Role of calmodulin in antidiuretic hormone mediated water transport

Several classes of tricyclic antidepressants inhibit the action of antidiuretic hormone (ADH) and cyclic adenine monophosphate (cAMP) on osmotic water flow across toad urinary bladder without any effect on sodium transport. This finding suggests that calmodulin is involved in the hydroosmotic action of ADH (and of serosal hypertonicity), possibly in inducing exocytosis at the luminal border of vesicles rich in water channels.     

Localization of barriers to water flow in toad urinary bladder

Although it is well accepted that vasopressin (ADH) increases the permeability to water of the toad bladder granular cell's luminal membrane, recent studies have suggested that regulation also takes place at an additional "postluminal" site within the epithelial granular cell. These studies are based upon the observation that a number of experimental maneuvers can alter tissue permeability to water, but do not change the number of particle aggregates observed on the protoplasmic face of the granular cell's luminal membrane with freeze-fracture electron microscopy. These aggregates are believed by many investigators to mediate the transport of water across the luminal membrane. The dissociation between permeability and aggregate frequency described above has been variously interpreted as the consequence of changes in the permeability of the aggregates themselves, or of changes in the permeability of a "postluminal" barrier that is functionally in series with the luminal membrane. We attempted to distinguish between these 2 possibilities by studying paired toad bladders during 3 protocols that alter vasopressin-stimulated water flow across the intact tissue without altering aggregate frequency. Estimates of the permeability of postluminal barriers were obtained by exposing the luminal surface to amphotericin B, an antibiotic that forms water-permeant channels in the luminal membrane. Of the 3 protocols, only diminishing bladder filling volume decreased the water flow elicited by luminal amphotericin B, suggesting that only that protocol indeed decreased the permeability of some postluminal barrier. The other 2 protocols, increasing PCO2 and repeatedly stimulating the bladder with vasopressin, did not alter amphotericin B-elicited flow, suggesting that postluminal barriers were not altered by these 2 protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the sodium permeability of the luminal border of toad bladder by intracellular sodium and calcium: role of sodium-calcium exchange in the basolateral membrane

Sodium movement across the luminal membrane of the toad bladder is the rate-limiting step for active transepithelial transport. Recent studies suggest that changes in intracellular sodium regulate the Na permeability of the luminal border, either directly or indirectly via increases in cell calcium induced by the high intracellular sodium. To test these proposals, we measured Na movement across the luminal membrane (th Na influx) and found that it is reduced when intracellular Na is increased by ouabain or by removal of external potassium. Removal of serosal sodium also reduced the influx, suggesting that the Na gradient across the serosal border rather than the cell Na concentration is the critical factor. Because in tissues such as muscle and nerve a steep transmembrane sodium gradient is necessary to maintain low cytosolic calcium, it is possible that a reduction in the sodium gradient in the toad bladder reduces luminal permeability by increasing the cell calcium activity. We found that the inhibition of the influx by ouabain or low serosal Na was prevented, in part, by removal of serosal calcium. To test for the existence of a sodium- calcium exchanger, we studied calcium transport in isolated basolateral membrane vesicles and found that calcium uptake was proportional to the outward directed sodium gradient. Uptake was not the result of a sodium diffusion potential. Calcium efflux from preloaded vesicles was accelerated by an inward directed sodium gradient. Preliminary kinetic analysis showed that the sodium gradient changes the Vmax but not the Km of calcium transport. These results suggest that the effect of intracellular sodium on the luminal sodium permeability is due to changes in intracellular calcium.     

Effect of distension on ADH-induced osmotic water flow in toad urinary bladder

Summary We recently described a method by which the resistance to water flow of the luminal membrane of ADH-stimulated toad bladder can be quantitatively distinguished from that of barriers lying in series with it. This method requires estimates of both total bladder water permeability (assessed by transbladder osmotic water flow at constant gradient) and luminal membrane water permeability (assessed by quantitation of the frequency of ADH-induced luminal membrane particle aggregates). In the present study we examined the effect of bladder distension on transepithelial osmotic water flow before and during maximal ADH stimulation. Base-line water flow was unaffected by bladder distension, but hormonally stimulated flow increased systematically as bladders became more distended. Distension had no effect on the frequency of ADH-induced intramembranous particle aggregates. By comparing the relationships between aggregate frequency and hormonally induced water permeability in distended and undistended bladders, we found that distension appeared to enhance ADH-stimulated water flow by decreasing the resistance of the series permeability barrier while the apparent water permeability associated with each single luminal membrane aggregate was unaffected. In that bladder distension causes tissue thinning, the series resistance limiting ADH-stimulated water flow appears to be accounted for by deformable barriers within the bladder tissue itself, probably unstirred layers of water.     

