scholarly journals Electron microscopic cytochemical localization of adenylate cyclase in amphibian urinary bladder epithelium: effects of antidiuretic hormone.

1987 ◽  
Vol 35 (1) ◽  
pp. 103-111 ◽  
Author(s):  
W L Davis ◽  
R G Jones ◽  
D B Goodman
Keyword(s):  
Urinary Bladder ◽  
Adenylate Cyclase ◽  
Antidiuretic Hormone ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Bladder Epithelium ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Amphibian Urinary Bladder ◽  
Hormone Sensitive

A cytochemical technique for electron microscopic localization of adenylate cyclase was used to identify this enzyme in quiescent and hormone-stimulated toad urinary bladder epithelium. In the absence of vasopressin (antidiuretic hormone), adenylate cyclase was detected along the outer surface of the basolateral plasma membranes of granular cells, mitochondria-rich cells, and basal cells, the major cell types comprising the hormone-sensitive urinary epithelium. In the presence of antidiuretic hormone, the basolateral precipitates were markedly increased. The latter was true for both tissues incubated in the presence of an osmotic gradient and those stimulated in the absence of such a gradient. A significant mucosal reaction was never seen. Such data indicate that the hormone receptors for vasopressin are located along the basolateral membranes of all epithelial cells comprising the mucosal hormone-sensitive epithelium. All cells of the epithelium also demonstrate a vasopressin-sensitive adenylate cyclase. We discuss possible mechanisms that attempt to integrate the cytochemical data into an overall scheme for the physiological action of this hormone on amphibian urinary bladder.

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

1987 ◽  
Vol 35 (1) ◽  
pp. 39-48 ◽  
Author(s):  
W L Davis ◽  
R G Jones ◽  
D B Goodman
Keyword(s):  
Urinary Bladder ◽  
Atpase Activity ◽  
Reaction Medium ◽  
Ion Concentration ◽  
Toad Urinary Bladder ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Atpase Reaction ◽  
Amphibian Urinary Bladder ◽  
Free Media

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.

Fluorescent markers to study membrane retrieval in antidiuretic hormone-treated toad urinary bladder

1986 ◽  
Vol 251 (2) ◽  
pp. C274-C284 ◽  
Author(s):  
H. W. Harris ◽  
J. B. Wade ◽  
J. S. Handler
Keyword(s):  
Electron Microscopy ◽  
Urinary Bladder ◽  
Horseradish Peroxidase ◽  
Antidiuretic Hormone ◽  
Apical Membrane ◽  
Granular Cell ◽  
Cell Uptake ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Apical Surface

Antidiuretic hormone (ADH) stimulation of toad urinary bladder causes fusion of intracellular vesicles called aggrephores with the apical plasma membrane of granular cells. Aggrephores contain intramembrane particle aggregates whose appearance in the apical membrane is believed to produce a large increase in its water permeability. ADH removal (ADH washout) is thought to cause the retrieval of aggrephores into granular cell cytoplasm. We studied granular cell uptake of dextran and horseradish peroxidase conjugated with fluorescein, rhodamine, or both during ADH washout. Granular cell uptake of fluorescent dextran was dependent on prior exposure to ADH, a linear function of dextran concentration, and increased by a transepithelial osmotic gradient. Immediately after removal of ADH, granular cell fluorescence was finely dispersed and located near the apical surface. Subsequently, it coalesced into larger bodies. This change was most apparent when a single bladder was subjected to two cycles of ADH stimulation and removal using a dextran containing a different fluorophore for each cycle. The ultrastructural correlate for these fluorescent patterns was identified using rhodamine-labeled horseradish peroxidase. Electron microscopy showed that after detachment from the apical membrane, label was initially in tubular-shaped vesicles near the apical surface. Later, these vesicles clustered near multivesicular bodies and transferred their label to these structures. These tubular vesicles closely resemble the morphology of aggrephores visualized by freeze-fracture electron microscopy. We conclude that these fluorescent compounds can be used as markers for the luminal contents of membrane retrieved during ADH washout and allow detailed study of its intracellular processing.

