Metabolic dependence of the offset of antidiuretic hormone-induced osmotic flow of water across the toad urinary bladder

Summary The elevated osmotic permeability to water induced by antidiuretic hormone (ADH) in the isolated urinary bladder of the toad is rapidly reversed by removal or washout of the ADH. This return to normal water permeability is delayed by the suppression of production of metabolic energy by any of three maneuvers: (i) low temperature (2°C); (ii) inhibition of oxidative phosphorylation (10mm azide or 0.5mm 2,4 dinitrophenol); or (iii) inhibition of glycolysis (10mm iodoacetate or 10mm 2-deoxyglucose). Moreover, exposure to cytochalasin B, 2.1×10^–5 m, either before or after initiation of the hormonal effect also delays the return of water permeability to normal following removal of ADH. When considered within constraints imposed by models which predict ADH's action on water permeability to be either via modulation of the fluidity of lipids in the membrane or via the figuration of proteins (pores) in the lipid membrane, these observations on the inhibition of the reversal of ADH stimulation of water flow are more consistent with the protein (pore) theory and place limitations on the mechanisms by which proteins in such pores can return to the resting or impermeable state.     

Nature of the water permeability increase induced by antidiuretic hormone (ADH) in toad urinary bladder and related tissues

In artificial lipid bilayer membranes, the ratio of the water permeability coefficient (Pd(water)) to the permeability coefficient of an arbitrary nonelectrolyte such as n-butyramide (Pd(n-butyramide)) remains relatively constant with changes in lipid composition and temperature, even though the individual Pd's increase more than 100- fold. I propose that this is a general rule that also holds for the lipid bilayers of cells and tissues, and that therefore if Pd(water)/Pd(solute greatly exceeds the value found for artifical lipid bilayers (where "solute" is a molecule, such as 1,6 hexanediol or n- butyramide, that crosses the cell membrane by a solubility-diffusion mechanism without the aid of a special transporting system), then water crosses the cell membrane via aqueous pores. Applying this criterion to the toad urinary bladder, we find that even in the unstimulated bladder, water probably crosses the luminal membrane primarily through small aqueous pores, and that this almost certainly the case after antidiuretic hormone (ADH) stimulation. I suggest that ADH stimulation ultimately leads either to formation (or enlargement) of pores, by the rearrangement of preexisting subunits, or to an unplugging of these pores.     

The membrane action of antidiuretic hormone (ADH) on toad urinary bladder.

Radioactive tracer and electrical techniques were used to study the transport of nonelectrolytes and sodium, respectively, across toad urinary bladders in the presence and absence of ADH. The permeability of lipophilic molecules was roughly proportional to bulk phase oil/water partition coefficients both in the presence and absence of hormone; i.e., ADH elicited a general nonselective increase in the permeation of all nine solutes tested. The branched nonelectrolyte, isobutyramide, was less permeable than its straight-chain isomer, n-butyramide, in control tissues. ADH reduced the discrimination between these structural isomers. Hydrophilic solutes permeated more rapidly than expected. In the presence of hormone, there was no change in the permeation of large hydrophilic solutes considered to move via an extracellular pathway, but there was a marked increase in the permeability of water and other small hydrophilic solutes. Collectively, these results suggest that ADH acts to increase the motional freedom or fluidity of lipids in the cell membrane which is considered to be the preferred pathway for the permeation of lipophilic and small hydrophilic molecules. At concentrations of cAMP and ADH which elicit equivalent increments in the shortcircuit current, the effects of these agents on nonelectrolyte transport and membrane electrical conductance are divergent. Such observations suggest that some membrane effects of ADH may not be directly dependent upon cAMP. ADH in the mucosal solution increased the permeability of the toad bladder when the surface charge on the outer surface of the apical membrane was screened with the polyvalent cation, La-3+. These experiments emphasize that interaction of ADH with membranes of toad urinary bladder may account for at least some effects of this hormone.     

Microviscosity of mucosal cellular membranes in toad urinary bladder: Relation to antidiuretic hormone action on water permeability

Summary The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad,Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23°C, 5mm dibutyryl cyclic 3,5-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P<0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.     

Regulation of membrane permeability by vasopressin; activation of the water permeability pathway in toad urinary bladder by N-ethyl-maleimide

1. Vasopressin induces a rapid increase in water permeability and stimulates net sodium transport in responsive epithelia through the mediation of cAMP. 2. In amphibian urinary bladder, the increase in water permeability is dependent on an intact cytoskeleton and is associated with the exocytotic insertion of tubular vesicles containing particle aggregates (the putative water channels) into the apical membrane of the granular epithelial cells. 3. In the toad bladder, mucosal addition of NEM, 0.1 mM, elicits a slow and irreversible increase in transepithelial water flow, whilst decreasing net sodium transport. 4. The hydrosmotic response to mucosal NEM is inhibited by cellular acidification, by pretreatment with cytoskeleton-disruptive drugs, and by agents that increase cytosolic calcium. 5. Mucosal NEM potentiates the hydrosmotic response to a submaximal, but not a maximal, dose of vasopressin. 6. Mucosal NEM, like vasopressin, induces both vesicle fusion and the appearance of particle aggregates at the granular cell apical surface. 7. NEM, unlike vasopressin, does not increase cellular cAMP content. 8. Mucosal NEM appears to increase transcellular water flow by activating cellular processes normally triggered by vasopressin, at a step beyond cAMP.     