Effect of hypertonicity and colchicine on intramembranous particle aggregation in toad urinary bladder

Summary Colchicine, an agent which disrupts microtubules, inhibits the vasopressin (VP)-induced increase in water permeability as well as intramembranous particle (IMP) aggregation in the luminal plasma membrane of granular cells of toad urinary bladder. However, the hydroosmotic response induced by serosal hypertonicity is not affected by colchicine. The present investigation was initiated to establish whether serosal hypertonicity is associated with IMP aggregation and whether the aggregation, if present, is altered by colchicine. The experimental half of paired hemibladders from the toad, Bufo marinus, treated with 0.1 mM colchicine for 4 h prior to exposure to serosal mannitol (240 mM) demonstrated no significant difference in osmotic water How (Jv) (1.03 × 0.18 vs. 1.13 ± 0.22l · min^–1 · cm^–2; p>0.20) when compared with control hemibladders. Similarly, comparison of control and colchicine-treated bladders revealed no difference in the number of IMP aggregation sites per area of membrane (17.8 ± 2.0 vs. 24.7 ± 3.5/100^m; p>0.10), the relative area of membrane occupied by these sites (0.30 ± 0.06 vs. 0.39 ± 0.07%; p>0.10) or the mean size of the aggregates (17.0 ± 1.4 vs. 15.8 ± 1.0 × 10³ m²; p > 0.20). These results indicate that in toad bladder the increase in J_v induced by serosal hypertonicity is associated with IMP aggregation. Secondly, an intact microtubule system is not required to induce the hydroosmotic or the aggregation responses. If, as has been proposed, the cellular actions of VP and serosal hypertonicity share a common pathway to bring about an increase in osmotic water permeability and cause IMP aggregation in the luminal membrane of the granular cell, the present results suggest that the pathway begins at a step subsequent not only to the generation of cAMP, but also beyond the involvement of the microtubule system.This work was supported in part by U.S. Public Health Service Grant AM 13845. Dr. Dratwa was supported through a U.S. Public Health Service International Research Fellowship F05TW2447. The authors gratefully acknowledge the technical assistance of Mrs. Helen Parks, Mr. Isaiah Taylor, Mrs. Betty Waller, and Mrs. Jessie Calder     

Ultrastructure of the apical plasma membrane of the granular cells in the frog bladder during cobalt-ion decrease in the vasopressin effect

Simultaneous studies were performed on changes in water permeability and on the ultrastructural organization of the frog urinary bladder epithelium in the presence of Co-ions under vasopressin-stimulated water flow. A possible inhibition of the vasopressin-stimulated water flows by Co-ions is supposed from the extracellular surface of the apical membrane of granular cells responsible for water permeability of this epithelium. Using the freeze-fracture technique for studying the apical membrane ultrastructure, it was shown that with the maximum water flow the square occupied by intramembrane particle aggregates was as much as 1.8% of the total square of membranes, to reduce to 0.3% with the smaller water flow, the average sizes of aggregates being 0.35 mkm and 0.08 mkm in both these cases, respectively. Application of 1 x 10(-3)-1 x 10(-4) M CoCl2 from the mucose part inhibits the vasopressin-stimulated water flow. In this case no aggregates are actually seen on the P-face of the apical membrane, the number of intramembrane particles of the E-face being similar to that when the water permeability was originally low. It is concluded that Co-ion may influence the structure and function of the apical plasma membrane from its extracellular surface.     

Activation of cyclic AMP-dependent protein kinase A common step in vasopressin- and hypertonicity-induced water flow

Water flow across the amphibian urinary bladder can be induced by either vasopressin or serosal hypertonicity. In an effort to determine the common intracellular steps mediating both responses, we determined the in situ activation of cyclic AMP-dependent protein kinase in bladders stimulated by vasopressin or hypertonicity. Treatment of bladders with vasopressin (1 mU/ml) caused in situ activation of cytosolic cyclic AMP-dependent protein kinase of epithelial cells, with a rise in the kinase ratio and cyclic AMP content. Similarly, hyperonicity increased the kinase ratio, but this occured without a measurable increase in cyclic AMP content per mg protein. Because of the hypertonicity-induced cell shrinkage, epithelial cell water decreased by 20%, which may result in a proportionate increase in cyclic AMP concentration (per ml cell water). Furthermore, cell shrinkage also increases intracellular electrolyte concentration, which, in turn, should delay reassociation and consequent inactivation of the predominant Type II cyclic AMP-dependent protein kinase of the epithelial cells. Thus activation of cyclic AMP-dependent protein kinase during hypetonicity may be the result of cell shrinkage, with an associated increase in cyclic AMP and electrolyte concentrations. Studies with prostaglandin synthesis inhibitors and colchicine, a microtubule disrupting agent, also indicated common pathways for vasopressin and hypertonicity. Both naproxen and meclofenamate significantly enhanced the hypertonicity response. Colchicine pretreatment, on the other hand, caused a small (18%) but significant inhibition of the hypertnicity response, similar to its effect on the vasopressine response (25% inhibition). Thus, the increased water permeability of the toad bladder in response to both vasopressin and hypertonicity follows a similar pathway. Activation of cyclic AMP-dependent protein kinase represents the first common step yet identified.     

