Antidiuretic hormone-dependent membrane capacitance and water permeability in the toad urinary bladder

Antidiuretic hormone (ADH) increased the electrical capacitance of apical membrane of the toad bladder; this effect was modulated by the osmotic gradient across the tissue. Capacitance was measured from the transepithelial voltage response to constant-current pulses using bladders depolarized with KCl-sucrose serosal solution to reduce basolateral resistance and with Na-free mucosal solution to increase apical membrane resistance. Addition of ADH (20 mU/ml) increased capacitance by 28 +/- 9% (mean +/- SD) in the absence and by 8 +/- 3% in the presence of an osmotic gradient (200 mosM, mucosal side hypotonic). With bladders stimulated in the absence of an osmotic gradient, rapidly imposing a gradient resulted in a peak rate of water flow that declined to 40% of the peak value after 15-20 min. ADH-dependent capacitance also decreased with a similar time course. Removal of ADH reversed the capacitance change (t1/2 = 10-15 min), but the reversal was slower than the decline in water flow to basal levels (t1/2 less than 5 min). Colchicine and cytochalasin B also inhibited the ADH-induced capacitance increase. The capacitance change was also inhibited when the mucosal solution was made hypertonic with raffinose. The results are interpreted within the framework of a previously proposed model of ADH-stimulated water transport in which cytoplasmic vesicular structures fuse with the apical plasma membrane.     

The effect of hormones and of an osmotic gradient on the structure and properties of mammalian foetal urinary bladder in vitro.

1. Water and isotope fluxes were measured by incubating urinary bladders of foetal pigs and sheep in vitro in the presence and absence of a concentration and osmotic gradient. The structure of the urinary bladder of foetal pigs under various conditions was studied by electron microscopy. Its ultrastructure was found to be closely similar to that of foetal sheep. 2. Antidiuretic hormone (ADH) (0-2 U. ml-1) enhanced the enlargement of intercellular spaces caused by dilute mucosal medium in pig bladders; prolactin (1 u. ml-1) prevented osmotic dilatation of the intercellular spaces. 3. The hydraulic conductivity, Lp, was estimated to be 0-5 X 10 (-7) cm.s-1atm-1 in sheep and pigs at about 100 days gestation; the ratio of osomotic to diffusional permeability, (LpRT/VW)/PD, in the presence and absence of ADH, was 2-1 and 1-6 respectively. These are similar to the values found in fish gills. 4. Prolactin reduced bulk flow of water to zero in seven out of eight bladders investigated. Incubation with ADH or vasotocin (55 mu. ml-1) in the presence of prolactin restored water flux to 22% and 45% of control values respectively. 5. There was no significant net flux of sodium from mucosa to serosa in pig bladder except in the presence of prolactin. No net flux of sodium occurred from mucosal to serosal side of pig or sheep bladders in the presence of an adverse electrochemical gradient, although in sheep the permeability ratio was significantly greater than one. 6. The diffusional flux ratio for water remained unity under all conditions; vasotocin increased unidirectional fluxes and prolactin reduced them. The flux ratios were unaffected by the direction of bulk fluid flow, probably because diffusion was rapid compared to flow: the ratio of diffusional flux to volume flow was between 11 and 18.     

Does the gradual hydroosmotic response to antidiuretic hormone depend on intracellular cAMP accumulation or on the formation of intramembrane particle aggregates?

In experiments on frog urinary bladder the mechanisms behind the gradual development of a hydroosmotic reaction to antidiuretic hormone (ADH) were investigated. It was suggested that the velocity of hydroosmotic reaction may be limited by (a) formation and insertion of particle aggregates into the apical membrane or (b) by velocity of cAMP formation. The urinary bladders were exposed to 23 nM ADH for different times (from 1 to 20 min) and water flow was measured over a period of 40 min. It was found that the value of the full hydroosmotic response increased progressively with the time of exposure to the hormone; however, the enhancement of water flow was equal during each time interval before reaching the reaction maximum. A direct correlation between the value of ADH-stimulated water flow, cAMP content in bladder tissue and frequency of particle aggregates in the granular cell apical membrane was observed. The content of cAMP in ADH-treated bladders was higher by 80% in the absence than in the presence of an osmotic gradient. Pretreatment of urinary bladders with 50 M cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, significantly accelerated the development of the hydroosmotic reaction and increased the magnitude of water flow in comparison with the effect of ADH only. No changes in cyclic AMP phosphodiesterase activity were found in the urinary bladder homogenates under the action of ADH, so it seems likely that accumulation of cAMP depends only on the increase of adenylate cyclase activity. The data obtained allow one to conclude that the gradual hydroosmotic response to ADH depends on the accumulation of cAMP, which may be considered as the main limiting factor of the velocity of the hydroosmotic reaction.     

