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

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.     

8-P-Chlorophenylthio-cyclic AMP: a potent partial simulator of antidiuretic hormone action.

In the toad urinary bladder 8-p-chlorophenylthio-cyclic AMP mimics the stimulatory effects of antidiuretic hormone on osmotic water permeability, 3H2O diffusion, and transepithelial sodium transport; but unlike the hormone does not cause an increase in urea permeability. Trheshold activation for the hydroosmotic response is observed at 1 micrometer and full activation at 100 micrometer. These results suggest that cyclic AMP may not mediate all the physiological effects of antidiuretic hormone and that this highly potent cyclic AMP analog may be useful in elucidating the precise role of cyclic AMP in other biomediate hormone action.     

Role of substance P in water homeostasis.

The effect of the naturally occurring peptide substance P on release of antidiuretic hormone (ADH) was studied in anesthetized dogs. Intravenous infusions of substance P in doses of 0.5, 5.0, and 50 ng/kg/min increased plasma concentrations of ADH in a dose-related fashion. At the two low doses, this increase occured in the absence of changes in urine volume, sodium excretion, free water clearance, and urinary cyclic AMP excretion. In addition, when substance P was administered in a concentration of 0.5 ng/kg/min, plasma levels of ADH were increased even though blood pressure did not change, suggesting that substance P may release antidiuretic hormone by a direct mechanism. Intrarenal infusions at a rate of 0.5 and 5 ng/kg/min caused dose-related decreases in free water clearance and significant increases in urinary cyclic AMP excretion. These data suggest that substabce P may play an important role in the regulation of water balance.     

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.     

Systems analysis of the renal function

A previous model of the renal function and compartmented whole body, which incorporated actions of antidiuretic hormone on urine flow and composition, is extended to include the influences of additional phenomena, many exogenous to the kidney. The phenomena incorporated were selected by orderly trial from among those mechanisms suggested in the literature and found by operation of the model to be important for adequately representing renal responses to various stresses. Specifically, these phenomena are: the intrinsic-osmotic effect of water; details of the body's antidiuretic hormone cycle; gastrointestinal exchanges; changes in glomerular filtration caused by alteration in blood volume and pressure; and resistance to flow across kidney tubular walls. Most of these phenomena involve non-steady-state, irreversible processes.The modeled renal function was found not to be sensitive to certain mechanisms sometimes associated with its operation. The most important is volume sensing, whereby extracellular volume changes are postulated to impose control on antidiuretic hormone production through stretch receptors in the circulatory system or through other volume sensors. Model investigations show that blood volume changes that alter blood pressure during stress can operate to affect kidney tubular-fluid flow in a manner which, when combined with other modeled kidney processes, yields correct urinary and other responses. No additional control on the antidiuretic hormone cycle through special volume receptors is required.The stresses to which the present model was exposed and responds correctly are: water loading through ingestion; hypertonic saline infusion; hypertonic urea solution ingestion; antidiuretic hormone dysfunction, as in diabetes insipidus; controlled rehydration after dehydration; and two combined stresses—hypertonic saline solution ingestion followed by water ingestion and the converse experiment. Most of these stresses involve transients in which the body becomes far removed from steady state. Different aspects of the total renal function, depending upon the specific stress, become more or less important in aiding the body to return to its original state.     

A possible role of atrial natriuretic peptide in ethanol-induced acute diuresis.

The acute effects of ethanol on plasma atrial natriuretic peptide levels were investigated in 4 clinically healthy males, aged 24-26 years, consumed either 750 ml of water as a control study, or the same beverage with 1 ml/kg alcohol added, which increased the plasma alcohol concentration to 99.12 +/- 15.10 mg/dl at 60 min. Plasma atrial natriuretic peptide levels were significantly higher in the alcohol study compared to the control study at each time point (10, 20, 30, 60, 120 min after drinking onset), and with a peak at 10 min. Atrial natriuretic peptide levels showed a positive significant correlation with plasma antidiuretic hormone in the control group, while no relationship was found between the two peptides in the alcohol study. Moreover, a significant correlation exists between plasma atrial natriuretic peptide levels and systolic arterial blood pressure, and heart rate, and between the variations in atrial natriuretic peptide values and the variations in plasma sodium, serum ethanol, and plasma osmolality in the alcohol study. Acute ethanol intake causes an increase in urinary volume, and a decrease in urinary potassium excretion and urinary osmolality, and no change in urinary sodium excretion. These data suggest that acute ethanol administration causes a rapid increase in plasma levels of atrial natriuretic peptide, which could be an important factor of ethanol-induced diuresis. The main mechanisms for increased atrial natriuretic peptide release from atria after acute ethanol ingestion seem to be atrial stretch, due to the increase in arterial blood pressure, in heart rate, in sympathetic tone, and in plasma osmolality, and to a direct secretory effect by antidiuretic hormone.     

