Membrane structural specialization of the toad urinary bladder revealed by the freeze-fracture technique

Summary Examination of the toad urinary bladder by freeze-fracture electron microscopy reveals that the mitochondria-rich cells of the epithelium possess distinctive and characteristic membrane structural specialization. Unique rod-shaped intramembrane particles are found in luminal and basal membranes as well as certain intracellular vesicles of this cell type. The consistent finding of two discrete patterns of luminal membrane structural organization supports the possibility that two morphological forms of mitochondria-rich cell exist within the toad bladder epithelium.     

Intramembrane particle structures in epithelial cells of the toad urinary bladder: a quantitative freeze-fracture study

Freeze-fracture electron microscopy reveals intramembrane particle arrays in basal membranes of granular epithelial cells as well as both upper and lower plasma membranes of the underlying basal cells in the toad urinary bladder. These particle arrays are morphologically indistinguishable from the luminal membrane aggregates which are known to be associated with antidiuretic hormone (ADH)-stimulated water transport. In both granular and basal cells particle arrays are frequently located in and/or around the openings of vesicular and/or tubular structures fused to the plasma membranes, suggesting that they may be transferred from the cytoplasm by membrane fusion. Quantification of cytoplasmic aggrephores in control granular cells shows that they can be numerous and as close to the basolateral membrane as they are with the luminal membrane, to which they are known to fuse and deliver aggregates upon ADH stimulation. Aggrephore-like tubules were also found in the basal cells. Particle array densities were quantified for 6 pairs of control and ADH-stimulated hemibladders. At least 1440 microns 2 area of plasma membrane for each membrane domain was examined. Results indicate that the presence of these particle arrays in granular and basal cell membranes is highly variable and that exposure to ADH does not cause a statistically significant increase in their frequency.     

Insulin-stimulated sodium transport in toad urinary bladder

Mammalian and teleost insulins increase active sodium transport by the toad urinary bladder at subnanomolar concentrations. This stimulation is evident within 15 min and persists for hours. Porcine proinsulin and a cross-linked derivative of bovine insulin are less effective than porcine insulin in stimulating the short-circuit current (SCC), indicating the specificity appropriate for activation of sodium transport through an insulin receptor. The initial stimulation by insulin of the SCC is not blocked by pretreatment with actinomycin D, puromycin, cycloheximide, or tunicamycin. However, in the presence of any one of these inhibitors the sustained increase in SCC is blocked and the rise is short-lived, lasting only 45 to 90 min. In amphotericin-treated bladders, the addition of insulin did not further stimulate SCC.     

Stretch induces granule exocytosis in toad urinary bladder

Evidence from thin sections and freeze-fracture is presented showing that stretch induces a burst of exocytosis in granular cells of the toad urinary bladder epithelium. Since the role of granule exocytosis in hormonally-induced permeability changes in this tissue has not yet been clarified, we propose that the stretch factor is an important parameter to consider and standardise in future physiological and morpho-functional studies using this model transporting system.     

Amide transport channels across toad urinary bladder

Summary Urea and other small amides cross the toad urinary bladder by a vasopressinsensitive pathway which is independent of somotic water flow. Amide transport has characteristics of facilitated transport: saturation, mutual inhibition between amides, and selective depression by agents such as phloretin. The present studies were designed to distinguish among several types of transport including (1) movement thought a fixed selective membrane channel and (2) movement via a mobile carrier. The former wold be characterized by co-transport (acceleration of labele amide flow in the direction of net flow in the opposite direction). Mucosal to serosal (MS) and serosal to mucosal (SM) permeabilities of labeled amides were determined in paired bladers. Unlabeled methylurea, a particularly potent inhibitor of amide movement, was added to either the M or S bath, while osmotic water flow was eliminated by addition of ethylene glycol to the opposite bat. Co-transport of labeled methylurea and, to a lesser degree, acetamide and urea with unlabeled methylurea was observed. Co-transport of the nonamides ethylene glycol and ethanol could not be demonstrated. Methylurea did not alter water permeability or transmembrane electrical resistance. The demonstration of co-transport is consistent with the presence of ADH-sensitive amide-selective channcels rather than a mobile carrier.     

Role of osmotic forces in exocytosis: studies of ADH-induced fusion in toad urinary bladder

Antidiuretic hormone (ADH) treatment of toad urinary bladder activates an exocytotic-like process by which intramembrane particle aggregates are transferred from membranes of elongated cytoplasmic tubules to the luminal-facing plasma membrane. We find that the number of these ADH- induced fusion events, and the number of aggregates appearing in the luminal membrane, are reduced when the luminal bathing medium is made hyperosmotic. As an apparent consequence of the inhibition of their fusion with the luminal membrane, the elongated cytoplasmic tubules become enormously swollen into large, rounded vesicles. These results are consistent with the view that osmotic forces are essential to the basic mechanism of exocytosis.     

