Electron microscopic cytochemical localization of adenylate cyclase in amphibian urinary bladder epithelium: effects of antidiuretic hormone

A cytochemical technique for electron microscopic localization of adenylate cyclase was used to identify this enzyme in quiescent and hormone-stimulated toad urinary bladder epithelium. In the absence of vasopressin (antidiuretic hormone), adenylate cyclase was detected along the outer surface of the basolateral plasma membranes of granular cells, mitochondria-rich cells, and basal cells, the major cell types comprising the hormone-sensitive urinary epithelium. In the presence of antidiuretic hormone, the basolateral precipitates were markedly increased. The latter was true for both tissues incubated in the presence of an osmotic gradient and those stimulated in the absence of such a gradient. A significant mucosal reaction was never seen. Such data indicate that the hormone receptors for vasopressin are located along the basolateral membranes of all epithelial cells comprising the mucosal hormone-sensitive epithelium. All cells of the epithelium also demonstrate a vasopressin-sensitive adenylate cyclase. We discuss possible mechanisms that attempt to integrate the cytochemical data into an overall scheme for the physiological action of this hormone on amphibian urinary bladder.     

Antidiuretic hormone response in the amphibian urinary bladder: time course of cytochalasin-induced vacuole formation, an ultrastructural study employing ruthenium red

Cytochalasin is known to inhibit the antidiuretic hormone-induced hydro-osmotic response (bulk water flow) in the amphibian urinary bladder without altering hormone-stimulated diffusional water permeability or short-circuit current. In addition, histological studies have shown that the mold metabolite induces the formation of large intracellular vacuoles or lakes in the epithelial cells. We report here a transmission electron microscopic time-course study which indicates that during the early phases of the ADH response cytochalasin causes the formation of numerous multivesicular bodies or aggregates derived from individual basolateral pinocytotic vesicles. Because of their apparent hypertonic nature, the vesicles, as well as the vesicular aggregates, accumulate water during hormone-stimulated hydro-osmotic flow. As a result, the multivesicular bodies dilate and fuse to form the large intracellular lakes characteristic of cytochalasin treatment in the presence of both an applied osmotic gradient and vasopressin. In the presence of mucosal ruthenium red, the luminal glycocalyx was heavily stained with this tracer. At no time, however, even in the presence of hormone, was there any evidence for the uptake of this dye at the apical epithelial border. In the presence of serosal ruthenium red, the lateral intercellular spaces, basolateral pinocytotic vesicles, basal lamina, and collagen, as well as other subepithelial structures, were ruthenium positive. With cytochalasin D, vasopressin, and serosal ruthenium red, both the pinocytotic vesicles and the multivesicular bodies demonstrated an apparent membrane associated ruthenium positive coat. The tracer data indicates that the basolateral pinocytotic vesicles, increased by the presence of hormone, are indeed endocytotic in nature. The mucopolysaccharide coat associated with these structures may be involved in ionic and/or fluid transport.     

Neutral aminopeptidase: a potential marker enzyme of the amphibian urinary bladder epithelial cell apical membrane

Antidiuretic hormone induces, in the apical plasma membrane of amphibian urinary bladder epithelial cells, the exocytotic insertion of intramembranous particle aggregates that probably contain water channels. Purification of the apical membrane is a way to characterize the aggregates. The isolation of such purified membranous fractions involves the use of specific exogenous or endogenous markers. One of them could be the neutral aminopeptidase (AP), whose activity was detected in urinary bladder. Enrichment in AP activity was observed in plasma membrane preparations compared to cell homogenates (X2.7). However, a large part of the enzyme activity was also recovered in the soluble fraction of the preparation, suggesting large proteolysis of the protein. The enzyme presents a low optimal pH (6.4) and a high specificity for proline-p-nitroanilide as compared to the AP present in kidneys and intestines. To localize the protein in the amphibian bladder epithelium, an immunological approach was necessary due to the low activity of the enzyme in this tissue. The low enzymatic activity also prevented the purification of sufficient amounts of the urinary bladder AP as antigen, and we prepared antibodies against purified AP from frog or toad kidneys where the activity is 60 times higher than in the bladder. The serum specificity was verified by spot immunodetection, Western blot, inhibition capacity of antibodies, and immunoadsorption on a solid support with the renal enzyme. The sera were found to be able to react with native as well as denatured forms of the kidney enzyme. Antibodies cross-reacted with several peptides of low molecular weight (40-60 kDa) from urinary bladder plasma membrane proteins (Western blot).(ABSTRACT TRUNCATED AT 250 WORDS)     