Tetracycline-induced inhibition of Na+ transport in the toad urinary bladder.

The effects of three tetracyclines, demethylchlortetracycline (DMC), minocycline (MNC), and oxytetracycline (OTC), on Na+ transport (measured as short-circuit current) were examined in toad urinary bladders mounted in modified Ussing chambers. During a 1-h incubation period serosal DMC (but not MNC or OTC) inhibited basal Na+ transport, whereas MNC (but not DMC or OTC) inhibited ADH-stimulated Na+ transport. MNC also inhibited cyclic AMP-stimulated Na+ transport. During longer incubation periods all three drugs inhibited basal Na+ transport. The DMC-induced inhibition of basal Na+ transport and the MNC-induced inhibition of ADH-stimulated Na+ transport were paralleled by an inhibition of the active conductance of the bladders. Thus, although all three drugs inhibit basal Na+ transport, only MNC inhibits ADH-stimulated Na+ transport. This effect does not correlate with the known effects of the tetracyclines on ADH-stimulated water flow or with drug-protein binding, and may be related to the greater lipid solubility of MNC.     

Effects of prolonged saline exposure on water, sodium and urea transport and on electron-microscopical characteristics of the isolated urinary bladder of the toad Bufo bufo

1. A comparison was made of various transport properties and electron-microscopical characteristics of isolated urinary bladders from toads (Bufo bufo) maintained in either tap water or 0.7% saline (0.7 g NaCl in 100 ml. H(2)O) for 10 days to 2 months.2. In the absence of Pitressin, isolated bladders from saline-adapted toads showed:(a) markedly, and significantly, lower osmotic water flow;(b) moderately, but not significantly, lower urea permeability;(c) no significant change in net sodium transport (measured as short-circuit current, I(sc)); and(d) significantly smaller intercellular space/mucosal cell ratios in electron-micrographs.3. Differences in the transport and electron-microscopical characteristics between bladders from water-exposed and saline-adapted toads became more evident in the presence of exogenous Pitressin (10 m-u./ml. serosal solution):(a) the stimulating influence of Pitressin on osmotic water flow, short-circuit current and urea permeability was considerably smaller in bladders from saline-adapted toads than in those from water-exposed toads;(b) the influence of Pitressin on short-circuit current was reduced more profoundly than that on either water flow or urea permeability;(c) the Pitressin-induced increment in intercellular space/mucosal cell ratio was significantly smaller in electron-micrographs of bladders from saline-adapted toads than in those from water exposed toads.4. The effects of saline adaptation are discussed in relation to decreased permeability of mucosal membrane barriers.     

Activation energy for water transport in toad bladder

Activation energies (Ea) for water movement across vasopressin-(ADH) sensitive epithelia have been reported to be about 10 kcal/mol (1, 12). The present study shows that measurements of Ea for osmotic water flow across toad bladders are unreliable, because a temperature change induces marked alterations in membrane permeability to water within a 2.5-min interval. Thus bladders equilibrated with ADH either at room temperature or at 33 degrees C and then suddenly subjected to a lower temperature were found to exhibit a marked increase in membrane permeability to water. This observation suggests that there is a rapid turn-over of water permeability sites and that sudden exposure to cold inhibits the removal more than the induction of sites by ADH. To stabilize ADH-induced water channels for Ea measurements, bladders were exposed to ADH at room temperature, fixed with glutaraldehyde, and subjected to osmotic gradients at different temperatures. The Ea values for osmotic water flow across these ADH-permeabilized, glutaraldehyde-fixed bladders were 5.1 (4-12 degrees C), 4.3 (12-21 degrees C), 3.6 (21-36 degrees C), and 3.6 kcal/mol (30-38 degrees C). Ea values for shear viscosity of water in these temperature ranges were calculated to be 4.7, 4.2, 4.1, and 3.6 kcal/mol, respectively. The close correlation between Ea values for bulk water viscosity and osmotic water flow across the bladder wall suggests that an equivalent number of hydrogen bonds must be broken to achieve an increase in water flow through ADH-induced channels and an increase in fluidity of water in bulk solution.     

