The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium

The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 μg per calf bladder. Low levels of 5′ nucleotidase, Mg²⁺-ATPase and ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 12 000 and 22 000. These may associate to form a 30 000 apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ complex which could represent the individual ‘particles’ of the dodecameric subunits seen by electron microscopy in the plaque regions.     

A spin label study of the urinary bladder luminal membrane

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.     

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.     

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).     

The water permeability of toad urinary bladder. I. Permeability of barriers in series with the luminal membrane

Antidiuretic hormone (ADH) induces a large increase in the water permeability of the luminal membrane of toad urinary bladder. Measured values of the diffusional water permeability coefficient, Pd(w), are spuriously low, however, because of barriers within the tissue, in series with the luminal membrane, that impede diffusion. We have now determined the water permeability coefficient of these series barriers in fully stretched bladders and find it to be approximately 6.3 X 10(- 4) cm/s. This is equivalent to an unstirred aqueous layer of approximately 400 microns. On the other hand, the permeability coefficient of the bladder to a lipophilic molecule, hexanol, is approximately 9.0 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of only 100 microns. The much smaller hindrance to hexanol diffusion than to water diffusion by the series barriers implies a lipophilic component to the barriers. We suggest that membrane-enclosed organelles may be so tightly packed within the cytoplasm of granular epithelial cells that they offer a substantial impediment to diffusion of water through the cell. Alternatively, the lipophilic component of the barrier could be the plasma membranes of the basal cells, which cover most of the basement membrane and thereby may restrict water transport to the narrow spaces between basal and granular cells.     

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.     

Eosinophil peroxidase increases membrane permeability in mammalian urinary bladder epithelium

Eosinophilperoxidase (EPO), a cationic protein found in eosinophils, has beenreported to be cytotoxic independent of its peroxidase activity. Thisstudy investigated with electrophysiological methods whether EPO istoxic to mammalian urinary bladder epithelium. Results indicate thatEPO, when added to the mucosal solution, increases apical membraneconductance of urinary bladder epithelium only when the apical membranepotential is cell interior negative. The EPO-induced conductance wasconcentration dependent, with a maximumconductance of 411 µS/cm² and aMichaelis-Menten constant of 113 nM. The EPO-induced conductance wasnonselective for K⁺ andCl. The conductance waspartially reversed using voltage but not by removal of EPO from thebulk solution. Mucosal Ca²⁺reversed the EPO-induced conductance by a mechanism involving reversible block of the conductance. Prolonged exposure (up to 1 h) toEPO was toxic to the urinary bladder epithelium, as indicated by anirreversible increase in transepithelial conductance. These resultssuggest that EPO is indeed toxic to urinary bladder epithelium via amechanism that involves an increase in membrane permeability.     

The isolation and characterization of plasma membranes from calf thymocytes.

A simple method is described for the isolation and characterization of plasma membranes from calf thymocytes. The procedure involves extraction of thymocytes in a hypotonic medium containing borate and EDTA. Membrane ghosts, obtained by centrifugation of the cell lysate, are purified by passage through a column containing glass beads. The purity of plasma membranes was checked by chemical analysis, by assay of marker enzymes and also by electron microscopy. Polyacrylamide gel electrophoresis of the calf thymocyte plasma membrane produced a number of protein bands as well as a major band which stained for carbohydrate. The method is rapid and could be applied to isolate plasma membranes from nucleated cells of various types in large quantities.     

