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     

Cellular lithium and transepithelial transport across toad urinary bladder

Summary Toad urinary bladders were exposed on either their mucosal or serosal surfaces, or on both surfaces, to medium in which sodium was replaced completely by lithium. With mucosal lithium Ringer's, serosal sodium Ringer's, short-circuit current (SCC) declined by about 50 percent over the first 60 min and was then maintained over a further 180 min. Cellular lithium content was comparable to the sodium transport pool. With lithium Ringer's serosa, SCC was abolished over 60 to 120 min whether the mucosal cation was sodium or lithium. Measurements of cellular ionic composition revealed that the epithelial cells gained lithium from both the mucosal and serosal media. With lithium Ringer's mucosa and serosa, cells lost potassium and gained lithium and a little chloride and water, but these changes in cellular ions could not account for the current flow across the tissue under these conditions, which must, therefore, have been carried by a transepithelial movement of lithium itself. The inhibition by serosal lithium of SCC was overcome by exposure of the mucosal surface of the bladders to amphotericin B. Thus it reflected, predominantly, an inhibition of lithium entry to the cells across the apical membrane. It is suggested that this inhibition is a consequence of cellular lithium accumulation.     

Surface hydrophobicity and water transport of the toad urinary bladder: Effects of vasopressin

Summary The present study investigated whether the hydrophobic properties (wettability) of the luminal surface of the toad urinary bladder might play a role in modulating water transport across this epithelium. In the absence of vasopressin (ADH), water transport across the tissue was low, while luminal surface hydrophobicity (water contact angle) was relatively high. Following stimulation by ADH, water transport increased and surface hydrophobicity decreased. The addition of indomethacin to inhibit ADH-induced prostaglandin synthesis did not reduce these actions of ADH. In an attempt to alter water transport in this tissue, a liposomal suspension of surface-active phospholipids was administered to the luminal surface. This addition had no detectable influence on the low basal rates of water transport, but blocked the ADH-induced stimulation of water transport. We suggest that surface-active phospholipids on the toad bladder luminal membrane may contribute to the hydrophobic characteristics of this tissue. ADH may act to decrease surface hydrophobicity, facilitating the movement of water molecules across an otherwise impermeable epithelium. This surface alteration may be associated with the appearance of water channels in the apical membrane.     

Apical membrane K conductance in the toad urinary bladder

Summary The conductance of the apical membrane of the toad urinary bladder was studied under voltage-clamp conditions at hyperpolarizing potentials (mucosa negative to serosa). The serosal medium contained high KCl concentrations to reduce the voltage and electrical resistance across the basal-lateral membrane, and the mucosal solution was Na free, or contained amiloride, to eliminate the conductance of the apical Na channels. As the mucosal potential (V _m) was made more negative the slope conductance of the epithelium increased, reaching a maximum at conductance of the epithelium increased, reaching a maximum atV _m=–100 mV. This rectifying conductance activated with a time constant of 2 msec whenV _m was changed abruptly from 0 to –100 mV, and remained elevated for at least 10 min, although some decrease of current was observed. ReturningV _m to+100 mV deactivated the conductance within 1 msec. Ion substitution experiments showed that the rectified current was carried mostly by cations moving from cell to mucosa. Measurement of K flux showed that the current could be accounted for by net movement of K across the apical membrane, implying a voltage-dependent conductance to K (G _K). Mucosal addition of the K channel blockers TEA and Cs had no effect onG _K, while 29mm Ba diminished it slightly. Mucosal Mg (29mm) also reducedG _K, while Ca (29mm) stimulated it.G _K was blocked by lowering the mucosal pH with an apparent pK₁ of 4.5. Quinidine (0.5mm in the serosal bath) reducedG _K by 80%.G _K was stimulated by ADH (20 mU/ml), 8-Br-cAMP (1mm), carbachol (100 m), aldosterone (5×10^–7 m for 18 hr), intracellular Li and extracellular CO₂.     

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.     