Relation of ADH effects to altered membrane fluidity in toad urinary bladder

Membrane fluidity, urea permeability, and osmotic water permeability in toad urinary bladder are regularly enhanced by antidiuretic hormone (ADH). In addition, organized intramembranous particle aggregates, which correlate specifically with hormonally stimulated water permeability, are found in granular cell luminal membranes consequent to ADH stimulation. In this investigation ADH-stimulated changes in urea and osmotic water permeability and luminal membrane aggregates at room temperature (24.8 +/- 0.4 degrees C) and in the cold 10.6 +/- 0.2 degrees) were compared with corresponding changes in membrane fluidity, as assessed by n-butyramide permeability. Although a critical level of membrane fluidity is undoubtedly required, the occurrence of aggregates in the luminal membrane is independent of an accompanying hormonally induced change of membrane fluidity. ADH-stimulated osmotic water permeability in toad bladder is also independent of the coincident change in membrane fluidity, and as a process almost certainly involves membrane channels, not a solubility-diffusion process through membrane lipids. For ADH-stimulated transbladder urea movement, channels seem to be involved as well, and the change induced in membrane fluidity by ADH could be an underlying factor in their formation.     

Ca2+- and H+-dependent effects of crude bacterial phospholipase C on the hydroosmotic response of toad urinary bladder to serosal hypertonicity

Phospholipase C (EC 3.1.4.3.) from Clostridium perfringens (crude extracts) was used to study the role of phospholipids in the osmotic permeability of the urinary bladder of the toad. When added to the serosal bath (430 mU/ml) it inhibited the effects of antidiurectic hormone (ADH) and exogenous cyclic AMP. Under the same conditions the increase in osmotic flow produced by serosal hypertonicity (SH) was slightly enhanced by the lipase. The hydroosmotic effect of SH was greatly potentiated by the lipase by decreasing 10-fold the Ca²⁺ concentration. The SH-induced flow was inhibited by the lipase if the Ca²⁺ or the H⁺ concentration was increased 10-fold, but not if the increase in positive charges was produced by a concentration of Mg²⁺. Phospholipase C had no effect on the action of either ADH or SH if added to the mucosal bath. Serosal neuraminidase or phospholipase A₂ could not mimic the effect of phospholipase C on SH. The effect of phospholipase C on the response to SH was not modified if fatty acid-free bovine serum albumin was added to the bath. Therefore, the release of products of lipolysis into the bath do not seem to be responsible for the effects of phospholipase C on SH-induced water flow. The results suggest that the effects of the enzyme on the composition and rearrangement of lipids at the basolateral membrane produce modifications of the water flow. Ca²⁺ and H⁺ may modify the enzyme-substrate interaction, suggesting that different phospholipids may be differentially involved in the control of water permeability of the basolateral membrane. Changes in Ca²⁺ and H⁺ concentration may also activate or suppress different lipolytic enzymes of the nonhomogenous enzymatic complex employed. These results support the idea that ADH and SH converge in a final common site of action in order to increase the water permeability of the apical membrane, but they do so by activating different steps, SH acting mainly at a post-cyclic AMP site.     

Fluid movements across rabbit ileum coupled to passive paracellular ion movements.

1. Theophylline (10 mM) and choleragen (1 x 10(-6) g ml.-1) abolish net fluid absorption by everted sacs of rabbit ileum. Triaminopyrimidine (20 mM) and ethacrynate (0.1 mM) prevent this inhibition of net fluid movement. Replacing Ringer Cl- with isethionate prevents the theophylline-dependent decrease in fluid absorption also. 2. Ouabain (0.1 mM) abolishes net fluid movements in both control and theophylline-treated tissue. 3. With ouabain present, hypertonic NaCl (200 mM) in the mucosal solution causes net fluid secretion (serosal-mucosal flux). With theophylline added to both the mucosal and serosal solution, net fluid absorption (mucosal-serosal flux) is observed (P less than 0.001). Triaminopyrimidine (20 mM), or ethacrynate (0.1 mM), or replacement of Ringer Na+ with choline, or Ringer Cl- with isethionate all prevent the theophylline-induced reversal of osmotic flow. 4. Theophylline increases passive net flux of Na+ and Cl- from mucosal solution containing hypertonic (200 mM) NaCl+ ouabain (0.1 mM) across sheets of ileum into serosal solution containing mannitol Ringer + ouabain. The increased passive Na+ flux is blocked by triaminopyrimidine and the increased Na+ and Cl- fluxes are blocked by ethacrynate (0.1 mM). 5. The suggested route of increased NaCl leakage is via the paracellular pathway as it is inhibited by triaminopyrimidine. The increase, itself, is a consequence of the increased passive permeability of the mucosal border to Cl-, induced by theophylline or choleragen. Water is apparently electro-osmotically coupled to the paracellular Na+ leakage (100 mole water mole-1 Na+), hence increased passive leakage reverses osmotic flow. In active tissue the lateral intercellular space contains hypertonic NaCl, and hence increased leakage of NaCl across the tight-junction in theophylline or choleragen-treated tissue gives rise to net fluid secretion.     