Regulation of luminal membrane water permeability by water flow in toad urinary bladder

Intramembranous particle aggregates (presumed sites for water flow) which appear in the luminal membrane consequent to ADH treatment are derived from cytoplasmic membrane structures (now termed "aggrephores") which fuse with the luminal membrane. We have previously shown that bladders stimulated in the absence of an osmotic gradient have about twice as many aggregates and about three times as many sites of aggrephore fusion as bladders stimulated with ADH in the presence of a 175 milliosmolal gradient. The present studies show that the frequency of fused aggrephores and luminal membrane aggregates can be modified as a consequence of alterations in transmembrane water flow initiated by changing the transbladder osmotic gradient during hormone stimulation. Bladders treated with ADH for 1 hr without a gradient and then for 1 hr with a gradient had approximately 1/3 as many aggregates and fusion sites as paired bladders treated for 2 hr without a gradient. Conversely, bladders treated with ADH for 1 hr with a gradient and then for 1 hr without a gradient had approximately 2x as many aggregates and fusion sites as bladders treated for 2 hr with a gradient. In other experiments we demonstrate that the time course of hormone washout is greatly accelerated if carried out in the presence of an osmotic gradient. In paired bladders that were first stimulated with ADH for 30 min in the absence of a gradient, aggregates and fusion sites as well as osmotic water permeability determined in fixed bladders, persisted at near maximum levels for 15 min of washout in the absence of a gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation and characterization of a plasma membrane and a myelin fraction derived from oligodendroglia of calf brain.

—A method is described for the fractionation of bulk isolated oligodendroglial cells from calf brain to produce both a plasma membrane and an attached myelin fraction. The cells are homogenized in a sucrose solution containing Mg²⁺ and K+ at a pH of 6·5. Crude membrane fractions are obtained from this homogenate by discontinuous sucrose density gradient centrifugation. After being subjected to osmotic shock, these fractions are purified by continuous sucrose density gradient centrifugation. The plasma membrane fraction, which bands at 1·0 m -sucrose, was identified by its morphology and enzyme content. Electron microscopy showed it to be a homogeneous preparation of vesicles composed, for the most part, of smooth trilaminar membranes. Enzymatic analysis revealed the presence of high specific activities of Na+, K+-ATPase, 5′-nucleotidase and 2′,3′-cyclic AMPase. Lipid analysis showed a higher galactolipid and lower phospholipid content than has been reported for neuronal and synaptic membranes. The attached myelin fraction, which bands at 0·7 m -sucrose has the typical multilamellar appearance of myelin, but differs considerably from normal myelin in having high concentrations of plasma membrane marker enzymes, and a lipid composition intermediate between normal myelin and the plasma membrane fraction. The ganglioside content and protein patterns of these fractions have also been examined.     

Regular structures in membranes: the lumenal plasma membrane of the cow urinary bladder.

The ultrastructure of the lumenal plasma membrane of the cow urianry bladder has been studied in thin sections of glutaraldehyde- and glutaraldehyde-H2O2-fixed specimens, by negative staining and freeze fracture. A regular hexagonal array of particles confined to polygonal plaques 0-1-0-4-mum in diameter and separated by 0-02-mum interplaque areas is revealed by all 3 techniques. Cross-sections through particulate areas fixed with glutarayldehyde-H2O2 display a tetralaminar structure consisting of the usual approximately 8-nm-thick trilamellar unit membrane structure, on the external dense leaflet of which is located an additional approximately 4-nm-thick stratum which is occasionally resolved into a row of regulrly spaced approximately 4-nm-diameter particles. Non-particulate areas feature only the approximately 8-nm-thick trilamellar structure. Tangential sections reveal an hexagonal array of particles with a unit cell of approximately 16 nm. Four membrane faces can be revealed by freeze fracture and etching of membranes of the cow urinary bladder; 2 complementary split inner membrane faces (A and B) revealed by the cleaving process and the lumenal and cytoplasmic membrane surfaces exposed by etching. Face B, which belongs to the external membrane leaflet and faces the cytoplasm, displays plaques of particles arranged in a hexagonal lattice with a unit cell of approximately 16 nm. Face A, which belongs to the cytoplasmic membrane leaflet and faces the lumen, displays a complementary array of hexagonally packed pits. The hexagonally arranged particles also protrude into the lumenal membrane surface where they can occasionally be resolved into 6 approximately 5-nm-diameter subunits; the cytoplasmic surface appears smooth. Six approximately 5-nm-diameter subunits are also revealed in negatively stained preparations. The data are consistent with a model for the membrane in which the particles forming the hexagonal structure protrude above the lumenal membrane surface and also bridge most of the thickness of the membrane.     