Effects of potassium-free media and ouabain on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis

Summary The technique of X-ray microanalysis was used to study the composition of toad urinary bladder epithelial cells incubated in Na Ringer's and K-free medium, with and without ouabain. Following incubation under short-circuit conditions, portions of tissue were coated with an external albumin standard and plunge-frozen. Cryosections were freeze-dried and analyzed. In Na Ringer's, granular and basal cells, and also the basal portion of the goblet cells, had similar water and ion compositions. In contrast, mitochondria-rich cells contained less Cl and Na. On average, the granular cells and a subpopulation of the basal cells lost K and gained Na after ouabain and in K-free medium alone. However, there was considerable variation from cell to cell in the responses, indicating differences between cells in the availabilities of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control. In contrast, the compositions of both the low Cl, mitochondria-rich cells and a sub-population of the basal cells were little affected by the different incubation conditions. This is consistent with a comparatively low Na permeability of these cells. The results also indicate that (i) much, if not all, of the K in the dominant cell type, the granular cells, is potentially exchangeable with serosal medium Na, and (ii) Na is accumulated from the serosal medium under K-free conditions. They also provide information about the role of the (Na–K)-ATPase in the maintenance of cellular K in K-free medium, being consistent with other evidence that removal of serosal medium K inhibits transepithelial Na transport by decreasing Na entry to the cells from the mucosal medium, rather than solely by inhibiting the basolateral membrane (Na–K)-ATPase.     

Time course of vasopressin-induced formation of microvilli in granular cells of toad urinary bladder

Summary Vasopressin-induced transformation of ridges to microvilli on the surface of granular cells of toad urinary bladder occurs in conjunction with induced alterations in the water permeability of the luminal membrane. This study was designed to establish the relationship between the time course for induction of microvilli and the time course for induction of increased water permeability after vasopressin stimulation. Hemibladders were examined at 2.5, 5, 10, 20 and 30 min following exposure to 20 mU/ml of vasopressin and at 5, 10, 20, 30, 40, 50 and 60 min after washout of vasopressin. Within 2.5 min, vasopressin initiated complete transformation of ridges to microvilli on approximately 13% of the granular cells, while osmotic water flow (Jv) was 0.31±0.10 l·min^–1·cm^–2. Five minutes following vasopressin stimulation, microvilli were present on approximately 30% of granular cells andJv was 2.27±0.13 l·min^–1·cm^–2. At 10 minJv was maximum at 4.03±0.15 l·min^–1·cm^–2 and 50% of the granular cells were covered with microvilli. This percentage increased to 70% at 20 min and was maintained at 30 min, althoughJv decreased to 3.9±0.35 l·min^–1·cm^–2 at 30 min. Five minutes following vasopressin washout, ridges interspersed with microvilli reappeared asJv fell to 1.10±0.30 l·min^–1·cm^–2. At 10 min after vasopressin washout,Jv approached basal levels, but the reversal of microvilli to ridges remained incomplete. At 60 min after vasopressin washout, the granular cells had regained their original ridgelike surface structures. Thus, these studies establish a temporal relationship between the induction and reversibility of vasopressin-induced microvillous formation and alterations in the osmotic water permeability of the apical plasmalemma.     

Time course of active Na transport and oxidative metabolism following transepithelial potential perturbation in toad urinary bladder

Summary The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive currentI ^a and the rate of suprabasal CO₂ productionJ _CO2 ^sb were observed in 10 toad urinary bladders at short-circuit and after clamping at 100 mV, serosa positive. Following perturbation of (0100mV),I ^a declined sharply within 1/2 min, remained near constant 15 min, and then increased slightly.J _CO2 ^sb declined more gradually, remained near constant at 4–7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy ofJ _CO2 ^sb starting at 2.9±1.1 (sd) min and lasting 4.7 ±1.8 (sd) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state,I ^a was also nearly constant. Considering that in steady statesI ^a/FJ _Na ^a , the rate of transepithelial active Na transport, during the early quasi-steady state mean values ±se ofJ _Na ^a ,J _CO2 ^sb and (J _Na ^a /J _CO2 ^sb ) were, respectively, 29.9±1.7%, 59.4 ±3.2%, and 56.4±5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4±6.0%, 38.2±6.1%, and 111.3±8.6%. Thus the flow ratioJ _Na ^a /J _CO2 ^sb was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements ofI ^a andJ _CO2 ^sb in three hemibladders were qualitatively similar. In terms of a phenomenological black-box treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations.     