Water handling in the rat jejunum: effects of acidification of the medium

The minute-by-minute net water movement (J _w) in the rat jejunum was studied in relation to the diffusive water (P _w) and mannitol (P _s) permeabilities with the following results, (a) J _w was a linear function of the applied hydrostatic and osmotic transepithelial gradients (hydrostatic permeability coefficient, P _hydr=0.052±0.011 cm s^–1; osmotic permeability coefficient, P _osm= 0.0069±0.0014 cm s^–1. (b) A fraction of this absorptive J _w (transport-associated J _w, J _wt=0.086±0.024 l min^–1 cm^–2) was independent of the presence of any osmotic, hydrostatic or chemical gradient. (c) In the absence of Na⁺, J _wt was not significantly different from zero and there was an increase in P _hydr but no change in P _osm. (d) In the presence of a hydrostatic gradient (10 cm H₂O, mucosal side), acidification of the medium (95% CO₂ bubbling, pH 6.2) simultaneously and reversibly increased J _w and decreased P _w. (e) When an osmotic gradient was present (40 mM polyethyleneglycol on the serosal side) a net increase in J _w was observed. CO₂ bubbling in these conditions reversibly reduced J _w while increasing P _s. (f) These effects were not observed when the serosal or mucosal pH was reduced in the presence of a nonpermeant buffer (HEPES/TRIS; MES/TRIS). If we accept that P _s is a good marker of paracellular movements and that P _w mainly reflects transcellular water movements, we may conclude that acidification of the medium, in the presence of bicarbonate, modifies both paracellular and transcellular routes. The experimental evidence indicates that an increase in proton concentration opens the paracellular pathway and probably has a blocking effect on a transcellular route.     

Requirement of Na+ and K+ for the action of antidiuretic hormone on water permeability

Previous reports have shown that in the toad bladder the absence of serosal Na+ or K+ inhibits the action of antidiuretic hormone (ADH) on the osmotic permeability (Pf) but not on diffusional permeability (PDW) to water. This dissociation could be due to unstirred layers with low PDW, precluding detection of changes in the PDW of the cells. When serosal Na+ was replaced by choline, Pf (micrometer/s) was inhibited from 201 to 65. In the same conditions, if appropriate corrections were made to allow for the PDW of unstirred layers in the bulk phase and stroma, the PDW (micrometers/s) of the cellular pathway decreased from 19.8 to 15.9. The Pf/PDW ration then became 10 in the presence of serosal Na+ and 11 in its absence. When serosal K+ was deleted Pf decreased from 197 to 127 and PDW (corrected for unstirred layers) from 19.8 to 13.1 The Pf/PDW ratio was 10, both in the presence and absence of serosal K+. In conclusion, it is impossible to estimate the effect of any given experimental manipulation on the ADH-induced increase in PDW and compare it with the effect of Pf unless attention is paid to the restrictions upon measurements of diffusion imposed by unstirred layers. The deletion of Na+ or K+ from the serosa inhibits the effect of ADH on Pf and PDW to the same extent. Therefore, their presence in the serosa is essential for a full effect of ADH on the permeability to water of the apical membrane.     

