Electrokinetic energy conversion studies of aqueous solutions of oxalic acid and urea across urinary bladder membranes.

Electrokinetic studies namely hydrodynamic permeability, electroosmotic permeability and streaming potential measurements of aqueous solutions of oxalic acid and urea have been made across urinary bladder membranes of goat. Energy conversion maxima and degree of coupling for these permeants have computed. It has been found that these values increase with increases in concentration of the permeants. Since electro-osmotic flux tendency is quite opposite for oxalic acid in comparison to that of urea, energy conversion values increase with increase in concentration but in opposite directions. Such studies are expected to be of use in understanding electrophysiology of the bladder as inefficient functioning of the bladder leads to formation of urinary calculi and many other types of disorders. Methodology of non-equilibrium thermodynamics have been used to explain the data.     

Electrokinetic energy conversion studies of alkaline solutions of uric acid,oxalic acid,L-cystine and L-tyrosine across urinary bladder membranes

Electrokinetic studies of alkaline solutions of oxalic acid, cystine and tyrosine across urinary bladder membranes have been made. Data have been analysed in the light of non-equilibrium thermodynamics. Maximum energy conversion efficiency (η_max), kinetic energy term (α₁) and polarization term (α₂) have been computed. It has been found that η_max., α₁, and α₂, etc., are maximum for uric acid among the permeants used. Since η_max, and α₁ and α₂ are related with membrane interface, such studies are relevant in understanding the comparative effect of permeants on bladder interface.     

Electrokinetic energy conversion studies of aqueous solution of urea, glucose and their mixtures across urinary bladder membranes

Electrokinetic studies of aqueous solutions of urea, glucose, urea-glucose mixture (urea concentration increasing and glucose fixed) and glucose-urea mixture (glucose concentration increasing and urea concentration fixed) have been carried out across urinary bladder membranes of goat. Results have been analysed using methodology of non-equilibrium thermodynamics. It has been found that energy conversion maxima and degree of coupling for mixtures is higher than urea and glucose solutions. It has also been found that in the case of urea-glucose mixtures, the value of maxima and degree of coupling first decreases and then increases with increase in concentration while in the case of glucose-urea mixture, the trend is not definite. With urea solutions only, both these values increase with increase in concentration. It has been observed that energy conversion maxima and degree of coupling for urine is much higher as compared to other permeants. It appears that second order phenomenological coefficient L112 is related with degree of coupling (qe) as the trend of two is quite similar.     

Electrokinetic energy conversion by aqueous oxalic acid, citric acid, ascorbic acid, hippuric acid, and acetyl salicylic acid across urinary bladder membranes

Efficiency of energy conversion for electro-osmosis and streaming potential and the degree of coupling of acids across urinary bladder membranes of goat have been computed using non-equilibrium thermodynamic theory. The energy conversion maxima and degree of coupling for acids responsible for the formation of urinary calculi are found to be much low as compared to urea and urine.     

The nature of urea transport across the luminal membrane of Bufo bufo urinary bladder

By using the washing-out technique, counterflow acceleration for urea was demonstrated on the luminal membrane of Bufo bufo urinary bladder, in the absence of ADH. This phenomenon completely disappears in the presence of phloretin 10-4 M on the luminal side and is consistent with the presence of a mobile carrier mechanism for urea transport across the luminal membrane, in basal conditions. In the presence of ADH, counterflow acceleration is completely absent. This result is in agreement with the presence of urea selective channels, induced by ADH, as proposed by Levine & Worthington (1976).     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na+ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal 22Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na+ flux was a linear function of mucosal Na+ concentration. Ouabain-sensitive Na+ flux and ouabain-sensitive, Na+-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na+ with the basolateral transport process is assumed to involve the interaction of some number of Na+ ions, n, with a maximal flux, MMAX, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value for MMAX of 287.8 pM cm-2 sec-1 with an intracellular Na concentration of 2.0 mM Na+ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na+-induced current, we calculate a Na+ to K+ coupling ratio of 1.40 +/- 0.07 for the transport process.     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

Summary The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na⁺ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal²²Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na⁺ flux was a linear function of mucosal Na⁺ concentration. Ouabain-sensitive Na⁺ flux and ouabain-sensitive, Na⁺-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na⁺ with the basolateral transport process is assumed to involve the interaction of some number of Na⁺ ions,n, with a maximal flux,M _max, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value forM _max of 287.8pm cm^–2 sec^–1 with an intracellular Na concentration of 2.0mm Na⁺ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na⁺-induced current, we calculate a Na⁺ to K⁺ coupling ratio of 1.40±0.07 for the transport process.     

