The participation of intracellular membranes in forming highly permeable domains in the plasma membrane of epithelial cells during the vasopressin stimulation of water transport]

Using different electron microscopic techniques, parallel studies of structural alterations in the apical membrane and specific granules of the frog urinary bladder granular cells were made. The results obtained suggest the participation of granule membranes in the formation of highly permeable domains in the apical membranes. After ADH action, the domains with high water permeability are internalized bringing cell membrane retrieval.     

Electron microscopic study of colonic epithelial cells from the grass frog Rana temporaria under different intensity of water absorption

Three cell types have been revealed in the epithelium of the frog large intestine: granular, mitochondria-rich, and mucosal cells. Under a low water permeability (0.12 +/- 0.10 mkl/(min.cm2)) the distribution of intramembrane particles (IMP) in the apical cell membrane was the same as in the most cell plasma membranes studied with freeze-fracture method. Under rising osmotic permeability and water absorption (0.43 +/- 0.05 mkl/(min.cm2)) the IMP distribution did not change. In these conditions, the quantity of fusion sites between granule membranes and the apical membrane increased, and the intercellular spaces in basolateral epithelial region were diluted. A a low water permeability, in addition to usual microtubules, bundles of noncentrosomal microtubules with associated osmiophilic globules were revealed. A comparative analysis has been made of the present evidence and previously obtained data on the frog urinary bladder epithelium.     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

Cholesterol localization in the membranes of granular cells in the bladder epithelium of the frog during stimulated water transport

The polyene antibiotic filipin has been used to characterize the cholesterol distribution in the membranes of resting and ADH-stimulated frog urinary bladder in freeze-fracture replicas. In general, the intracellular membranes takes up filipin only insignificantly. An exception is the cholesterol rich granule membrane. Both density and polarity of filipin-induced deformations were evaluated, and the asymmetry in membrane cholesterol was analysed. Upon ADH-stimulation of water flow both density and polarity of filipin-induced deformations altered differently in apical and basolateral regions of the plasma membrane. This difference is presumably due to the stretching of the basolateral membrane as a result of swelling, on the one hand, and to incorporation of aggregate containing membranes into the apical membrane, on the other one. The results obtained may suggest that the appearance of ADH-induced intramembranous particle aggregates in the apical membrane be accompanied with a relative cholesterol decrease in this apical membrane.     

Structural and functional characteristics of the cellular reaction of the bladder epithelium in the frog to calcium extraction from the apical and basolateral membranes

Extraction of Ca++ ions from cells of the frog urinary bladder serosa side is followed by an increase in the bladder wall permeability for water and inulin. Ultrastructural changes were observed, such as destruction of cell junctions, swelling of the cell and their organelles, reconstruction of the cytoskeleton elements. The free calcium Ringer solution injected in the bladder lumen does not change the permeability of the wall for water and sodium ions. In this case the cell response to the antidiuretic hormone decreases; the ultrastructure of cells and intercellular junctions is not disturbed; the distribution of intramembrane particles on the P- and E-faces of the apical membrane is normal. The above results indicate that there are qualitative differences in the cell response towards the extraction of Ca++-ions between the serosal and mucosal membranes. This also suggests that on the external surface of the apical membrane Ca++ ions may play a very important role in redistribution of intramembrane particles under the action of the antidiuretic hormone.     

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.     

Subcellular redistribution of AQP5 by vasoactive intestinal polypeptide in the Brunner's gland of the rat duodenum

Aquaporin (AQP)5, an exocrine-type water channel, was detected in the rat duodenum by Western blot analysis, and was localized by immunohistochemistry in the secretory granule membranes as well as in the apical and lateral aspects of the plasma membrane of Brunner's gland cells. Incubation of duodenal slices with vasoactive intestinal polypeptide (VIP) in vitro significantly increased the amount of AQP5 in the apical membrane fraction in a dose- and time-dependent manner with the amount reaching a plateau at 100 nM VIP and becoming near maximal after a 30-s incubation. Protein kinase inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 50 muM), and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; PKA-specific, 1 muM) blocked this increase, but PKC-specific inhibitor calphostin C did not, implying the involvement of PKA but not PKC in this cellular event. Intravenous injection with VIP (40 mug/kg body wt) provoked dilation of the lumen of the Brunner's gland at 2 and 7 min and increased the staining intensity of AQP5 in the apical and lateral membranes. AQP1 (both nonglycosylated and glycosylated forms) was also found to localize in the apical and basolateral membranes of cells of Brunner's gland. VIP, however, did not provoke any significant change in the AQP1 level in the apical membrane, as judged from the results of the above in vitro and in vivo experiments. These results suggest that VIP induced the exocytosis of granule contents and simultaneously caused translocation of AQP5 but not of AQP1 to the apical membrane in Brunner's gland cells.     