Effects of medium hypertonicity on water permeability in the mammalian rectum: ultrastructural and molecular correlates

Minute-by-minute net water fluxes (Jw) were measured across the isolated rectal epithelium in rats and rabbits. Five minutes after a serosal (but not mucosal) hypertonic challenge (plus 200 mosmol/l) a significant increase in the basal Jw was recorded in both species [deltaJw, microl min(-1) cm(-2): 0.40+/-0.06 (rats); 0.45+/-0.10 (rabbits)]. At the same time, most epithelial cells shrank markedly while the intercellular spaces were wide open (electron microscopy studies). In freeze-fracture studies multi-strand tight-junction structures (only slightly modified by serosal hypertonicity in rabbits) were observed in control conditions. No structural changes were observed after mucosal hypertonicity (both in rats and rabbits). Immunohistochemical studies showed the expression of aquaporin 3 (AQP3) at the basolateral membrane of epithelial cells in the rat. A first conclusion is that the epithelium of the mammalian rectum is a highly polarized, aquaporin-3-containing, water permeability structure. The Jw increase induced by serosal hypertonicity was sensitive to mercurial agents in both species and no changes in unidirectional [14C]mannitol fluxes (Ps) or transepithelial resistance (RT) were observed during this Jw increase. These observations suggest a transcellular route for the osmotically induced increase in water fluxes. In the rabbit rectum the initial Jw response, associated with serosal hypertonicity, was a transient one. It was followed by a second, slow and HgCl2-sensitive Jw increase (a transient peak in paracellular mannitol permeability was also observed). A second conclusion is that serosal hypertonicity induces an increase in transcellular water permeability in both rat and rabbit rectum.     

Calcium release in relation to permeability changes in toad bladder epithelium following antidiuretic hormone

1. Methods for measuring the release of (45)Ca from isolated urinary bladders of toads (Bufo marinus) pre-loaded with this isotope have been devised. One method allowed separate collection from the mucosal and serosal surfaces of the bladders.2. Reducing the ambient calcium concentration reduced the rate of (45)Ca efflux suggesting that efflux of radiolabel represents calcium exchange.3. Antidiuretic hormone, theophylline and prostaglandin E(1) all increased calcium efflux, while lanthanum and amphotericin were without effect. Cyclic AMP caused only an inhibition of calcium release.4. The increase in (45)Ca efflux due to antidiuretic hormone came exclusively from the mucosal side. Experiments with EGTA suggest that the calcium entering the mucosal solution arises mainly from superficial sites in the mucosal membrane.5. The release of (45)Ca by hormone was not influenced by removal of sodium from the bathing solution. Low pH and amiloride reduced or abolished calcium release to hormone.6. The time course of calcium release from the mucosal surface due to hormone was rapid (commencing between 0.5 and 1.5 min after hormone application). Thus calcium release precedes the increase in sodium transport and hydro-osmotic flow following hormone, and appears to be at least as rapid as cyclic AMP generation in the tissue.7. The relationship between calcium release or exchange and the permeability changes in the bladder to water and to sodium, following hormone, are discussed.     

Solvent drag of sucrose during absorption indicates paracellular water flow in the rat kidney proximal tubule

Single convoluted proximal tubules of the rat kidney were lumen perfused in situ with isosmotic solutions containing C¹⁴-sucrose and H³-inulin as tracers, to evaluate whether the extracellular marker sucrose is entrained by water during proximal tubular reabsorption. Inulin was used as volume marker. The absorptive rate was varied by using as luminal perfusion fluids either a solution made up of (in mmole/l) 120 NaCl, 5 glucose, 25 NaHCO₃ and altering the perfusion rate, or a solution containing 110 NaCl and 70 raffinose.J ^S, the net sucrose efflux is found to be a function of the net volume flow,J _V, such that atJ _V=0,J ^S is very small and at high rates ofJ _V,J ^S is over 60-fold the value observed at lowJ _V values. In addition, the transported to luminal sucrose concentrations decreased withJ _V in a hyperbolic manner.Unstirred layers affect the diffusive component ofJ ^S, but only to a small extent. Therefore, the large remaining dependency ofJ ^S withJ _V must be due to drag of sucrose by water, within the paracellular pathway. This leads to the conclusion that water flows through the paracellular pathway during absorption in the rat proximal tubule, in addition to transcellular water flow. Using equations for molecular sieving and the measured value of _s for sucrose of 0.76–0.91, it is calculated that the pathway where entrainment of solute by water occurs must be 1.0–1.1 nm wide. This calculation is only tentative since _s depends on the as yet unknown relative contribution of transcellular and paracellular pathways to transepithelial water osmotic permeability.     