Apical material extracted from amphibian urinary bladder epithelium by enzymes and detergent treatment

The apical plasma membrane of epithelial cells of frog and toad urinary bladder is subject to large modifications during the induction of water permeability by the antidiuretic hormone. A better characterization of the apical membrane is necessary for a clear understanding of the mechanisms of hormone action. Towards this end, apical material was extracted by enzymatic treatment and by incubation with detergent. Proteolytic enzyme alone had little effect under our conditions. A pretreatment with several glycosidases (alpha-mannosidase or endo-beta-N-acetylglucosaminidase H) increased the hydrolytic action of papain, elastase, proteinase K or Staphylococcus aureus V8 protease and allowed the detection of a major 76 kD in SDS gel electrophoresis. The n-octyl-beta-D-glucopyranoside (0.2%) led to the extraction after 150 mn of 1 to 5 micrograms proteins per cm2 of amphibian urinary bladder apical surface. The extracted proteins migrated as several bands on SDS gels. One of them probably corresponds to the 76 kD fragment obtained after proteolysis. The absence of alteration of the water permeability after extraction and the good preservation of the ultrastructure are evidence for the localisation of the 76 kD at the apical membrane surface. This protein may be the best candidate as antigen to raise antibodies against the apical surface of amphibian urinary bladder epithelial cells.     

The ultrastructural immunocytochemical localization of superoxide dismutase in the amphibian urinary bladder: Effect of aldosterone

Summary Transmission electron microscopy and immunohistochemistry, the latter employing the avidin—biotin—peroxidase (ABC complex) technique, were utilized to localize copper—zinc superoxide dismutase (CuZn—SOD) enzyme activity in the epithelial cells of the toad urinary bladder mucosa. This scavenger enzyme catalyses the dismutation (reduction—oxidation) of the superoxide anion (O₂ ^–), a toxic free radical generated during normal cellular respiration. In unstimulated epithelial cells, enzyme activity was seen in the cytosol of granular, mitochondrial-rich and goblet cells. The basal cells were generally devoid of enzyme activity. In addition to the cytosol, SOD activity was also seen in association with the apical plasma membrane of the epithelial cells. In the presence of the steroid hormone aldosterone (10^–7 m, 30 min—6 h), CuZn—SOD activity was markedly increased along the luminal mucosal membrane of granular, mitochondrial-rich and goblet cells. This increase was seen as early as 30 min after the addition of hormone, and as long as 6 h after treatment. The cytosolic reaction was usually decreased or absent under these conditions. From the data presented, it appears that CuZn—SOD is involved in electrolyte (sodium) transport in the epithelial cells of the toad urinary bladder. The latter may involve hormone-induced alterations in luminal cell membrane structure and chemistry.To whom reprint requests should be addressed.     

Mitochondrial migration and Ca-ATPase modulation in secretory ameloblasts of fasted and calcium-loaded rats

Migration of mitochondria and modulation of Ca-ATPase activity in secretory ameloblasts were investigated ultrastructurally and ultracytochemically by using lower incisors taken from normally fed, 30-hr-fasted, and calcium (Ca)-loaded rats. In normally fed rats, almost all mitochondria were localized in a narrow infranuclear compartment between the nucleus and proximal cell webs of secretory ameloblasts. In 30-hr-fasted rats, a prominent migration of many mitochondria into the supranuclear region of the cells was noted. Mitochondria returned to the infranuclear compartment and seldom appeared in the supranuclear region when fasted rats were Ca-loaded by transcardiac perfusion with physiological Ca solution. Normally, the mitochondria of secretory ameloblast exhibited moderate Ca-ATPase activity along their inner membranes. This mitochondrial Ca-ATPase was decreased by a 30-hr fast and became prominent again after Ca loading. Plasma-membrane Ca-ATPase was demonstrated in the entire cell surface of secretory ameloblasts. An especially abundant reaction was found along the invaginated cell surface of the Tomes process. This Ca-ATPase also became very weak and was almost abolished from the Tomes process after fasting, but Ca loading caused reappearance of an intense Ca-ATPase activity on the entire cell surface, including along Tomes's processes. These results suggest that 1) mitochondrial localization in secretory ameloblasts is influenced by the Ca concentration of the extracellular milieu, and 2) the level of mitochondrial and cell-membrane ATPase activity is responsive to the concentration of extracellular calcium.     

Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder: Effects of bradykinin,kallidin, eledoisin,and physalaemin

Summary It has been shown by means of Bentley'sin vitro preparation of the isolated urinary bladder of the toad,Bufo marinus paracnemis Lutz, that bradykinin reversibly inhibited the increase brought about by vasopressin on the permeability to water of the toad bladder. The increased hydro-osmotic response of the bladder to oxytocin was also inhibited by the kinin. The effect on water permeability was observed when bradykinin was added either to the serosal Ringer's solution or to the mucosal solution. The addition of bradykinin alone did not alter the basal osmotic water transfer across the bladder. In this context, bradykinin acted as a competitive antagonist of vasopressin (and oxytocin). Although lacking intrinsic activity, bradykinin exhibited affinity for receptor sites that are also common to the neurohypophysial hormones, causing a parallel shift of the log-dose/response curve for vasopressin without changing the maximal responses. The effects of other kinins (namely kallidin, eledoisin and physalaemin) on the toad bladder were also tested. Each of these drugs alone did not change the basal water flux across the bladder wall. Like bradykinin, these peptides inhibited the increase in water permeability evoked by vasopressin and oxytocin in the bladder. In view of the importance of neurohypophysial hormones and their target tissues to the osmotic homeostasis of amphibians, and the observation of antagonism between the kinins and the pituitary hormones coupled to the abundance of kinins in the amphibian organism, particularly in the skin and urinary bladder, teleological reasoning predicts a physiological role for the kinins, possibly functioning to dampen excesses and oscillations in membrane permeability that could occur in face of a constant and variable secretion of neurohypophysial hormone, thus adding to the homeostatic response of the amphibian organism.     

Comparison of K(+)-p-nitrophenyl phosphatase activity in the urinary bladder of the frog Rana esculenta during hibernation and active life.

We have studied effects of hibernation on the frog urinary bladder, an organ involved in water and ion transepithelial transport and taking part in osmoregulation. We have demonstrated K(+)-p-nitrophenyl phosphatase activity (an enzyme involved in ion and water transport) both in active and hibernating frogs. Most of the reaction product deposition was found on basolateral membranes of granular cells of the urinary bladder epithelium during all seasons. Therefore, it seems likely that this organ, unlike organs studied previously (skin, kidney and lung), maintains its function in the osmoregulatory process during hibernation.     

The effects cytochalasin B on the surface morphology of the toad urinary bladder epithelium: a scanning electron microscopic study.

Cytochalasin B depresses the hydroosmotic response of the toad urinary bladder to vasopressin without affecting basal (bulk flow) permeability, diffusional permeability, or the hormone induced increase in short circuit current. Fine structural studies demonstrated that this macrolide fungal metabolite, in the presence of both an osmotic gradient and vasopressin, induces the formation of large intracellular vacuoles or 'lakes' in epitelial cells lining the bladder mucosa. Some surface changes (shortening and irregularity of microvilli, clumping of the glycocalyx, etc.) were reported by transmission electron microscopy. Scanning electron microscopy demonstrates that cytochalasin B drastically alters the mucosal surface morphology of the hormone stimulated bladder. Lesser changes were seen in the absence of vasopressin. In the presence of arginine vasopressin, excessive cellular swelling and possible rupturing, as well as surface membrane infolding and rippling, were seen in the cytochalasin treated tissues, The specific entity most affected by this treatment is the granular cell.     

Water and sodium transport: effects of calcium channel blocker and calmodulin antagonists on the apical and basolateral membranes of amphibian epithelia

Ca2+ channel blocker (sensit) and calmodulin antagonists (thioridazine, perphenazine, oxyprothepine) applied to the mucosal side of frog urinary bladder, weakened the response of epithelial cells to vasopressin. Thioridazine (2.7 X 10(-5) mol X l-1) and sensit (1.7 X 10(-4) mol X l-1) applied to the serosal side rapidly increased the permeability of the epithelia for sodium and potassium ions along the concentration gradient (from serosa to mucosa). The same concentrations of these blockers when applied to the mucosal side of frog urinary bladder selectively decreased vasopressin stimulated water permeability and did not influence ionic permeability. Both thioridazine and sensit decreased the short-circuit current across frog skin. The results show that the Ca2+ channel blocker and the calmodulin antagonists tested influenced water and ionic transport across the epithelial cell membranes, and had different effects upon the apical and the basolateral cell membranes.     

Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells

In order to elucidate the problem of which cells are involved in calcium transport and to estimate the role of mitochondria in calcium transport in the avian shell gland, the fine structure and the Ca-ATPase, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD+)-dependent isocitrate dehydrogenase (NAD+-ICDH) activity of the shell gland of egg-laying Japanese quails were examined. The surface epithelial cells, consisting of ciliated cells with cilia and microvilli and non-ciliated cells with microvilli, had many large and electron-dense granules. The tubular-gland cells occupied the proprial layer and lacked secretory granules. When an egg was in the shell gland, the well-developed mitochondria of tubular-gland cells characteristically tended to accumulate in the apical cytoplasm, while they were scattered throughout the cytoplasm when an egg was not in the shell gland. Intense Ca-ATPase activity was found on the microvilli of tubular-gland cells, and moderate activity was found on the lateral-cell surface. In the surface epithelial cells, the basolateral cell surface showed moderate enzymatic activity. Both SDH and NAD+-ICDH activity were found in tubular-gland cells when an egg was in the shell gland. These results strongly suggest that calcium for eggshell calcification is actively transported by the tubular-gland (depending on Ca-ATPase activity) and that the mitochondria of gland cells may play an important role in this process as an energy source.     

The effect of aldosterone on cyclic nucleotide phosphodiesterase activity in toad urinary bladder

Cyclic AMP phosphodiesterase activity was assayed in the 700 xg supernatant solution of homogenates of epithelial cells scraped from toad urinary bladders. The activity of the enzyme was lower in cells obtained from bladders incubated with aldosterone for 24 hours than in cells from paired tissue incubated without aldosterone. This difference may well account for the permissive effect of aldosterone on the physiologic and biochemical responses of the toad bladder to vasopressin.     

Antidiuretic response in the urinary bladder of Xenopus laevis: Presence of typical aggrephores and apical aggregates

The urinary bladder of the aquatic toad Xenopus laevis is known to exhibit a low permeability to water and a poor sensitivity to antidiuretic hormone. In order to precise the characteristics and the specific cellular mechanisms of this reduced hydroosmotic response we used a sensitive volumetric technique to monitor net water flow and studied the correlation between the anti-diuretic hormone (ADH)-induced net water flow and the fine ultrastructural appearence of the urinary bladder epithelium. Transmural net water flow was entirely dependent on the osmotic gradient across the preparation and not on the hydrostatic pressure difference. We observed the existence of a low but significant hydro-osmotic response to arginine vasopressin. Freeze-fracture electron microscopy demonstrated the presence of typical aggrephores in the subapical cytoplasm. The response to the hormone was accompanied by the appearance of typical intramembrane aggregates into the apical plasma membrane. Water permeability increase and apical aggregate insertion were both slowly but fully reversible. Except for the multilayered structure of the epithelium and the particularly low response to antidiuretic hormone, all the studied permeability and ultrastructural characteristics of the bladder were thus very similar to those observed in other sensitive epithelia such as the amphibian bladder and skin and the mammalian collecting duct which exhibit a high hydro-osmotic response to the hormone.     

Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells

Summary In order to elucidate the problem of which cells are involved in calcium transport and to estimate the role of mitochondria in calcium transport in the avian shell gland, the fine structure and the Ca-ATPase, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD⁺)-dependent isocitrate dehydrogenase (NAD⁺-ICDH) activity of the shell gland of egg-laying Japanese quails were examined. The surface epithelial cells, consisting of ciliated cells with cilia and microvilli and non-ciliated cells with microvilli, had many large and electron-dense granules. The tubular-gland cells occupied the proprial layer and lacked secretory granules. When an egg was in the shell gland, the well-developed mitochondria of tubular-gland cells characteristically tended to accumulate in the apical cytoplasm, while they were scattered throughout the cytoplasm when an egg was not in the shell gland. Intense Ca-ATPase activity was found on the microvilli of tubular-gland cells, and moderate activity was found on the lateral-cell surface. In the surface epithelial cells, the basolateral cell surface showed moderate enzymatic activity. Both SDH and NAD⁺-ICDH activity were found in tubular-gland cells when an egg was in the shell gland. These results strongly suggest that calcium for eggshell calcification is actively transported by the tubular-gland (depending on Ca-ATPase activity) and that the mitochondria of gland cells may play an important role in this process as an energy source.     