Enhancing effects of angiotensin I on the vasopressin-stimulated water flow of toad bladder through increased cyclic AMP in mucosal cells

The effects of angiotensins I and II on 10 mU/ml vasopressin-stimulated water flow across toad bladder were examined. Angiotensin I at concentrations of 10(-6) and 10(-7) M enhanced the water flow, but angiotensin II failed to do so at these concentrations. Angiotensin I had no effect on 5 mM cyclic AMP-stimulated water flow. After being preincubated for 30 min with angiotensin II, angiotensin I failed to have any stimulatory effect on vasopressin-stimulated water flow. At 10(-6) M angiotensin I significantly enhanced vasopressin-stimulated cyclic AMP content in bladder mucosal cells. These results indicate that angiotensin I enhances vasopressin-stimulated water flow by increasing cyclic AMP production in bladder cells and that angiotensin II may possibly interfere with angiotensin I in a competitive manner.     

Effects of colchicine and cytochalasin B on hypertonicity-induced changes in toad urinary bladder

Summary Coincident with an increase in the water permeability of toad urinary bladder induced by serosal hypertonicity, a transformation of the ridge-like surface structures of the granular cells into individual microvillous structures occurs. This study was initiated to establish whether the transformation is mediated by the cytoskeletal network and, thus, can be prevented by disruption of microtubulemicrofilament function with colchicine or cytochalasin B (CB). Scanning electron microscopy revealed the characteristic branching ridges on granular cells of control bladder incubated with colchicine or CB. In contrast, transformation of ridges to discrete microvilli was observed in experimental bladders exposed to serosal hypertonicity alone or in combination with either colchicine or CB. These results suggest that the mechanism underlying hypertonicity-induced surface changes which are associated with increased water permeability does not involve either microtubules or microfilaments.     

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.     

Barriers to water flow in vasopressin-treated toad urinary bladder

Summary Unstirred layers of water complicate the measurement of water permeability across epithelia. In the toad urinary bladder, the hormone vasopressin increases the osmotic water permeability of the granular epithelial cell's luminal membrane, and also leads to the appearance of aggregates of particles within this membrane. The aggregates appear to be markers for luminal membrane osmotic water permeability. This report analyzes the relationship between transbladder osmotic water flow and aggregate frequency, and demonstrates that flow across the bladder is significantly attenuated by unstirred layers of water or by structural barriers other than the luminal membrane when the luminal membrane is made permeable by vasopressin. This analysis in addition yields unique values for the permeabilities of both the luminal membrane and the barriers to water flow which lie in series with it.     

Regulation of ADH-stimulated water flow at a post-luminal barrier in toad bladder

Recent studies show that ADH-stimulated water flow across toad bladder may be regulated at a site other than the luminal membrane. In these studies luminal membrane particle aggregate frequency has been used as a measure of luminal membrane water permeability. In fully stretched bladders the relationship between total tissue permeability and aggregate frequency is curvilinear, rather than linear. This implies a resistance in series with the luminal membrane that can become rate-limiting for water flow during ADH stimulation. The possibility that transtissue water movement is actually regulated at such a post-luminal membrane resistance is suggested by the finding that within 30 min following exposure to hormone, water flow becomes attenuated without any change in aggregate frequency. Supporting this possibility, recent data from follow-up studies suggest that the apparent water permeability per luminal membrane aggregate is not reduced with time. Finally, for bladders in which prostaglandin synthesis is inhibited (by naproxen), increases in both base-line water flow and water flow consequent to treatment with a submaximal dose of ADH (0.125 mU/ml), are much less than expected from simultaneously observed changes in luminal membrane aggregate frequency. In parallel experiments to these, moreover, direct measurements of luminal membrane water permeability from the rate of change of cell volume consequent to a transluminal membrane osmotic challenge, confirm that luminal membrane water permeability increases to the extent expected from changes in aggregate frequency. All of the data taken together argue for a post-luminal membrane barrier in toad bladder which regulates tissue permeability during ADH stimulation.     