On the large error introduced in the estimate of the density of membrane pores from permeability measurements when diffusion in "unstirred layer" within the cells is disregarded

After the development of the "black lipid membrane" techniques, studies of the permeability of labeled water and nonelectrolytes across these artificial membranes have yielded permeability constants comparable in magnitude to those obtained from tracer studies of living cell membranes. This general agreement has affirmed the belief that the living cell membranes are indeed closely similar to these bilayer phospholipid membranes. In this report, we draw attention to a hidden assumption behind such comparisons made: the assumption that labeled material passing through the cell membrane barriers instantly reaches diffusion equilibrium inside the cell. The permeability constants to labeled water (and nonelectrolytes) across lipid layers were obtained using setups in which the lipid membrane was sandwiched between aqueous compartments both of which were vigorously stirred. In studies of permeability of living cell membranes only the outside solution was stirred, the intracellular water remained stationary. Yet the calculations of permeability constants of the cell membrane were made with the tacit assumption, that once the labeled materials pass through the cell membrane, they were instantly mixed with the entire cell contents as if a stirrer operating at infinite speed had been present inside the cells. Ignoring this unstirred condition of the intracellular water, in fact, lumped all the real-life delay due to diffusion in the cytoplasm and added it to the resistance to diffusion of the membrane barrier. The result is an estimated membrane permeability to labeled water (and nonelectrolytes) many times slower than it actually is. The present report begins with a detailed analysis of a specific case: tritiated water diffusion from giant barnacle muscle fibers and two non-living models, one real, one imagined.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodi-phosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

GRAMP 100: a membrane protein concentrated on secretory granule membranes and the apical cell surface in exocrine acinar cells.

Using a monoclonal antibody (SG10A6) raised against secretion granule membranes of the rat parotid gland, we have identified an antigen that is a common component of both exocrine pancreatic and parotid granule membranes. SG10A6 (an IgM) immunoprecipitates antigen that migrates as a single band (M(r) approximately 80 KD unreduced; M(r) approximately 100 KD reduced) and immunoblots at least two polypeptides that are similar to the reduced and nonreduced immunoprecipitated antigen. This granule-associated membrane polypeptide (GRAMP 100; named for the apparent M(r) in reduced form) is also a prominent component of plasma membrane fractions. Immunocytochemical localization at the electron microscopic level demonstrates the presence of GRAMP 100 on granule membranes, especially condensing vacuoles and exocytotic figures, and the apical plasma membrane. Lower levels of antigen are detected on basolateral plasma membrane and on peri-Golgi membranes that may be part of the endosomal system. Both the cell fractionation and immunocytochemical localization indicate that GRAMP 100 differs in distribution from GRAMP 92 and 30K SCAMPs, two other components of exocrine granule membranes identified with monoclonal antibodies. To date, no polypeptides have been identified with this approach that are exclusive components of exocrine granule membranes.     

Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.     

Identification of SNAREs involved in regulated exocytosis in the pancreatic acinar cell.

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca(2+), suggesting that they are controlled by different Ca(2+)-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.     

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.     

Evidence for proteic water pathways in the luminal membrane of kidney proximal tubule

The osmotic permeability of the apical membrane of proximal tubule cells was studied on rat brush-border membrane vesicles by following their rate of shrinkage with a stopped-flow device coupled to light transmission recording. The mercuric sulfhydryl reagent para-chloromercuribenzenesulfonic acid (PCMBS) reduced the water permeability of the membrane, in a time- and dose-dependent manner, to 35% of the control value. Mercuric chloride was a more potent inhibitor and decreased the osmotic water permeability of the brush-border membrane to 15% of the control. This inhibition was reversed by an excess of cysteine, while cysteine per se did not modify the rate of vesicle shrinkage. These results suggest that most of the osmotic water movements across kidney brush-border membranes are through polar pathways which involve the integrity of the membrane proteins.     