Passive sugar flux across frog jejunum in vitro

Summary Transmural diffusion of 3-O-methylglucose (3-MG) shows symmetry in bullfrog jejunum perfused in vitro. In both the mucosal to serosal (M to S) and serosal to mucosal (S to M) direction, the permeability coefficient is 1–2×10^–6 cm/sec as measured in isosmotic Na₂SO₄ Ringer's solution containing phlorizin, or K₂SO₄ Ringer's. The coefficient—both from M to S and S to M—increases several fold when osmotic water flow is induced from M to S. This is believed to be due to the opening of intercellular spaces. Furthermore, M to S osmotic flow exerts a solvent drag effect on 3-MG. With S to M osmotic flow which is smaller for the same osmotic driving force and induces no space opening, no permeability change is observed and no significant solvent drag effect. The data, along with measurements of mucosal cell and submucosal tissue concentrations during stationary 3-MG diffusion, indicate that the main resistance to sugar diffusion resides in the mucosal cells, especially in their luminal border. By some isosmotic non-electrolyte Ringer's solutions and moderately hyperosmotic media, the epithelium is progressively destroyed, leading to a time-dependant increase of sugar permeability and a rapid loss of active transport capacity.This work was presented, in part, at the 38. Tagung der Deutschen Physiologischen Gesellschaft, Erlangen, September 1970 [Pflügers Arch.319, R 95 (1970)].     

Influence of mucosal and serosal pH on antidiuretic action in frog urinary bladder.

Mucosal acidification to pH 6.5 reduced by 88% the oxytocin- (2.2 x 10(-8) M) elicited increase of water permeability in frog urinary bladder. Mucosal alkalinization (pH 10.5) increased by as much as 200% the response to the same concentration of oxytocin. These effects were not observed when supramaximal concentrations of oxytocin were imployed. Similar changes were found when the serosal pH was modified. The hydrosmotic responses elicited by serosal hypertonicity or cyclic AMP plus theophylline were also affected by mucosal or serosal changes of the hydrogen in concentration, suggesting an effect at a post-cyclic AMP level. Important interactions were found between luminal pH and serosal hypertonicity when experimental conditions were employed similar to those observed in the collecting duct of mammalian nephron. Freeze-fracture studies showed that the number of intramembranous aggregates of particles induced by ADH in the luminal membrane was reduced by mucosal acidification and augmented by an increase in medium pH.     

Structure and function in urinary bladder of foetal sheep

1. The structure and function of the epithelial lining of the urinary bladder of sheep foetuses was investigated by electron microscopic studies made in conjunction with a series of experiments in which the permeability of the bladder to sodium and water was measured in vitro. Measurements were made at gestational ages ranging from 50 to 141 days (term = 147 days) Osmolarity and electrolyte concentrations of urine found in the foetal bladder were also measured.2. The development of tight junctions between the bladder epithelial cells was investigated by incubating the tissue with solutions containing 1 mM-LaCl(3) on the mucosal surface. No penetration of the junctions by lanthanum was observed in foetuses of 90 days or older. In younger bladders, the epithelial layer was stripped by treatment with lanthanum, but tight junctions appeared to be fully developed in early bladders incubated without lanthanum.3. The surface structure of the luminal (mucosal) plasmalemma was fully developed at 50 days.4. Unidirectional fluxes of labelled sodium and water were measured with identical solutions bathing the two surfaces of the bladder wall. No net water movement occurred; the mean ratio of efflux to influx in nine bladders was 1.002 +/- 0.039 (S.E. of mean). Under these conditions, the flux ratio for sodium was 1.735 +/- 0.143 (S.E. of mean) in twelve bladders.5. Antidiuretic hormone (ADH) had no effect on net water movement but reduced the net efflux of sodium so that the flux ratio became 1.285 +/- 0.255 (S.E. of mean) n = 8. ADH also had a striking effect on the structure of the epithelium, causing marked swelling of the intercellular spaces. The tight junctions remained an effective barrier to lanthanum penetration under these conditions; lanthanum was not observed in the enlarged spaces.     