Nocodazole inhibition of the vasopressin-induced water permeability increase in toad urinary bladder

Nocodazole is a synthetic antitumor drug that binds rapidly to tubulin. When this drug is applied to toad bladder prior to vasopressin stimulation it inhibits the vasopressin response. A maximum inhibition (68%) is reached with a dose level of 10 μ/ml applied one-half hour prior to vasopressin stimulation (20 mU/ml). This compares with an inhibition of 50% seen with a 3-h exposure of the tissue to colchicine (0.1 mM) prior to stimulation with vasopressin. Application of nocodazole (1 μ/ml) 3 min after hormonal stimulation shows no inhibition of the response at one-half hour past stimulation. These data support the view that microtubules are involved in the vasopressin-induced increase in water permeability in toad bladder and also indicate that this involvement is limited to the period prior to or directly after stimulation.     

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)     

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.     

Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone

Summary Antidiuretic hormone (ADH) increases the apical (external facing) membrane water permeability of granular cells that line the toad urinary bladder. In response to ADH, cytoplasmic vesicles called aggrephores fuse with the apical plasma membrane and insert particle aggregates which are visualized by freeze-fracture electron microscopy. Aggrephores contain particle aggregates within their limiting membranes. It is generally accepted that particle aggregates are or are related to water channels. High rates of transepithelial water flow during ADH stimulation and subsequent hormone removal decrease water permeability and cause the endocytosis of apical membrane and aggrephores which retrieve particle aggregates. We loaded the particle aggregate-rich endocytic vesicles with horseradish peroxidase (HRP) during ADH stimulation and removal. Epithelial cells were isolated and homogenized, and a subcellular fraction was enriched for sequestered HRP obtained. The HRP-enriched membrane fraction was subjected to a density shifting maneuver (Courtoy et al.,J. Cell Biol. 98:870, 1984), which yielded a purified membrane fraction containing vesicles with entrapped HRP. The density shifted vesicles were composed of approximately 20 proteins including prominent species of 55, 17 and 7 kD. Proteins of these molecular weights appear on the apical surface of ADH-stimulated bladders, but not the apical surface of control bladders. Therefore, we believe these density shifted vesicles contain proteins involved in the ADH-stimulated water permeability response, possibly components of particle aggregates and/or water channels.     

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.     

Effect of temperature on nonelectrolyte permeation across the toad urinary bladder

Summary The permeability of the toad urinary bladder to 22 nonelectrolytes was obtained from measurements of radioactive tracer fluxes. The permeability coefficients (P's), after suitable corrections for unstirred layers, were proportional to the olive oil/water partition coefficients for the majority of the molecules (P K_oil ^1.3). In the absence of chain branching, inductive effects, and intramolecular hydrogen bonding effects, a hydroxyl group reducedP an average 500-fold and a methylene group increasedP an average four fold. Branched chain solutes were less permeable than their straight chain isomers, and small solutes, polarand nonpolar, exhibited higher rates of permeation than expected from the relationship betweenP and K_oil. (Over the molecular size range 18–175 cc/moleP (Molecular Volume)^–2.7.) The high rates of permeation of small molecules are consistent with diffusion through a highly organized lipid structure. Large polar solutes, e.g., sucrose, appear to pass across the epithelium via an extracellular shunt pathway. The apparent activation energies (E _a ) for the permeation of 16 select molecules were obtained from permeability measurements over the temperature range 2–32°C. Linear Arrhenius plots (i. e., logP/T ^–1) were obtained for all molecules after unstirred layer corrections. In the absence of these corrections phase transitions were seen for molecules with very highP's (P>300×10^–7 cm/sec), but these are simply due to diffusion limited permeation.E _a increased by 2.5–3.6 kcals/mole with the introduction of each additional methylene group into a molecule, and decreased by up to 9 kcals/mole for the addition of a hydroxyl group. Qualitatively similar results were obtained in preliminary studies of olive oil/water partition coefficients. Arrhenius plots of the toad bladder conductance over the temperature range 2–32°C yield apparent activation energies of 4–5 kcals/mole which is identical to that found previously for leaky epithelia.     

Ultrastructural correlates of the antidiuretic hormone-dependent and antidiuretic hormone-independent increase of osmotic water permeability in the frog urinary bladder epithelium

Electron and confocal microscopy, using immunocytochemical methods, was employed to assess osmotic water permeability of the frog (Rana temporaria) urinary bladder during transcellular water transport, induced by antidiuretic hormone (ADH) or by wash-out of autacoids from serosal, ADH-free Ringer solution. The increase of osmotic water permeability of the urinary bladder was accompanied by relevant ultrastructural changes, the most remarkable being: (1) the appearance of aggregates of intramembranous particles in the apical membrane of granular cells, and the extent of the membrane area covered by the aggregates proportional to that of the water flow; (2) redistribution of actin filaments in the cytoplasm of granular cells; judging from the anti-actin label density, the number of actin filaments in the apical region of cytoplasm was reduced by 2.5–4 times compared with normal; (3) a decrease in the total electron density of the cytoplasm due to the increased water content of granular cells.     