Amide transport channels across toad urinary bladder

Summary Urea and other small amides cross the toad urinary bladder by a vasopressinsensitive pathway which is independent of somotic water flow. Amide transport has characteristics of facilitated transport: saturation, mutual inhibition between amides, and selective depression by agents such as phloretin. The present studies were designed to distinguish among several types of transport including (1) movement thought a fixed selective membrane channel and (2) movement via a mobile carrier. The former wold be characterized by co-transport (acceleration of labele amide flow in the direction of net flow in the opposite direction). Mucosal to serosal (MS) and serosal to mucosal (SM) permeabilities of labeled amides were determined in paired bladers. Unlabeled methylurea, a particularly potent inhibitor of amide movement, was added to either the M or S bath, while osmotic water flow was eliminated by addition of ethylene glycol to the opposite bat. Co-transport of labeled methylurea and, to a lesser degree, acetamide and urea with unlabeled methylurea was observed. Co-transport of the nonamides ethylene glycol and ethanol could not be demonstrated. Methylurea did not alter water permeability or transmembrane electrical resistance. The demonstration of co-transport is consistent with the presence of ADH-sensitive amide-selective channcels rather than a mobile carrier.     

Lumenal plasma membrane of the urinary bladder. II. Isolation and structure of membrane components

A technique has been devised for isolation of lumenal plasma membranes from transitional epithelial cells lining the urinary bladder in rabbits and for subsequent separation of particle-bearing plaque regions from particle-free areas of the membranes. The success of the procedures employed and their effects on the isolates were assessed by electron microscopy of conventional plastic sections, negatively stained preparations, and freeze-etch replicas. When bladders are distended with a solution of 0.01 M thioglycolic acid, which reduces sulfhydryl bridges, cytoplasmic filaments are disrupted, and large segments of the lumenal membranes rupture and float free into the lumen. A centrifugation procedure was developed for isolating a fraction enriched with the large fragments. A comparison of membranes isolated in the presence of thioglycolate with those isolated from epithelial cells homogenized in sucrose medium indicates that thioglycolate has little effect on their fine structure except for the removal of filaments which are normally associated with their cytoplasmic surface. The curved plaques of hexagonally arrayed particles and the particle-free interplaque regions, both characteristic of membranes before exposure to thioglycolate, are well preserved. Subsequent treatment of thioglycolate-isolated lumenal membranes with 1% sodium desoxycholate (DOC) severs many of the interplaque regions, releasing individual plaques in which the particles are more clearly visible than before exposure to desoxycholate. Presumably, DOC acts by disrupting the hydrophobic bonds within the membrane; therefore, this type of cohesive force probably is a major factor maintaining the structural integrity of interplaque regions. This conclusion is consistent with the observation that interplaque regions undergo freeze-cleaving like simple bilayers with a plane of hydrophobic bonding.     

Cellular lithium and transepithelial transport across toad urinary bladder

Summary Toad urinary bladders were exposed on either their mucosal or serosal surfaces, or on both surfaces, to medium in which sodium was replaced completely by lithium. With mucosal lithium Ringer's, serosal sodium Ringer's, short-circuit current (SCC) declined by about 50 percent over the first 60 min and was then maintained over a further 180 min. Cellular lithium content was comparable to the sodium transport pool. With lithium Ringer's serosa, SCC was abolished over 60 to 120 min whether the mucosal cation was sodium or lithium. Measurements of cellular ionic composition revealed that the epithelial cells gained lithium from both the mucosal and serosal media. With lithium Ringer's mucosa and serosa, cells lost potassium and gained lithium and a little chloride and water, but these changes in cellular ions could not account for the current flow across the tissue under these conditions, which must, therefore, have been carried by a transepithelial movement of lithium itself. The inhibition by serosal lithium of SCC was overcome by exposure of the mucosal surface of the bladders to amphotericin B. Thus it reflected, predominantly, an inhibition of lithium entry to the cells across the apical membrane. It is suggested that this inhibition is a consequence of cellular lithium accumulation.     