Modulation of antidiuretic hormone-dependent capacitance and water flow in toad urinary bladder

1984 ◽  
Vol 246 (4) ◽  
pp. F501-F508
Author(s):  
L. G. Palmer ◽  
N. Speez
Keyword(s):  
Urinary Bladder ◽  
Antidiuretic Hormone ◽  
Water Flow ◽  
Water Permeability ◽  
Apical Membrane ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Apical Surface ◽  
Membrane Area ◽  
Close Relationship

To test the hypothesis that antidiuretic hormone- (ADH) dependent water permeability is associated with changes in apical membrane area, hormone-dependent water flow and capacitance changes were measured in the toad urinary bladder under a number of different conditions. Dose-response relationships for water flow (Jv) and capacitance increases (delta C) were similar from 1 to 20 mU/ml ADH. At higher concentrations, Jv reached a plateau, while delta C decreased. The decrease in delta C was prevented by elimination of the osmotic gradient across the tissue. Serosal hydrazine (10 mM) increased Jv sevenfold and delta C threefold in the presence of 1 mU/ml ADH. Mucosal NH4Cl, at constant mucosal pH, increased Jv by 50-100%, but did not significantly change delta C. In the absence of an osmotic gradient, mucosal NH+4 increased delta C by 50%. NH4Cl had no effect on hydroosmotic response to 8-bromo-adenosine 3',5'-cyclic monophosphate (cAMP). Mucosal CO2 (9%) decreased Jv by greater than 90%, and delta C by 60% with 20 mU/ml ADH. Mucosal CO2 also inhibited the hydroosmotic response to 8-bromo-cAMP. Removal of serosal Na diminished cAMP-dependent Jv and delta C. The results confirmed the close relationship between ADH-dependent water permeability and membrane capacitance. They indicate, however, that under some circumstances membrane may be retrieved from the apical surface without affecting water permeability.

Cytoplasmic dilution induces antidiuretic hormone water channel retrieval in toad urinary bladder

1992 ◽  
Vol 263 (1) ◽  
pp. F163-F170 ◽  
Author(s):  
H. W. Harris ◽  
B. Botelho ◽  
M. L. Zeidel ◽  
K. Strange
Keyword(s):  
Urinary Bladder ◽  
Antidiuretic Hormone ◽  
Apical Cell ◽  
Water Channel ◽  
Fluid Phase ◽  
Fluorescein Isothiocyanate ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Apical Cell Membrane

Antidiuretic hormone (ADH) increases the osmotic water permeability (Pf) of the toad urinary bladder by insertion of water channels into the apical cell membrane. Transepithelial water flow (Jv) reduces Pf by inducing endocytosis of apical water channels despite continuous ADH stimulation. This phenomenon is termed flux inhibition. We wished to determine whether cytoplasmic dilution or transcellular Jv causes flux inhibition because both have been proposed previously as a primary regulatory mechanism for this process. Apical membrane endocytosis was quantified by monitoring the uptake of the fluid phase marker fluorescein isothiocyanate dextran (FITC-dextran). FITC-dextran fluorescence was monitored in Triton X-100 extracts of epithelial cells as the ratio of total tissue fluorescence compared with background fluorescence. The background was defined as cellular autofluorescence and nonspecific tissue staining due to the presence of small amounts of free fluorescein contaminating the FITC-dextran. FITC-dextran uptake measured under symmetric isotonic (220 mosmol/kgH2O) conditions in either the absence (1.0 +/- 0.4 SD; n = 14) or presence (1.3 +/- 0.3; n = 4) of ADH was not statistically different from that of background. In contrast, flux inhibition induced by a 180 mosmol/kgH2O apical-to-basolateral osmotic gradient increased FITC-dextran uptake to 3.4 +/- 1.3 (n = 7). FITC-dextran uptake was identical in bladders exposed to symmetric hypotonic (150 mosmol/kgH2O) solutions during ADH (3.6 +/- 0.9; n = 6) or adenosine 3',5'-cyclic monophosphate (3.1 +/- 0.4 fold; n = 3) stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

OSMOTIC INHIBITION OF THE NATRIFERIC RESPONSE OF THE TOAD URINARY BLADDER TO VASOPRESSIN

1975 ◽  
Vol 67 (1) ◽  
pp. 119-125
Author(s):  
P. J. BENTLEY
Keyword(s):  
Urinary Bladder ◽  
Water Movement ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Toad Bladder ◽  
Toad Urinary Bladder ◽  
Electrical Potential Difference ◽  
Osmotic Gradient