Functional role of V1- and V2-receptors of the apical and basolateral membranes of the frog bladder epithelial cells

Arginine vasotocin, 0.02--1 nM, increases osmotic water permeability of frog urinary bladder, arginine vasotocin after a simultaneous addition to the mucosal and serosal Ringer solutions rises the water permeability to a lesser degree than on the hormone addition only to the serosal solution. 1 nM remestyp, an agonist of V1-receptors, from the apical membrane decreases the hydroosmotic effect of arginine vasotocin added to the serosal Ringer solution. When added to the mucosal solution, combination of the same concentrations of arginine vasotocin and SR 49059, an antagonist of V--receptors, or desmopressin, agonist of V2-receptor alone, increases the effect of the same concentration of arginine vasotocin added to the serosal solution. 1 nM arginine vasotocin at the luminal membrane increases secretion into the Ringer solution of prostaglandin E, and prostaglandin E1 but not of prostaglandin F2 alpha. The data obtained indicate the presence of the arginine vasotocin receptors responsible for the hydroosmotic effect only in the basolateral membranes, while arginine prostaglandin E, participation is shown in modulation of the arginine vasotocin effect.     

Water flow in the toad urinary bladder in response to vasopressin: role of potassium

In agreement with previous reports, we found that absence of K+ from the serosal bath of the toad urinary bladder substantially impairs vasopressin and cAMP-stimulated water flow. The decreased response to vasopressin appears unrelated to prostaglandin production since inhibition of endogenous prostaglandins by pretreatment with naproxen 10(-5) M failed to prevent the effect seen with K+-free Ringer's. The resistance to vasopressin does not appear to be directly related to epithelial K+ concentrations, in that maneuvers leading to decreased intracellular K+ failed to produce a similar effect. A more likely explanation appears to be that K+-free Ringer's induces an increased cytosolic Ca++ which, in turn, decreases the hydrosmotic effects of vasopressin. Several lines of evidence argue in favor of such an explanation: (a) Increased cytosolic Ca++ had been found in other tissues with low extracellular K+; (b) The resistance to vasopressin decreases with decreased serosal Ca++; (c) The effects of K+-free Ringer's are not additive in situations believed to have increased epithelial Ca++, i.e. replacement of serosal Na+ with choline; (d) The effects of K+-free serosal bathing medium could be both prevented and/or reversed if already established by increasing serosal bath, and presumably intracellular, pH, which is believed to decrease intracellular Ca++.     

Ultrastructural modifications of frog urinary bladder epithelium under the influence of hypertonic media

Summary The frog urinary bladder undergoes a marked increase in its water permeability when incubated in hypertonic media. Many similarities are found between this effect and the hydrosmotic action of antidiuretic hormone. The ultrastructural modifications of the epithelium observed under the influence of serosal hypertonicity (the intercellular spaces are dilated while the tight junctions remain closed) lead us to assume that the pathways of water movement across the epithelium could be the same in this case and in hydrosmotic response to the hormone. In contrast, when the mucosal medium is made hypertonic, the ultrastructure is differently altered: the intercellular spaces are closed, the tight junctions show small vesicles and numerous large vacuoles appearing in the cytoplasm.     

Reversible inhibition by lanthanum of the hydrosmotic response to serosal hypertonicity in toad urinary bladder

Summary In the urinary bladder of amphibia, hypertonicity of the serosal bath (SH) evokes an increase in transepithelial water permeability, the characteristics of which resemble the response to antidiuretic hormone (ADH). The ionic dependency, in particular for Ca²⁺, appears very similar forSH- and ADH-induced water fluxes. In the present experiments La³⁺ was used as a probe to study the Ca²⁺-dependency of the hydrosmotic response toSH in isolated urinary bladder of the toadBufo marinus.Addition of La³⁺ (5mm) on the serosal side of the membrane produced a significant and reversible increase in basal transepithelial water flux. The hydrosmotic response elicited by adding 250mm mannitol to the serosal Ringer's solution was inhibited by 30% in the absence of serosal Ca²⁺. Similarly, the hydrosmotic response toSH was inhibited by 37%, 30% and 40% when 5mm La³⁺ was added to the serosal medium 30 min before, concommitantly with, or 60 min after induction ofSH. The inhibition of transepithelial water flux observed in the absence of serosal Ca²⁺ or in the presence of serosal La³⁺ was reversible.The results support a critical role for Ca²⁺ in the modulation of transepithelial water permeability in the urinary bladder of amphibia. Ca²⁺ presumably exerts its effects at a post-cyclic AMP step.     

