Ultrastructural correlates of the antidiuretic hormone-dependent and antidiuretic hormone-independent increase of osmotic water permeability in the frog urinary bladder epithelium

Electron and confocal microscopy, using immunocytochemical methods, was employed to assess osmotic water permeability of the frog (Rana temporaria) urinary bladder during transcellular water transport, induced by antidiuretic hormone (ADH) or by wash-out of autacoids from serosal, ADH-free Ringer solution. The increase of osmotic water permeability of the urinary bladder was accompanied by relevant ultrastructural changes, the most remarkable being: (1) the appearance of aggregates of intramembranous particles in the apical membrane of granular cells, and the extent of the membrane area covered by the aggregates proportional to that of the water flow; (2) redistribution of actin filaments in the cytoplasm of granular cells; judging from the anti-actin label density, the number of actin filaments in the apical region of cytoplasm was reduced by 2.5–4 times compared with normal; (3) a decrease in the total electron density of the cytoplasm due to the increased water content of granular cells.     

The membrane action of antidiuretic hormone (ADH) on toad urinary bladder.

Radioactive tracer and electrical techniques were used to study the transport of nonelectrolytes and sodium, respectively, across toad urinary bladders in the presence and absence of ADH. The permeability of lipophilic molecules was roughly proportional to bulk phase oil/water partition coefficients both in the presence and absence of hormone; i.e., ADH elicited a general nonselective increase in the permeation of all nine solutes tested. The branched nonelectrolyte, isobutyramide, was less permeable than its straight-chain isomer, n-butyramide, in control tissues. ADH reduced the discrimination between these structural isomers. Hydrophilic solutes permeated more rapidly than expected. In the presence of hormone, there was no change in the permeation of large hydrophilic solutes considered to move via an extracellular pathway, but there was a marked increase in the permeability of water and other small hydrophilic solutes. Collectively, these results suggest that ADH acts to increase the motional freedom or fluidity of lipids in the cell membrane which is considered to be the preferred pathway for the permeation of lipophilic and small hydrophilic molecules. At concentrations of cAMP and ADH which elicit equivalent increments in the shortcircuit current, the effects of these agents on nonelectrolyte transport and membrane electrical conductance are divergent. Such observations suggest that some membrane effects of ADH may not be directly dependent upon cAMP. ADH in the mucosal solution increased the permeability of the toad bladder when the surface charge on the outer surface of the apical membrane was screened with the polyvalent cation, La-3+. These experiments emphasize that interaction of ADH with membranes of toad urinary bladder may account for at least some effects of this hormone.     

Increases in permeability of Escherichia coli outer membrane induced by polycations

The action of polycations (such as polylysine and compound 48/80) on Escherichia coli was studied with use of Ca2+, K+ and TPP+ ion-selective electrodes. Rapid efflux of Ca2+ was observed when a polycation was added in cell suspension. The polycation treatment promoted a drug-inducing K+ release from the cytoplasmic membrane. TPP+ uptake was also increased by addition of a polycation. Without the polycation treatment, the uptake of TPP+ was largely suppressed due to a permeability barrier of the outer membrane. The results show that a polycation disrupted the permeability barrier of the outer membrane.     

The influence of amlodipine and verapamil on ion and water transport in the nephron, skin and urinary bladder of amphibians.

1. The addition of amlodipine or verapamil into the lumen of the newt distal tubule led to the decrease of reabsorption of Na, Cl, Ca and of fluid. 2. The application of amlodipine to the outside of the frog skin caused large increases in potential difference (PD) and short circuit (SCC) similar to what is seen with Co2+. If both amlodipine and Co2+ were applied simultaneously to the outer surface the increases in PD and SCC were additive. 3. Verapamil added to the outer surface of the skin caused a reduction in PD which could be overcome by subsequent addition of amlodipine. 4. After addition of amlodipine to serosal or mucosal surfaces of the frog urinary bladder, the ability of vasopressin to increase osmotic permeability was markedly attenuated. 5. It is likely that the calcium channel blockers used here not only affect intracellular calcium levels by inhibiting entry through calcium channels, but they may also alter calcium dependent processes within the plasma membranes which modulate sodium transfer across epithelia.     

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.     

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.     

Cation-selective channels in amphibian epithelia: electrophysiological properties and activation

1. Na+ as well as Li+ move across the apical membrane through amiloride-sensitive ionic channels. 2. K+ movements across the apical membrane occur through Ba2+- and Cs+-sensitive channels which do not allow the passage of Na+ or Li+. 3. A third pathway in the apical membrane is permeable for Na+, K+, Cs+, Rb+, NH+4 and Ti+. The currents carried by these monovalent cations are blocked by Ca2+ and divalent cations as well as La3+. 4. In the urinary bladder, the Ca2+-sensitive currents are stimulated by oxytocin, activators of cytosolic cAMP and cAMP analogues. Also the oxytocin activated currents are blocked by divalent cations and La3+. 5. Nanomolar concentrations of mucosal Ag+ activate the third channel and open the pathway for movements of Ca2+, Ba2+ and Mg2+, which are known to permeate through Ca2+ channels in excitable tissues.     

Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.     

New evidence about the relationship between water channel activity and calcium in salinity-stressed pepper plants

This study, of how Ca2+ availability (intracellular, extracellular or linked to the membrane) influences the functionality of aquaporins of pepper (Capsicum annuum L.) plants grown under salinity stress, was carried out in plants treated with NaCl (50 mM), CaCl2 (10 mM), and CaCl2 (10 mM) + NaCl (50 mM). For this, water transport through the plasma membrane of isolated protoplasts, and the involvement of aquaporins and calcium (extracellular, intracellular and linked to the membrane) has been determined. After these treatments, it could be seen that the calcium concentration was reduced in the apoplast, in the cells and on the plasma membrane of roots of pepper plants grown under saline conditions; these concentrations were increased or restored when extra calcium was added to the nutrient solution. Protoplasts extracted from plants grown under Ca2+ starvation showed no aquaporin functionality. However, for the protoplasts to which calcium was added, an increase of aquaporin functionality of the plasma membrane was observed [osmotic water permeability (Pf) inhibition after Hg addition]. Interestingly, when verapamil (a Ca2+ channel blocker) was added, no functionality was observed, even when Ca2+ was added with verapamil. Therefore, calcium seems to be involved in plasma membrane aquaporin regulation via a chain of processes within the cell but not by alteration of the stability of the plasma membrane.     

Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride (50 microM), indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+). Lowering pHb in the presence of oxytocin (50 mU/ml) produced a transepithelial current (3 microA.cm-2 at pHb 5.5) which was blocked by 100 microM of Hg2+, Zn2+, or Ni2+ at the basolateral side, and by DCCD (10(-5) M) or Hg2+ (100 microM) from the apical side. The net hydroosmotic water flux (JH2O) induced by oxytocin in frog bladder sacs was blocked by inhibitors of H(+)-adenosine triphosphatase (ATPase). Diethylstilbestrol (DES 10(-5) M), oligomycin (10(-8) M), and DCCD (10(-5) M) prevented JH2O when present in the lumen. These effects cannot be attributed to inhibition of metabolism since cyanide (10(-4) M), or 2-deoxyglucose (10(-3) M) had no effect on JH2O.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Morphometric analysis of epithelial cells of frog urinary bladder. I. Effect of antidiuretic hormone, calcium ionophore (A23187) and PGE2

Changes in epithelial cell morphology, especially at the apical plasma membrane, are frequently cited as initial evidence for antidiuretic hormone (ADH)-induced increase in membrane permeability. The effects of ADH and agents that alter and modify calcium and prostaglandin concentrations on the morphology and cytology of the epithelial cells of frog (Rana pipiens) urinary bladder are presented using the techniques of transmission and scanning electron microscopy. It was found that, like ADH, calcium ionophore, A23187, produce intense microvilli formation, microfilament mobilization and an increase in the density of granules and membrane associated vesicles, suggesting a prominent role of calcium in these processes. Moreover, our results suggest that these membrane and cytosolic transformations may be mediated in part through prostaglandin formation, as exogenous PGE2 mimicked these effects, and indomethacin, a prostaglandin synthesis inhibitor, attenuated ionophore's effect on luminal cytomorphology. However, unlike ADH, prostaglandins and ionophore inhibit hormonal-induced increase in transepithelial water flow. These results suggest that other components more distal to the luminal membrane, perhaps the basolateral membrane, may be rate-limiting for transepithelial water flow and possibly are regulated by either changes in calcium concentrations or prostaglandins.     

Peculiarities of Signal Transduction from Arginine-Vasopressin Receptors Located on the Apical Surface of Epithelial Cells of the Frog Urinary Bladder and on the Amoeba Outer Membrane

Mechanisms of action of arginine-vasopressin (AVP) on water transport across the wall of urinary bladder of the frog R. temporaria L. and the outer membrane of the amoeba A. proteus were considered. AVP and its functional analogs were added to the basolateral and apical surfaces of the frog urinary bladder membrane and to the amoeba outer surface. In amoeba the AVP effect was evaluated from action on frequency of contractions of contractile vacuole that represents its organ of water—electrolyte homeostasis. It was shown that the system of signal transduction from the apical AVP receptors differed considerably from that located on the basolateral membrane and had a marked similarity with the system of signal transduction from AVP-sensitive structures on the outer membrane of amoeba. The urinary bladder apical membrane as well as the amoeba outer membrane has a mixed type of sensitivity to AVP with unidirectional (not antagonistic like on the basolateral membrane) involvement of systems coupled to receptors of the V₁ and V₂ type. We suggest that the system of signal transduction from apical AVP receptors corresponds to the earlier stage of evolutionary development.     