Lithium absorption by the rabbit gall-bladder

Lithium (Li+) absorption across the low-resistance epithelium of the rabbit gall-bladder was studied in order to elucidate possible routes and mechanisms of Li+ transfer. Li+ at a concentration of 0.4 mM in both mucosal and serosal media did not affect isosmotic mucosa-to-serosa fluid absorption. At this low concentration net mucosa-to-serosa Li+ absorption was insignificant when the ambient Na+ concentration was 115 mM, although the gall-bladder had a significant Li+ permeability (2.7 X 10(-5) cm s-1) and a significant mucosa-to-serosa Li+ gradient developed as a result of fluid absorption. Net Li+ absorption was induced at reduced mucosal Na+ concentrations (by lowering the Na+ concentration down to 50 mM with or without substitution with sucrose, or by adding sucrose to the mucosal medium). This Li+ absorption occurred even in the absence of a mucosa-to-serosa Li+ gradient. Na+ and Li+ absorptions occurring at 50 mM Na+ were inhibited to the same degree by mucosal 1 mM amiloride. Substitution of 5-50 mM (44%) Na+ by Li+ in the external medium dose-dependently depressed Na+ absorption by up to 76%, while substitution by 50 mM choline had no significant effect. Li+ inhibition of Na+ absorption was elicited from the mucosal side and was not accounted for by compensatory Li+ absorption; water and Na+ absorption rates decreased nearly in parallel. The effects of 0.4 mM amiloride and of substitution with 20 mM Li+ were only partly additive. It is concluded that Li+ absorption in the rabbit gall-bladder cannot be explained by passive (paracellular) transport, but must be the result of transcellular, active transport. Both at low and at high concentrations Li+ may enter the cell via an Na+/H+ exchanger in the apical cell membrane. At high concentrations Li+ may inhibit Na+ absorption by interference with the exchange mechanism and/or via effects at the cytoplasmic level. The Li+ transfer mechanism across the basolateral cell membrane remains unknown.     

Effects of changes in the composition of the serosal solution on the electrical properties of the toad urinary bladder epithelium.

1. The potential profile and the cellular and paracellular transepithelial resistances of the toad urinary bladder were measured, by means of micro-electrode techniques, as functions of the osmolality of the serosal solution. 2. Reductions in serosal osmolality (that increase the rate of active sodium transport) produced proportional decreases in the electrical resistances of the apical and basal-lateral cell membranes, while the changes in resistance of the paracellular pathway were more complex. The apical membrane potential increased. 3. Increases in serosal osmolality (that decrease sodium transport) produced increases in the electrical resistances of both cell membranes, and moderate reduction in the paracellular resistance. The polarity of the apical membrane potential reversed. 4. These results indicate that reductions in serosal solution osmolality stimulate sodium transport by increasing both the sodium permeability of the luminal cell membrane (thus increasing sodium entry), and the electromotive force generated at the serosal border of the cell, thus enhancing the rate of sodium pumping. Conversely, increases in osmolality reduced sodium transport by reducing both the sodium permeability of the luminal membrane and the serosal membrane electromotive force.     

AVP-independent high osmotic water permeability of frog urinary bladder and autacoids

 In the isolated frog urinary bladder a 20- to 50-fold increase of the osmotic water permeability has been revealed in the absence of arginine vasopressin (AVP) as a result of several successive changes of the serosal Ringer solution. This increase of the osmotic water permeability was of the same magnitude as that of the effect of 1 nM AVP. Similarly to the effect of AVP, the amount of adenosine 3′,5′-cyclic monophosphate (cAMP) in the cells rose, and aggregates of intramembraneous particles were formed in the apical plasma membrane of granular cells (as shown by the freeze-fracture method). Immunocytochemical studies using anti-actin monoclonal antibodies indicated depolymerization of F-actin following the AVP-independent change in water permeability. It was possible to decrease the high level of osmotic permeability to the initial level if 10 μl/ml of frog blood serum or a lipid extract of this blood serum, or 1 μM arachidonic acid or 1 nM prostaglandin E₂ was added to the serosal Ringer solution. The rapid restoration of the osmotic water impermeability of the epithelium after the AVP- evoked effect was achieved by the addition to the serosal Ringer solution of Ringer solution in which intact frog urinary bladders had been previously incubated for 1 h. The data obtained indicate that the maintenance of the impermeability to water of the osmoregulating epithelium and the restoration of the initial low level of the osmotic permeability after the effect of AVP are due to participation of prostaglandin E₂ and other autacoids as well as, probably, some physiologically active substances of a lipid nature that are present in the blood serum. Received: 5 December 1995 / Accepted 22 July 1996