Urea uptake and translocation in toad urinary bladder: the effect of antidiuretic hormone.

The uptake of C14-urea into everted and noneverted bladder sacs was compared, over short time periods (up to 2 min), with the transepithelial urea fluxes. This method allowed the study of the time course of urea uptake and distribution, while previously this problem was only studied in steady-state conditions. When mucosal uptake was studied no accumulation of C14-urea inside the tissue was observed, indicating that the mucosal border could be the limiting step. Comparative studies of urea and inulin uptake from the serosal side showed that urea equilibrated with the water epithelial cells in less than 30 sec. This accumulation suggested again that the mucosal border is an effective barrier for urea translocation. The kinetics of the increase in urea permeability induced by antidiuretic hormone was also studied and it was similar (T1/2:4.3 min) to the kinetics of the increase in water permeability induced by the hormone (T1/2:5.6 min). A strong parallelism was also observed between the time course of the increases in water and urea permeabilities induced by medium hypertonicity (T1/2 25 and 26 min, respectively). The values obtained for the permeability coefficient ktrans), either at rest or under ADH were similar to those previously reported employing steady-state techniques (28+/-8 and 432+/-25 cm-sec-1-10(-7), respectively).     

Urea uptake and translocation in toad urinary bladder: The effect of antidiuretic hormone

Summary The uptake of C¹⁴-urea into everted and noneverted bladder sacs was compared, over short time periods (up to 2 min), with the transepithelial urea fluxes. This method allowed the study of the time course of urea uptake and distribution, while previously this problem was only studied in steady-state conditions. When mucosal uptake was studied no accumulation of C¹⁴-urea inside the tissue was observed, indicating that the mucosal border could be the limiting step. Comparative studies of urea and inulin uptake from the serosal side showed that urea equilibrated with the water epithelial cells in less than 30 sec. This accumulation suggested again that the mucosal border is an effective barrier for urea translocation. The kinetics of the increase in urea permeability induced by antidiuretic hormone was also studied and it was similar (T1/2:4.3 min) to the kinetics of the increase in water permeability induced by the hormone (T1/2:5.6 min). A strong parallelism was also observed between the time course of the increases in water and urea permeabilities induced by medium hypertonicity (T1/2 25 and 26 min, respectively). The values obtained for the permeability coefficientk _trans), either at rest or under ADH were similar to those previously reported employing steady-state techniques (28±8 and 432±25 cm·sec^–1·10^–7, respectively).     

Ionic and metabolic requirements for the hydroosmotic response to antidiuretic hormone in toad urinary bladder

Summary A study has been conducted to determine the ionic and metabolic requirements for full expression of the hydroosmotic response to antidiuretic hormone in the toad urinary bladder. By appropriate manipulation of incubation conditions it can be shown that there is a pool of serosal sodium necessary for a full hormone response. This serosal sodium pool is not related to the transepithelial sodium transport pool. A full hydroosmotic response also requires serosal potassium; however, no specific anion requirement was demonstrated. Additionally, anaerobic or aerobic metabolism support a full hydroosmotic response equally well.     

Extracellular Ca2+ and the effect of antidiuretic hormone on the water permeability of the toad urinary bladder: An example of flow-induced alteration of flow

Summary The extracellular Ca²⁺ requirement for antidiuretic hormone (ADH) stimulation of water permeability in the toad urinary bladder has been critically examined. The polarity of the tissue was maintained with 1mm Ca²⁺ in the mucosal bathing medium and a serosal bath nominally free of Ca²⁺. Under these condition, ADH-induced osmotic water flow was inhibited by more than 60% while enhancement of the diffusional permeability to water was unaffected. Structural studies revealed that low serosal Ca²⁺ led to parallel alterations in epithelial architecture that amounted to a significant distorition of the osmotic water pathway. Prevention of these alterations, or restoration of normal cell-cell contact showed that the reduction of serosal Ca²⁺ did not restrict hormonal action,per se, but that it resulted in a weakening of cell-cell junctions such that intercellular space distension during water flow occurred to a point where the geometric conditions for maintenance of osmotic flow were compromised. We conclude that extracellular Ca²⁺ is not a requirement for the molecular aspects of ADH action but that, in its absence, a direct measurement of ADH-induced osmotic flow proves to be an inaccurate index of the hormone-generated changes in epithelial transport characteristics. Under certain conditions the ADH-effect on the tissue's hydraulic permeability is probably best assessed by measurement of the diffusional permability to water; although accuracy in this determination is difficult, it is not as strongly dependent on tissue geometry.