Influence of temperature and membrane lipid composition on the osmotic water permeability of teleost gills.

Osmotic water uptake was measured gravimetrically in isolated, ligated gill arches from trout (acclimated to and incubated at 5 degrees and 20 degrees C) and tilapia (21.5 degrees and 33 degrees C). For both species, incubation of arches at the higher temperature led to 1.5- to 3-fold greater measures of water weight gain. However, gills from warmer-acclimated trout and tilapia had 1- to >3-fold lower the initial rate and 1.5- to >2.5-fold lower the extent of water uptake seen in colder-acclimated conspecifics. Both the incubation temperature sensitivity and the acclimation effects are consistent with transmembrane water permeation. Calcium-free incubations (permitting paracellular water movement) also indicated that interfacial cell membranes contribute to gill permeability characteristics; without calcium, trout gill osmotic water uptake values increased 1.5- to 2-fold, and the temperature dependence of water uptake decreased (initial rate) or was eliminated (extent). The specific contribution of cholesterol to restricting barrier membrane water permeability was indicated by concentration-dependent increases in water uptake in the presence of either nystatin (a cholesterol-complexing, pore-forming agent) or methyl-beta-cyclodextrin (which selectively depletes membrane cholesterol). In addition, a cholesterol-specific cytochemical probe (filipin) intensely labeled the apical surface membranes of trout and tilapia gill epithelium. In summary, these studies implicate membrane cholesterol in determining water permeability in fish gills.     

Permeability properties of apical and basolateral membranes of the guinea pig caecal and colonic epithelia for short-chain fatty acids

Unidirectional fluxes of short-chain fatty acids (SCFA) indicated marked segmental differences in the permeability of apical and basolateral membranes. The aim of our study was to prove these differences in membrane permeability for a lipid-soluble substance and to understand the factors affecting these differences. Apical and basolateral membrane fractions from guinea pig caecal and colonic epithelia were isolated. Membrane compositions were determined and the permeability of membrane vesicles for the protonated SCFA was measured in a stopped-flow device. Native vesicles from apical membranes of the caecum and proximal colon have a much lower permeability than the corresponding vesicles from the basolateral membranes. For the distal colon, membrane permeabilities of native apical and basolateral vesicles are similar. In vesicles prepared from lipid extracts, the permeabilities for the protonated SCFA are negatively correlated to cholesterol content, whereas no such correlation was observed in native vesicles. Our findings confirm that the apical membrane in the caecum and proximal colon of guinea pig is an effective barrier against a rapid diffusion of small lipid-soluble substances such as SCFAH. Besides cholesterol and membrane proteins, there are further factors that contribute to this barrier property.     

Plasma membrane of the rat parotid gland: preparation and partial characterization of a fraction containing the secretory surface

A plasma membrane fraction from the rat parotid gland has been prepared by a procedure which selectively enriches for large membrane sheets. This fraction appears to have preserved several ultrastructural features of the acinar cell surface observed in situ. Regions of membrane resembling the acinar luminal border appear as compartments containing microvillar invaginations, bounded by elements of the junctional complex, and from which basolateral membranes extend beyond the junctional complex either to contact other apical compartments or to terminate as free ends. Several additional morphological features of the apical compartments suggest that they are primarily derived from the surface of acinar cells, rather than from the minority of other salivary gland cell types. Enzymatic activities characteristically associated with other cellular organelles are found at only low levels in the plasma membrane fraction. The fraction is highly enriched in two enzyme activities--K+ -dependent p-nitrophenyl phosphatase (K+ -NPPase, shown to be Na+/K+ adenosine triphosphatase; 20-fold) and gamma-glutamyl transpeptidase (GGTPase; 26-fold)--both known to mark plasma membranes in other tissues. These activities exhibit different patterns of recovery during fractionation, suggesting their distinct distributions among parotid cellular membranes. Secretion granule membranes also exhibit GGTPase, but no detectable K+ -NPPase. Since Na+/K+ adenosine triphosphatase and GGTPase, respectively, mark the basolateral and apical cellular surfaces in other epithelia, we hypothesize that these two enzymes mark distinct domains in the parotid plasmalemma, and that GGTPase, as the putative apical marker, may signify a compositional overlap between the two types of membranes which fuse during exocytosis.     