Dobutamine inhibits vasopressin-mediated water transport across toad bladder epithelium

This study aimed to investigate the effect of dobutamine on water transport across toad bladder epithelium. Water flow through the membrane was measured gravimetrically in bladder sac preparations. Dobutamine had no effect on basal water transport, but partially inhibited transport stimulated by vasopressin. Similarly, dobutamine exerted no influence on the hydrosmotic response to 8-chlorophenylthio-cAMP, but interfered with the response to phosphodiesterase inhibitor 1-methyl-3-isobutyl-xanthine. These results demonstrate that this catecholamine may inhibit vasopressin-stimulated water transport at a site prior to cAMP formation. The use of propranolol was ineffective in blocking the effect of dobutamine on transport stimulated by vasopressin, indicating that beta-adrenoceptors play no role in this effect. On the other hand, phentolamine significantly reduced the effect of dobutamine, indicating the involvement of alpha-adrenoceptors in such event. Rauwolscine also inhibited the effect of dobutamine, pointing to the specific contribution of the alpha(2)-adrenoceptors to this effect. Taken together, the results of this study demonstrate that dobutamine inhibits vasopressin-stimulated water transport in toad bladders through a mechanism mediated by the stimulation of alpha(2)-adrenoceptors, thus suggesting that such a drug may exert a direct cellular effect on membrane permeability to water in transporting epithelia. The current study may provide a better understanding of the effects of dobutamine on renal function by contributing towards the elucidation of its action mechanism.     

Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders

Summary A Na/Ca exchange system has been described in the plasma membrane of several tissues and seems to regulate the concentration of calcium in cytosol. Replacement of extracellular Na by sucrose increases calcium uptake into and decreases calcium efflux from the cell, leading to an increase in cytosolic calcium. The effect of an increase in cytosolic calcium mediated by the Na/Ca exchange system on H⁺ and Na transport in the turtle and toad bladder was investigated by replacing serosal Na isosmotically by sucrose or choline. Replacement of serosal by sucrose was associated with a significant inhibition of H⁺ secretion or Na transport which was reversible by addition of NaCl. Replacement of mucosal Na by sucrose failed to alter H⁺ secretion. Removal of serosal Na was associated with a significant increase in⁴⁵Ca uptake which could be blocked by pretreatment with lanthanum chloride. Pretreatment with lanthanum chloride blunted the inhibitory effect of replacement of serosal Na by sucrose on H⁺ and Na transport, thus suggesting that the increase in calcium uptake and the inhibition of transport are causally related. Under anaerobic conditions the rate of H⁺ or Na transport are linked to the rate of lactate production. The inhibition of Na or H⁺ transport by removal of serosal Na was accompanied by a proportional decrease in lactate production, thus suggesting that an increase in cytosolic calcium does not inhibit transport by uncoupling glycolysis from transport. Replacement of serosal Na by sucrose did not alter the force of the H⁺ or Na pump but led to an increase in resistance of the active pathway of H⁺ and Na transport. The inhibition of Na transport by replacement of serosal Na with sucrose could be reversed by addition of amphotericin B, an agent which increases luminal permeability to Na, thus suggesting that decreased Na entry across the apical membrane is the mechanism responsible for the inhibition of Na transport. The results of the present studies strongly suggest that an increase in cytosolic calcium through the serosal Na/Ca exchange system inhibits H⁺ and Na transport in the turtle and toad bladder probably by increasing the resistance of the luminal membrane.     

Evidence that monensin inhibits vasopressin-stimulated water flow at an early step in the receptor-adenylate cyclase sequence

Monensin, a highly selective sodium ionophore, inhibits vasopressin-stimulated water flow in toad urinary bladder pretreated with naproxen, an inhibitor of prostaglandin synthesis. Inhibition is partially dependent on the presence of sodium in the serosal medium, but not on serosal calcium. We have found that monensin does not inhibit water flow generated by forskolin, cyclic AMP, or isobutyl methyl xanthine (MIX); indeed, an enhancement of water flow was seen following cAMP and MIX, as well as following 0.2 microM forskolin. Our findings suggest that monensin uncouples the vasopressin-receptor-G protein-adenylate cyclase sequence at some early step, by a mechanism that remains unknown, but that may directly or indirectly involve intracellular sodium.     

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.