Intrinsic characteristics of the proton pump in the luminal membrane of a tight urinary epithelium. The relation between transport rate and delta mu H

A number of tight urinary epithelia, as exemplified by the turtle bladder, acidify the luminal solution by active transport of H+ across the luminal cell membrane. The rate of active H+ transport (JH) decreases as the electrochemical potential difference for H+ [delta mu H = mu H(lumen) - mu H(serosa)] across the epithelium is increased. The luminal cell membrane has a low permeability for H+ equivalents and a high electrical resistance compared with the basolateral cell membrane. Changes in JH thus reflect changes in active H+ transport across the luminal membrane. To examine the control of JH by delta mu H in the turtle bladder, transepithelial electrical potential differences (delta psi) were imposed at constant acid-base conditions or the luminal pH was varied at delta psi = 0 and constant serosal PCO2 and pH. When the luminal compartment was acidified from pH 7 to 4 or was made electrically positive, JH decreased as a linear function of delta mu H as previously described. When the luminal compartment was made alkaline from pH 7 to 9 or was made electrically negative, JH reached a maximal value, which was the same whether the delta mu H was imposed as a delta pH or a delta psi. The nonlinear JH vs. delta mu H relation does not result from changes in the number of pumps in the luminal membrane or from changes in the intracellular pH, but is a characteristic of the H+ pumps themselves. We propose a general scheme, which, because of its structural features, can account for the nonlinearity of the JH vs. delta mu H relations and, more specifically, for the kinetic equivalence of the effects of the chemical and electrical components of delta mu H. According to this model, the pump complex consists of two components: a catalytic unit at the cytoplasmic side of the luminal membrane, which mediates the ATP-driven H+ translocation, and a transmembrane channel, which mediates the transfer of H+ from the catalytic unit to the luminal solution. These two components may be linked through a buffer compartment for H+ (an antechamber).     

Eosinophil major basic protein increases membrane permeability in mammalian urinary bladder epithelium

The eosinophilgranule protein major basic protein (MBP) is toxic to a wide variety ofcell types, by a poorly understood mechanism. To determine whether theaction of MBP involves an alteration in membrane permeability, wetested purified MBP on rabbit urinary bladder epithelium usingtransepithelial voltage-clamp techniques. Addition of nanomolarconcentrations of MBP to the mucosal solution caused an increase inapical membrane conductance only when the voltage across the apicalmembrane was cell interior negative. The magnitude of the MBP-inducedconductance was a function of MBP concentration, and the rate of theinitial increase in conductance was a function of the transepithelialvoltage. The MBP-induced conductance was nonselective forK⁺ andCl. MucosalCa²⁺ reversed the inducedconductance, whereas mucosal Mg²⁺partially blocked the induced conductance and slowed the rate of theincrease in conductance. The induced conductance was partially reversedby changing the voltage gradient across the apical membrane to cellinterior positive. Prolonged exposure resulted in an irreversible lossof the barrier function of the urinary bladder epithelium. Theseresults suggest that an increase in cell membrane ion permeability isan initial step in MBP-induced loss of barrier function.

     

Adenylate cyclase and phosphodiesterase in transitional epithelium of the urinary bladder.

Adenylate cyclase activities in cell-free preparations of isolated transitional epithelium from rabbit urinary bladders were shown to be stimulated by epinephrine, prostaglandin E₁ (PGE₁), 5-guanylyl imidodiphosphate (GMP-PNP), and NaF. ACTH, aldosterone, insulin, glucagon, oxytocin, parathyroid hormone and vasopressin were without effect at the concentrations tested. The effects of epinephrine, PGE₁, and GMP-PNP appeared to be additive. Essentially all of the adenylate cyclase activity was particulate, while approximately 70% of the cyclic nucleotide 3':5'-phosphodiesterase activity was soluble. Single reciprocal plots of the phosphodiesterase data revealed non-linear kinetics.