Osmotic water permeability of Necturus gallbladder epithelium

An electrophysiological technique that is sensitive to small changes in cell water content and has good temporal resolution was used to determine the hydraulic permeability (Lp) of Necturus gallbladder epithelium. The epithelial cells were loaded with the impermeant cation tetramethylammonium (TMA+) by transient exposure to the pore-forming ionophore nystatin in the presence of bathing solution TMA+. Upon removal of the nystatin a small amount of TMA+ is trapped within the cell. Changes in cell water content result in changes in intracellular TMA+ activity which are measured with intracellular ion-sensitive microelectrodes. We describe a method that allows us to determine the time course for the increase or decrease in the concentration of osmotic solute at the membrane surface, which allows for continuous monitoring of the difference in osmolality across the apical membrane. We also describe a new method for the determination of transepithelial hydraulic permeability (Ltp). Apical and basolateral membrane Lp's were assessed from the initial rates of change in cell water volume in response to anisosmotic mucosal or serosal bathing solutions, respectively. The corresponding values for apical and basolateral membrane Lp's were 0.66 x 10(-3) and 0.38 x 10(-3) cm/s.osmol/kg, respectively. This method underestimates the true Lp values because the nominal osmotic differences (delta II) cannot be imposed instantaneously, and because it is not possible to measure the true initial rate of volume change. A model was developed that allows for the simultaneous determination of both apical and basal membrane Lp's from a unilateral exposure to an anisosmotic bathing solution (mucosal). The estimates of apical and basal Lp with this method were 1.16 x 10(-3) and 0.84 x 10(-3) cm/s.osmol/kg, respectively. The values of Lp for the apical and basal cell membranes are sufficiently large that only a small (less than 3 mosmol/kg) transepithelial difference in osmolality is required to drive the observed rate of spontaneous fluid absorption by the gallbladder. Furthermore, comparison of membrane and transepithelial Lp's suggests that a large fraction of the transepithelial water flow is across the cells rather than across the tight junctions.     

Permeabilities of teleost and elasmobranch gill apical membranes: evidence that lipid bilayers alone do not account for barrier function

Teleosts and elasmobranchs faced with considerable osmotic challenges living in sea water, use compensatory mechanisms to survive the loss of water (teleosts) and urea (elasmobranchs) across epithelial surfaces. We hypothesized that the gill, with a high surface area for gas exchange must have an apical membrane of exceptionally low permeability to prevent equilibration between seawater and plasma. We isolated apical membrane vesicles from the gills of Pleuronectes americanus (winter flounder) and Squalus acanthias (dogfish shark) and demonstrated approximately sixfold enrichment of the apical marker, ADPase compared to homogenate. We also isolated basolateral membranes from shark gill (enriched 2.3-fold for Na-K-ATPase) and using stopped-flow fluorometry measured membrane permeabilities to water, urea, and NH(3). Apical membrane water permeabilities were similar between species and quite low (7.4 +/- 0.7 x 10(-4) and 6.6 +/- 0.8 x 10(-4) cm/s for shark and flounder, respectively), whereas shark basolateral membranes showed twofold higher water permeability (14 +/- 2 x 10(-4) cm/s). Permeabilities to urea and NH(3) were also low in apical membranes. Because of the much lower apical to basolateral surface area we conclude that the apical membrane represents an effective barrier. However, the values we obtained were not low enough to account for low water loss (teleosts) and urea loss (elasmobranchs) measured in vivo by others. We conclude that there are other mechanisms which permit gill epithelia to serve as effective barriers. This conclusion has implications for the function of other barrier epithelia, such as the gastric mucosa, mammalian bladder, and renal thick ascending limb.     

On the role of calcium in the mechanism of ADH action

With an increased influx of Ca2+ in the cytoplasm, the response of cells to ADH in the urinary bladder of the frog was lowered by addition of ionophore A23187 from the side of the basolateral cell membrane, but inhibited when it was added from the apical cell membrane. The removal of calcium by EGTA from the serosal surface was accompanied by a sharp increase of osmotic permeability not only to water, but also to inulin; while when calcium was removed from the mucosal surface of the urinary bladder, osmotic permeability was not changed. After being added to the Ringer solution from the outer surface of the apical cell membrane, the inhibitors of Ca2+ channels (verapamil, Ni2+, Mn2+, Co2+) decreased the effect of ADH. These data indicate that Ca2+ applied onto the outer surface of apical plasma membrane plays an important role in the action of ADH.