Phenamil: An irreversible inhibitor of sodium channels in the toad urinary bladder

Summary Several new amiloride analogues and two reported photoaffinity analogues were tested for irreversible inhibition of short-circuit current,I _sc, in toad bladder. Bromoamiloride, a photoaffinity analogue, induced 40% irreversible inhibition at 500 m after irradiation with ultraviolet light 320 nm. Iodoamiloride caused no irreversible inhibition. Of the new analogues tested, only 3,5-diamino-6-chloro-N-[(phenylamino) aminomethylene] pyrazinecarboxamide,phenamil, irreversibly inhibitedI _sc at concentrations of 0.05 to 5 m when added to the mucosal solution. Irreversible inhibition ofI _sc by phenamil may be attributed to specific blockage of the mucosal sodium channels, which depended on: 1) time of exposure; 2) mucosal pH: 3) mucosal sodium concentration. For example, 5 m phenamil irreversibly inhibitedI _sc by 38% in 103mm Na at pH 8.6 and nearly 75% in 30mm Na at pH 6.4 after a 40-min exposure. Irreversible inhibition occurred in two phases with time constants of 10 min and approximately 140 min. Due to its irreversible nature, phenamil may be used to measure channel density.     

Single-channel recordings from the apical membrane of the toad urinary bladder epithelial cell

Summary The patch-clamp technique for the recording of single-channel currents was used to investigate the activity of ion channels in the intact epithelium of the toad urinary bladder. High resistance seals were obtained from the apical membrane of tightly stretched tissue. Single-channel recordings revealed the activity of a variety of ion channels that could be classified in 4 groups according to their mean ion conductances, ranging from 5 to 59 pS. In particular, we observed highly selective, amiloridesensitive Na channels with a mean conductance of 4.8 pS, channels with a similar conductance that were not Na-selective and channels with mean conductance values of 17–58 pS that were mostly seen after stimulation of the tissue with vasopressin or cAMP. When inside-out patches from the apical membrane were exposed to 110mm fluoride, large conductances (86–490 pS) appeared.     

Ion selectivity of the apical membrane Na channel in the toad urinary bladder

Summary The ion selectivity of the apical membrane Na channel in the toad urinary bladder was investigated. The electrical potential difference and resistance across the basal-lateral membrane were reduced using high concentrations of KCl in the serosal bathing medium, and gradients for various ions were imposed across the apical membrane by altering the composition of the mucosal bathing medium. Ion fluxes through the channel were measured as the transepithelial current inhibited by amiloride, a specific blocker of the channel's Na conductance. The selectivity sequence for alkali metal cations was H>Li>NaK. K, permeability was barely detectable; the selectivity for Na over K was about 1000:1. Ammonium, hydroxyl ammonium and hydrazinium ions were, like K, virtually impermeant. The results suggest that the size of the unhydrated ion is an important factor in determining permeability in this channel.     

Differential effects of aldosterone and ADH on intracellular electrolytes in the toad urinary bladder epithelium

Summary Quantitative electron microprobe analysis was employed to compare the effects of aldosterone and ADH on the intracellular electrolyte concentrations in the toad urinary bladder epithelium. The measurements were performed on thin freeze-dried cryosections utilizing energy dispersive x-ray microanalysis. After aldosterone, a statistically significant increase in the intracellular Na concentration was detectable in 8 out of 9 experiments. The mean Na concentration of granular cells increased from 8.9±1.3 to 13.2±2.2 mmol/kg wet wt. A significantly larger Na increase was observed after an equivalent stimulation of transepithelial Na transport by ADH. On average, the Na concentration in granular cells increased from 12.0±2.3 to 31.4±9.3 mmol/kg wet wt (5 experiments). We conclude from these results that aldosterone, in addition to its stimulatory effect on the apical Na influx, also exerts a stimulatory effect on the Na pump. Based on a significant reduction in the Cl concentration of granular cells, we discuss the possibility that the stimulation of the pump is mediated by an aldosterone-induced alkalinization.Similar though less pronounced concentration changes were observed in basal cells, suggesting that this cell type also participates in transepithelial Na transport. Measurements in mitochondria-rich cells provided no consistent results.     

Binding of3H-phenamil,an irreversible amiloride analog,to toad urinary bladder: effects of aldosterone and vasopressin

Summary Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al.,J. Membrane Biol. 87:45–54, 1985). In this paper,³H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455±102 channels/m² was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of shortcircuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82±5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that³H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4×10^–7 m. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations.     