Mode of action of amiloride in toad urinary bladder

Summary The effect of amiloride on the sensitivity to Na of the mucosal border of toad urinary bladder was investigated by recording Na concentration-dependent transepithelial potential difference (V _t ) and the intracellular potential. When mucosal Na concentration was normal, amiloride added to the mucosal solution at 10^–4 m markedly reduced the mucosal membrane potential (V _m ) and altered the potential profile from a two-step type to a well type. Similar changes were observed when Na was totally eliminated from the mucosal medium. The serosal membrane potential was insensitive to amiloride and elimination of mucosal Na. In the absence of amiloride, theV _t could be described by the Goldman-Hodgkin-Katz equation in the range of mucosal Na concentration from 0 to 16mm, and amiloride extended this concentration range. By using the Goldman-Hodgkin-Katz equation, Na permeability was calculated from the data ofV _t 's obtained in the allowed ranges of Na concentration and compared before and after the addition of amiloride. The results show that Na permeability decreases to 1/600 of control when the maximum dose of amiloride (10^–4 m) is applied. The relationship between Na permeability and amiloride concentration is well explained on the basis of assumptions that amiloride binds to the Na site of the mucosal border in one-to-one fashion and in a competitive manner with Na and that Na permeability reduces in proportion to increase in number of the sites bound with amiloride.     

Reserve of vasopressin-sensitive adenylate cyclase in toad urinary bladder

Studies have been performed on the effect of vasopressin on cyclic AMP content of toad bladders. A prompt increase in cyclic AMP content occurred after exposure to vasopressin, which reached maximal values within 8 min and remained elevated up to 30 min. By a comparison of the dose-response characteristics of vasopressin on cyclic AMP content, with those Na⁺ transport and osmotic water flow, it was shown that supramaximal concentrations of vasopressin with respect to physiological function generate more cyclic AMP than is required for maximal stimulation of Na⁺ transport and water flow. Thus, it would seem that a reverse of hormone-sensitive adenylate cyclase is present in this tissue.     

Metabolic cost of sodium transport in toad urinary bladder

Summary The metabolic cost of active sodium transport was determined in toad bladder at different gradients of transepithelial potential, , by continuous and simultaneous measurements of CO₂ production and of transepithelial electric current. Amiloride was used to block active sodium transport in order to assess the nontransport-linked, basal, production of CO₂ and the passive permeability of the tissue. From these determinations active sodium transport,J _Na, and suprabasal CO₂ production, , were calculated. Since large transients inJ _Na and frequently accompanied any abrupt change in , steady state conditions were carefully defined.Some 20 to 40 min were required after a change in before steady state of transport activity and of CO₂ production were achieved. The metabolic cost of sodium transport proved to be the same whether the bladder expended energy moving sodium against a transepithelial electrical potential grandient of +50 mV or whether sodium was being pulled through the active transport pathway by an electrical gradient of –50 mV. In both cases the value of the ratio averaged some 20 sodium ions transported per molecule of CO₂ produced.When the Na pump was blocked by 10^–2 m ouabain, the perturbations of the transepithelial electrical potential did not elicit changes ofJ _Na nor, consequently, of .The independence of the ratio from over the range ±50 mV indicates a high degree of coupling between active sodium transport and metabolism.     

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.     

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.     

Electron microscopic cytochemical localization of Ca-ATPase in toad urinary bladder

The calcium-regulating enzyme calcium adenosine triphosphatase (Ca-ATPase) was localized in the epithelium of amphibian urinary bladder by the one-step electron microscopic cytochemical procedure. The enzyme was identified along the basolateral border of the epithelial cells that comprise the bladder mucosa. The electron-dense precipitate indicating Ca-ATPase activity was seen in association with the outer leaflet of the basolateral plasmalemmae. Intracellularly, Ca-ATPase activity was seen in association with the mitochondrial matrix of the mitochondria-rich cells. Ca-ATPase was not seen along the apical microvillated border. Enzyme activity was also not seen after incubation in substrate-free media, calcium-free media, or incubation in the presence of vanadate. However, Ca-ATPase activity was evident when the calcium in the standard reaction medium was deleted in favor of magnesium. Addition of antidiuretic hormone (ADH; vasopressin) increased both the basolateral Ca-ATPase reaction and the mitochondrial reaction. Such data appear to indicate further that changes in cytosolic calcium ion concentration take place during the response of amphibian urinary bladder to the polypeptide hormone vasopressin.