SUMMARY The electrical potential difference and short-circuit current (scc, reflecting active transmural sodium transport) across the toad urinary bladder in vitro was unaffected by the presence of hypo-osmotic solutions bathing the mucosal (urinary) surface, providing that the transmural flow of water was small. Vasopressin increased the scc across the toad bladder (the natriferic response), but this stimulation was considerably reduced in the presence of a hypo-osmotic solution on the mucosal side, conditions under which water transfer across the membrane was also increased. This inhibition of the natriferic response did not depend on the direction of the water movement, for if the osmotic gradient was the opposite way to that which normally occurs, the response to vasopressin was still reduced. The natriferic response to cyclic AMP was also inhibited in the presence of an osmotic gradient. Aldosterone increased the scc and Na+ transport across the toad bladder but this response was not changed when an osmotic gradient was present. The physiological implications of these observations and the possible mechanisms involved are discussed.

Antidiuretic hormone-dependent membrane capacitance and water permeability in the toad urinary bladder

1983 ◽  
Vol 244 (2) ◽  
pp. F195-F204
Author(s):  
L. G. Palmer ◽  
M. Lorenzen
Keyword(s):  
Antidiuretic Hormone ◽  
Water Flow ◽  
Time Course ◽  
Apical Membrane ◽  
Constant Current ◽  
Apical Plasma Membrane ◽  
Voltage Response ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Capacitance Change

Antidiuretic hormone (ADH) increased the electrical capacitance of apical membrane of the toad bladder; this effect was modulated by the osmotic gradient across the tissue. Capacitance was measured from the transepithelial voltage response to constant-current pulses using bladders depolarized with KCl-sucrose serosal solution to reduce basolateral resistance and with Na-free mucosal solution to increase apical membrane resistance. Addition of ADH (20 mU/ml) increased capacitance by 28 +/- 9% (mean +/- SD) in the absence and by 8 +/- 3% in the presence of an osmotic gradient (200 mosM, mucosal side hypotonic). With bladders stimulated in the absence of an osmotic gradient, rapidly imposing a gradient resulted in a peak rate of water flow that declined to 40% of the peak value after 15-20 min. ADH-dependent capacitance also decreased with a similar time course. Removal of ADH reversed the capacitance change (t1/2 = 10-15 min), but the reversal was slower than the decline in water flow to basal levels (t1/2 less than 5 min). Colchicine and cytochalasin B also inhibited the ADH-induced capacitance increase. The capacitance change was also inhibited when the mucosal solution was made hypertonic with raffinose. The results are interpreted within the framework of a previously proposed model of ADH-stimulated water transport in which cytoplasmic vesicular structures fuse with the apical plasma membrane.

Vasopressin-elicited refractoriness of the response to vasopressin in toad urinary bladder

1981 ◽  
Vol 240 (6) ◽  
pp. F551-F557 ◽  
Author(s):  
J. S. Handler ◽  
A. S. Preston
Keyword(s):  
Urinary Bladder ◽  
Adenylate Cyclase ◽  
Water Permeability ◽  
Recovery Period ◽  
Ringer Solution ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Toad Urinary Bladder ◽  
Camp Phosphodiesterase ◽  
Prolonged Incubation

Incubation of the urinary bladder of Bufo marinus with high concentrations of vasopressin produces refractoriness to subsequent stimulation of water permeability by low concentrations of vasopressin. Development of refractoriness is directly dependent on concentration of vasopressin and duration of incubation with the hormone. Refractoriness develops in the absence of transepithelial water flow, is evident following a 2-h recovery period of incubation in hormone-free Ringer solution, and is reversed after prolonged incubation in hormone-free Ringer solution. Development and reversal of refractoriness is not altered by actinomycin D or cycloheximide. The steps at which refractoriness develops have been identified partially. Under different conditions, refractoriness involves: 1) reduced vasopressin-sensitive adenylate cyclase activity, 2) reduced epithelial cell cAMP accumulation in response to vasopressin the absence of demonstrable change in vasopressin-sensitive adenylate cyclase activity, cAMP phosphodiesterase activity, or loss of cAMP into the Ringer solution, and 3) refractoriness of water permeability response to exogenous cAMP.

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