. The performance of modified blend membranes applied for Bovine Serum Albumin (BSA) separation by ultrafiltration technique using Box-Behnken design with three variables: additive, time and pressure. Three different levels was studied to identify a significant correlation between the effect of these variables on the amount of separation of BSA. The methodology identifies the principal experimental variables, which have the greatest effect on the separation process. The experimental values are in good agreement with predicted values, the correlation coefficient was found to be 0.9871.     

A model for dispersion experiments

Summary A new model is proposed for the dispersion of animals and other organisms and its use is discussed for the analysis of the data from experiments on dispersion. The model is a generalisation of the random walk model, but because of its flexibility it should be much more widely applicable than the random walk model.The new model has been found to fit the results of many dispersion experiments and examples are given of its use with data for millipedes and Drosophila.     

Characterization of BADS-binding proteins in epithelial plasma membranes

Summary When a fluorescent stilbene was added to epithelial plasma membrane suspension the emission spectrum showed a broad peak containing overlapping emissions resulting from different adducts. By focusing on a specific emission wavelength a common site having a dissociation constant of 5m was calculated in the rat kidney, small intestine, pancreatic islets and shark rectal gland. This binding could be displaced by loop diuretics, (e.g., furosemide with an IC₅₀ of 40 m), DIDS (k _i 1 m) and thiocyanate. These results pose certain questions such as: (i) whether the evidence for multiple peaks are due to specific interactions representing multiple binding affinities and (ii) whether the binding of stilbene and the observed displacement can be identified on a specific protein. Separating the proteins present in the purified basolateral and brush-border membranes by SDS-PAGE, transfer of these proteins onto introcellulose paper and labeling of the nitrocellulose strips by radioactive BADS (4-benzamido-4 aminostilbene-2-2 disulphonic acid) and bumetanide could identify labeled proteins. These experiments showed that whereas some proteins bound either BADS or bumetanide, one protein with a molecular weight of 100 or 130,000 D appeared to bind both. This protein was found on the basolateral membrane in the rat kidney cortex and medulla and the shark rectal gland and in the basolateral and brush-border membranes of the small intestine. Displacement of the protein-bound stilbene by loop diuretics could not be quantitated on the nitrocellulose transfer strips for this protein. Antibodies raised against the cytoplasmic fragment of band 3 reacted with the stilbene-labeled 100–130,000 D proteins indicating sufficient immuno-cross-reactivity between the separate species. These experiments involving binding of BADS and bumetanide and cross-reactivity with the human band 3 antibody suggest that these kilodalton proteins could structurally resemble human band 3.     

Channels in epithelial cell membranes and junctions.

Epithelia may be classified as "tight" or "leaky," depending on whether there is a significant pathway for transepithelial ion permeation via the junctions and bypassing the cells. The resistance of this paracellular channel may depend partly on structures visible in the electron microscope, partly on wall charge. Permeability determinations in the leaky junctions of gallbladder epithelium, using many different organic cations, suggest that the critical barriers barriers to ion permeation are 5--8 A in radius and bind cations by up to four strongly proton-accepting oxygens. The apical cell membrane of tight epithelia contains a Na+-selective channel that is blocked by amiloride and Ca2+, subject to negative feedback control by the Na+ pump in the basolateral membrane, and somehow promoted by aldosterone. To determine the permeabilities of these two channels (the junctional channel of leaky epithelia, and the Na+ channel of tight epithelia) to water and nonelectrolytes remains a major unsolved problem.     

Bioadhesive properties of polygalacturonides against colonic epithelial membranes

Diseases of the gastrointestinal system are often related with irritations or pathological changes of mucous membranes. In an ex vivo system based on porcine colonic tissue various neutral and acidic polysaccharides were tested concerning their bioadhesive potential in order to form artificial mucin layers on colon epithelial membranes. Rhamnogalacturonans with a low degree of esterification and linear oligogalacturonids derived from pectin showed significant bioadhesion against colonic mucous membranes. In contrast highly esterified pectins and neutral polysaccharides were ineffective. Within a structure–activity relationship linear, strongly acidic homogalacturonides were shown to be most adhesive agents. Esterification, branching or non-linear backbone structures will reduce the adhesive properties. The bioadhesive effects were concentration-dependent. Polysaccharide layers, located exclusively on the apical membrane surface of colonic tissue, were visualized by fluorescent microscopy. The adhesion of the exogenous galacturonides on the tissue surface was mediated by interaction with the endogenous mucin, for the release of the endogenous mucines with a mucolytic agent resulted in a decreased bioadhesion of exogenous galacturonides. Additionally, mucin–galacturonide synergism was shown by rheological methods. The artificial mucin layers provide protective effects on colonic mucous membranes against toxic agents as shown by incubation of the tissue with TritonX-100.