Transport-related modulation of the membrane properties of toad urinary bladder epithelium.

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.     

Thermodynamic analysis of nonelectrolyte permeation across the toad urinary bladder

Summary Permeability coefficients (P's) and apparent activation energies (E _a s) for nonelectrolyte permeation across the toad urinary bladder have been analyzed in terms of the thermodynamics of partition between membrane lipids and water. Particular attention has been paid to the contributions made by –CH₂– and –OH groups: on the average, the addition of one –CH₂– group to a molecule increasesP fourfold, while the addition of one –OH group reducesP 500-fold. Using these changes inP, we have calculated the incremental free energies (F), enthalpies (H), and entropies (S) for partition, hydration, and solution in membrane lipids. The results for toad bladder have been compared and contrasted with those extracted from the literature for red blood cells, lecithin liposomes, and bulk phase lipid solvents. The partition of –CH₂– groups into toad bladder and red cell membranes is dominated by entropy effects, i.e., a decrease in entropy of the aqueous phase that pushes the group out of water, and an increase in entropy of the membrane lipid that pulls the group into the membrane. This process resembles that in frozen liposome membranes. In melted liposomes and bulk lipid solvents the free energy of solution in the lipid is controlled by enthalpy of solution. Partition of –OH groups in all systems is governed by hydrogen bonding between the –OH group and water. However, the solution of the –OH group in toad bladder membranes is complex, and processes such as dimer and tetramer formation in the lipid phase may be involved. The results presented in this and the previous paper are discussed in terms of the structure of phospholipid bilayer membranes. Attention is drawn to the possible role of structural defects in the quasi-crystalline structure of the lipid (so-called 2gl kinks) in the permeation of small molecules such as water, urea, methanol and acetamide.     

Pathways for bicarbonate transfer across the serosal membrane of turtle urinary bladder: Studies with a disulfonic stilbene

Summary Bicarbonate is transferred across the serosal (S) membrane of the epithelial cells of the turtle bladder in two directions. Cellular HCO ₃ ^– generated behind the H⁺ pump moves across this membrane into the serosal solution. This efflux of HCO ₃ ^– is inhibited by SITS (4-isothiocyano-4-acetamido-2,2-disulfonic stilbene). When HCO ₃ ^– is added to the serosal solution it is transported across the epithelium in exchange for absorbed Cl^–. This secretory HCO ₃ ^– flow traverses the serosal cell membrane in the opposite direction. In this study the effects of serosal addition of 5×10^–4 m SITS on HCO ₃ ^– secretion and Cl^– absorption were examined. The rate of H⁺ secretion was brought to zero by an opposing pH gradient, and 20mm HCO ₃ ^– was added toS. HCO ₃ ^– secretion, measured by pH stat titration, was equivalent to the increase inMS Cl^– flux after HCO ₃ ^– addition. Neither theSM flux of HCO ₃ ^– nor theMS flux of Cl^– were affected by SITS. In the absence of electrochemical gradients, net Cl^– absorption was observed only in the presence of HCO ₃ ^– in the media; under such conditions, unidirectional and net fluxes of Cl^– were not altered by serosal or mucosal SITS. H⁺ secretion, however, measured simultaneously as the short-circuit current in ouabain-treated bladders decreased markedly after serosal SITS. The inhibition of the efflux of HCO ₃ ^– in series with the H⁺ pump and the failure of SITS to affect HCO ₃ ^– secretion and Cl^– absorption suggest that the epithelium contains at least two types of transport systems for bicarbonate in the serosal membrane.     

Stimulation of Na+ transport across the toad urinary bladder by p-chloromercuribenzene sulfonate.

The sulfhydryl reagent p-chloromercuribenzene sulfonate increased the ISC across substrate-replete toad urinary bladder when applied to the mucosal (apical) surface. This increase was accounted for by an increased mucosal to serosal net flux of Na+. In the absence of substrate, the rise in ISC was accompanied by an irreversible increase in tissue conductance which was not apparent in the replete preparation. These findings suggest that p-chloromercuribenzene sulfonate may be useful in marking mucosal functions associated with the Na+ transport apparatus.