76 and 14 kDa polypeptides, two major components released from amphibian urinary bladder epithelium. Localization and potential role

Several experimental conditions such as antidiuretic hormone (ADH) challenge, apical treatment with phorbol myristate acetate (PMA), and mechanical stretching of the tissue are known to increase the insertion of intramembrane particle aggregates and/or granule exocytosis at the apical border of epithelial cells of amphibian urinary bladders. A constant release of 2 peptides of 76 and 14 kDa apparent molecular mass, respectively, was associated with these treatments. The localization of these 2 polypeptides was assessed by immunofluorescence and electron microscopy immunocytochemistry using fluorescent, peroxidase, and colloidal gold probes. The 76 kDa polypeptide appeared to be associated with the cell coat and with the granule content which is released at the apical cell surface. The 14 kDa peptide was also found in the cell coat, and postembedding immunocytochemistry indicates its presence in cytoplasmic subapical vesicles (aggrephores and/or granules). The migration of these 76 and 14 kDa polypeptides in SDS-polyacrylamide gel electrophoresis was modified neither by a treatment at 90 degrees C, nor by the presence or absence of calcium in the medium. Treatment with EGTA did not modify the fluorescence emission of the two peptides and, consequently, they are probably not among the major calcium binding proteins. The addition to the mucosal medium of the stretch extract or of antibodies raised against the 76 and 14 kDa peptides did not modify ADH-induced water permeability. However, a significant decrease of the hydrosmotic response to ADH occurred in subsequent stimulation-washout cycles when the anti-14 kDa peptide antiserum was applied to the mucosal bath. When the bladders were incubated with a stretch extract, we observed a slight alteration of the short-circuit current (Isc), an increase of the basal Na+ transport, and a decrease of the maximal Isc in response to ADH. The 76 kDa protein, released in the apical medium, could play a protective role in the cellular plasma membrane and could participate in the formation of the thick cell coat lining the apical membrane of the granular cells. The 14 kDa protein might be one of the proteins associated with the aggregates, but further studies will be necessary to clarify its exact role in the ADH-induced permeability modifications observed in amphibian urinary bladders.     

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.     

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.     

Spatial Organization of Calcium Signaling Involved in Cell Volume Control in the Fucus Rhizoid

Subprotoplasts prepared from different regions of rhizoid and thallus cells of Fucus zygotes displayed mechanosensitive plasma membrane channels in cell-attached patch-clamp experiments by using laser microsurgery. In excised patches, this channel was found to be voltage gated, carrying K+ outward and Ca2+ inward, with a relative permeability of Ca2+/K+ of 0.35 to 0.5, and an increased open probability at membrane potentials more positive than -80 mV. No significant difference was found in the density of this channel type from different regions of rhizoid or thallus cells. Hypoosmotic treatment of intact zygotes induced dramatic transient elevations of cytoplasmic Ca2+, initiating at the rhizoid apex and propagating in a wavelike manner to subapical regions. Localized initiation of the Ca2+ transient correlated with greater osmotic swelling at the rhizoid apex compared with other regions of the zygote. Ca2+ transients exhibited a refractory period between successive hypoosmotic shocks, during which additional transients could not be elicited and the ability to osmoregulate was impaired. Buffering the Ca2+ transients with microinjected Br2BAPTA similarly reduced the ability of rhizoid cells to osmoregulate. Ca2+ influx was associated with the initiation of the Ca2+ transient in apical regions, whereas intracellular sources contributed to its propagation. Thus, localized signal transduction is patterned by interactions of the cell wall, plasma membrane, and intracellular Ca2+ stores.     

Possible role of Ca2+-binding sites in the regulation of Na+ transport in toad urinary bladder

La3+ was used to assess the role of membrane-bound Ca2+ in the regulation of basal and antidiuretic hormone (ADH)-induced Na+ transport by the isolated toad urinary bladder. Na+ transport was monitored by means of a short-circuit current (Isc) device. Mucosal La3+ (0.5-5 mM) increased Isc, while serosal La3+ (5 mM) produced a biphasic response (stimulation followed by inhibition). The stimulatory effects of La3+ were additive when present on both sides and were suppressed by mucosal amiloride or serosal ouabain. The action of mucosal La+ was reversible but the inhibition produced by serosal La3+ was not. In the presence of serosal La3+ the natriferic effect of ADH was abolished, but Theophylline, dibutyryl-cAMP, Amphotericin B, mucosal La3+, mucosal low pH, and phospho(enol) pyruvate, were able to increase Isc. These results suggest that Ca2+ binding sites in apical and basolateral membranes may play a key role in the modulation of both basal and ADH-induced Na+ transport. Serosal La3+ apparently inactivates the hormone-receptor interaction and/or the link between the ADH-receptor complex and the activation of adenylate cyclase, but does not interfere with the operation of the Na+ "pump", the basal activity of adenylate cyclase or any of the intracellular events that mediate the effect of ADH on Na+ transport.     

Capacity of diphosphonates to bind calcium and their effect on the osmotic permeability of the frog bladder wall

Like EDTA, diphosphonates increase permeability of the frog urinary bladder wall for water osmotic gradient. Their effect is proportional to the stability of their binding with calcium ions. The efficacy of diphosphonates falls upon pH decline. The results indicate the significance of Ca binding in cells and membrane action of diphosphonates.     

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.