Ultrastructure and elemental composition of frog bladder granular epithelial cells in normal state and upon stimulation of water transport

Changes in the frog urinary bladder granular cell ultrastructure were analysed in parallel with those in element composition of these cells after induction of water transport across the urinary bladder wall. Two ultrastructural (ultrathin section and freeze-fracture) methods were used in addition to two methods of object preparation for electron microprobe analysis--freeze-drying and freeze-substitution. It has been shown that arginin-vasotocin stimulation of osmotic water flow across the urinary bladder wall causes certain morphological changes in the granular cells: decrease in electron density of the cytoplasm, depolymerization of the apical submembrane layer of actin microfilaments, increase in the number of sites of specific granules and apical membrane fusion, emergency of intramembrane particle aggregates in the apical membrane P-face. The quantitative electron microprobe analysis made it possible to reveal a statistically significant increase in sodium and calcium concentration and fall in that of potassium and chlorine in granular cells after water transport stimulation. A concentration gradient of sodium and potassium ions was seen to appear along the apical-basal axis in the cytoplasm of granular cells. Possible association between the obvious morphological transformations in granular cells and changes in their elemental composition has been discussed, in addition to some regulatory significance of calcium concentration increase in granular cells after arginin-vasotocin-induced osmotic water transport.     

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.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasoactive peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8-13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

Summary The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasocative peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8–13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Cell volume regulation in frog urinary bladder

We have studied the problem of cell volume homeostasis in toad and frog urinary bladder by using electrophysiological measurements and an optical measure of cell volume. After osmotically induced swelling, urinary bladder cells spontaneously regulate their volume through a net loss of potassium, chloride, and water. During inhibition of sodium transport by amiloride the cells swell to the same extent as controls, but the volume-regulatory process is blocked. Electrophysiological results under isosmotic conditions indicate that basolateral membrane resistance increases simultaneously with the amiloride-induced rise in apical membrane resistance during transport inhibition. These independent observations indicate that inhibition of apical membrane sodium entry results in a secondary decrease in basolateral membrane potassium permeability. When cells are exposed to calcium-free, hyposmotic Ringer's solution, cell volume regulation is blocked; subsequent addition of the calcium ionophore A23187 is ineffective in restoring the regulatory process. The ionophore does induce volume regulation, however, in amiloride-inhibited, osmotically swollen cells in the presence of external calcium. Calcium thus seems to control basolateral membrane potassium permeability and may be the intracellular mediator of apical and basolateral membrane interactions.     

Evidence of basolateral water permeability regulation in amphibian urinary bladder

It is well known that arginine vasopressin (AVP) produces up to a 40-fold increase (0.1 to 4,0 μL/min·cm²) in net water flux across the amphibian urinary bladder under an osmotic gradient (mucosal side 10% hypotonic). No AVP effect is observed when the gradient is in the opposite direction (serosal hypotonic). Similar asymmetrical behavior to osmotic gradients occurs in the frog corneal epithelium. This rectification phenomenon has not been satisfactorily explained. We measured net water fluxes in bladder sacs and confirmed that AVP has no effect when the serosal bath is hypotonic. We reasoned that the ‘abnormal’ serosal osmolarity was inducing changes in membrane water permeability, the very parameter being measured. Thus, we studied the effect of solution osmolarity on diffusional water flow (J_dw) across the frog bladder using ³H₂O. As expected, AVP doubled J_dw (in either direction from 12 to 21 μL/min·cm²) when the serosal solution was iso-osmolar regardless of mucosal osmolarity. However, in the AVP-stimulated bladders, hypo-osmolarity of the serosal solution reduced J_dw by 42%, an effect that was reversible when normal osmolarity was re-established. Amphotericin B (instead of AVP) was used to irreversibly increase the permeability to water of the apical membrane. Under these conditions, basolateral hypotonicity also reversibly decreased J_dw by 32%, suggesting the basolateral membrane as the site where permeability is reduced. SEM and TEM of the tissue shows extreme swelling when it was exposed to serosal hypotonicity with or without AVP and typical surface morphology changes following hormone stimulation. We conclude that this swelling may initiate a signaling mechanism that reduces basolateral water permeability. These findings constitute evidence of basolateral water channel permeability regulation, which can also contribute to cell volume regulation.