Effects of thyromimetric drugs on aldosterone-dependent sodium transport in the toad bladder

Summary Aldosterone increases transepithelial Na⁺ transport in the urinary bladder ofBufo marinus. The response is characterized by 3 distinct phases: 1) a lag period of about 60 min, ii) an initial phase (early response) of about 2 hr during which Na⁺ transport increases rapidly and transepithelial electrical resistance falls, and iii) a late phase (late response) of about 4 to 6 hr during which Na⁺ transport still increases significantly but with very little change in resistance. Triiodothyronine (T₃, 6nm) added either 2 or 18 hr before aldosterone selectively antagonizes the late response. T₃ per se (up to 6nm) has no effect on base-line Na⁺ transport. The antagonist activity of T₃ is only apparent after a latent period of about 6 to 8 hr. It is not rapidly reversible after a 4-hr washout of the hormone. The effects appear to be selective for thyromimetic drugs since reverse T₃ (rT₃) is inactive and isopropyldiiodothyronine (isoT₂) is more active than T₃. The relative activity of these analogs corresponds to their relative affinity for T₃ nuclear binding sites which we have previously described. Our data suggest that T₃ might control the expression of aldosterone by regulating gene expression, e.g. by the induction of specific proteins, which in turn will inhibit the late mineralocorticoid response, without interaction with the early response.     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.     

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

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

Dopaminergic inhibition of vasopressin-stimulated water flow in the toad bladder: Evidence for local formation of dopamine

Summary Dopamine administration increases renal excretion of water and Na. It remains uncertain whether these effects of dopamine are the result of a hemodynamic effect or the consequence of a direct cellular action. We investigated the effect of dopamine on water transport by the isolated toad bladderin vitro. Dopamine failed to alter baseline water flow but caused a significant inhibition of arginine vasopressin (AVP) or cyclic adenosine monophosphate (AMP) stimulated water flow. The effect of dopamine on stimulated water flow was not due to activation of adrenergic, adrenergic, or cholinergic receptors. The selective antagonists of dopamine, metoclopramide and apomorphine, prevented the effect of dopamine on AVP-stimulated water flow. These observations suggest the existence of a dopaminergic receptor in the toad bladder.l-Dopa also inhibited AVP-stimulated water flow. The effect ofl-Dopa could be prevented by metoclopramide, thus suggesting thatl-Dopa is converted to dopamine by an aromatic amino acid decarboxylase present in the toad bladder. To investigate this possibility we measured the effect of the decarboxylase inhibitor, carbidopa, on the¹⁴CO₂ production generated by decarboxylation of¹⁴Cl-Dopa in isolated toad bladder epithelial cells. Isolated toad bladder epithelial cells generated significant amounts of¹⁴CO₂ from¹⁴Cl-Dopa. This effect could be blocked by carbidopa, thus suggesting the existence of an aromatic amino acid decarboxylase system in the toad bladder. Carbidopa also prevented the inhibitory effect ofl-Dopa on AVP-stimulated water flow, suggesting thatl-Dopa needs to be converted to dopamine to inhibit water flow. These data suggest the existence of a dopaminergic receptor in the toad bladder. These data also suggest that dopamine can be formed locally in the toad bladder and can thus serve as a local modulator of water transport.     

Cytochalasin B and water transport

Summary A morpho-functional study of the effects of cytochalasin B (CB) on Na and water transport was made in amphibian epithelia. The functional studies confirmed the dissociation of the natriferic and hydrosmotic effects of vasopressin in toad urinary bladders exposed to CB and showed in addition that the block of the hydrosmotic effect was reversible and could still be induced in epithelia maximally stimulated with the hormone. Scanning electron microscopy revealed that CB, per se, did not alter the apical surface of the bladders. An almost total loss of microvilli of granular cells was seen, however, if CB was associated with vasopressin and an osmotic gradient. The results suggest two points: a) the block of the hydrosmotic flow induced by CB is due to factors beyond the apical membrane; b) microfilaments may be important mechanochemical transducers in the chain of events leading to the hydrosmotic effect of vasopressin.Supported by the grants Nos 3.1300.73 and 3.043-0.76 of the Swiss National Science FoundationThe authors are grateful to Miss C. Brücher, SEM operator of the Department of Physics, Ciba-Geigy, for skillful collaboration, to Mr. R. Mira for the illustrations and to Mrs. A. Cergneux for secretarial assistance     

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.