Ionic and metabolic requirements for the hydroosmotic response to antidiuretic hormone in toad urinary bladder

Summary A study has been conducted to determine the ionic and metabolic requirements for full expression of the hydroosmotic response to antidiuretic hormone in the toad urinary bladder. By appropriate manipulation of incubation conditions it can be shown that there is a pool of serosal sodium necessary for a full hormone response. This serosal sodium pool is not related to the transepithelial sodium transport pool. A full hydroosmotic response also requires serosal potassium; however, no specific anion requirement was demonstrated. Additionally, anaerobic or aerobic metabolism support a full hydroosmotic response equally well.     

Monitoring urea transport in rat kidney in vivo using hyperpolarized ¹³C magnetic resonance imaging

Urea functions as a key osmolyte in the urinary concentrating mechanism of the inner medulla. The urea transporter UT-A1 is upregulated by antidiuretic hormone, facilitating faster equilibration of urea between the lumen and interstitium of the inner medullary collecting duct, resulting in the formation of more highly concentrated urine. New methods in dynamic nuclear polarization, providing ～50,000-fold enhancement of nuclear magnetic resonance signals in the liquid state, offer a novel means to monitor this process in vivo using magnetic resonance imaging. In this study, we detected significant signal differences in the rat kidney between acute diuretic and antidiuretic states, using dynamic (13)C magnetic resonance imaging following a bolus infusion of hyperpolarized [(13)C]urea. More rapid medullary enhancement was observed under antidiuresis, consistent with known upregulation of UT-A1.     

Modulation of cytoskeletal organization and cytosolic granule distribution by verapamil in amphibian urinary epithelia

The present study examines the role of calcium in modulating epithelial cytomorphology by using verapamil, a calcium antagonist, and considering its effects on cytosolic granule distribution and exocytosis in toad urinary bladder. The effect of verapamil on the detection and distribution of microfilaments in toad urinary bladder using immunogold labeling techniques in toad urinary bladder epithelial cells was also examined. Verapamil, which inhibits antidiuretic hormone (ADH)-mediated water flow, increased the number, size and distribution of dense calcium-containing secretory granules in bladder epithelial cells. This calcium antagonist prevented granule exocytosis, such that, six-times the number of granules were present in verapamil-treated tissues. The normal cytomorphological changes that accompany the actions of ADH were attenuated by verapamil, including ADH-induction of microvilli. ADH increased the number of actin microfilaments as determined using protein A-gold by immunolabeling, whereas, verapamil treatment was unremarkable as compared to control. The results suggest that calcium may play a prominent role in mediating granule exocytosis and membrane fusion events that normally accompany hormone action.     

Role of calmodulin in antidiuretic hormone mediated water transport

Several classes of tricyclic antidepressants inhibit the action of antidiuretic hormone (ADH) and cyclic adenine monophosphate (cAMP) on osmotic water flow across toad urinary bladder without any effect on sodium transport. This finding suggests that calmodulin is involved in the hydroosmotic action of ADH (and of serosal hypertonicity), possibly in inducing exocytosis at the luminal border of vesicles rich in water channels.     

The effects of two analogues of arginine-vasopressin (ornithine-vasopressin and desamino-D-arginine-vasopressin) on kidney function in sheep.

The effects of intravenous infusion of ornithine-vasopressin (OVP) and desamino-D-arginine-vasopressin (dDAVP) were studied in normal and hydrated Merino sheep. In normal sheep, OVP resulted in a diuresis, increased urinary sodium and potassium excretion, and a fall in the plasma potassium concentration. Renal plasma flow remained constant but glomerular filtration rate and filtration fraction rose markedly. dDAVP in normal sheep was antidiuretic, but its only significant effect was a small decrease in plasma osmolality. In the hydrated sheep OVP was antidiuretic and resulted in increased urinary excretion of sodium and potassium, and a fall in the plasma potassium level. Renal plasma flow fell, but glomerular filtration and filtration fraction tended to rise. dDAVP in the hydrated sheep was also antidiuretic but urinary sodium and potassium excretion was reduced. Renal plasma flow and glomerular filtration fell, with a small decrease in filtration fraction. These results suggest that the diuretic effect in normal sheep and the electrolyte-excreting effects in both normal and hydrated sheep of OVP are related to the increase in glomerular filtration, which in turn is dependent on the vasopressor activity of the hormone. The increase in glomerular filtration caused by OVP is due to an increase in the filtration fraction of an unchanged renal plasma flow, which could be brought about by an increase in renal efferent arteriolar tone. The effects of hydration of the sheep were the conventional increased urine flow, decreased urine osmolality and decreased solute-free water reabsorption. Sodium and potassium excretion rose slightly and plasma osmolality fell. Renal plasma flow and glomerular filtration both increased with little change in filtration fraction. These effects could be brought about by suppression of endogenous vasopressin and a decrease in both afferent and efferent renal arteriolar tone.     

Urinary excretion of aquaporin-2 and inappropriate secretion of vasopressin in hyponatremic patients after cerebral infarction.

Aquaporin-2, a water-channel protein, is known to increase water permeability due to vasopressin binding to V2 receptors at the renal collecting duct and is excreted into the urine. It is still unclear whether a hyponatremic state is caused by vasopressin-dependent aquaporin-2 in patients clinically diagnosed with the syndrome of inappropriate secretion of antidiuretic hormone. To determine this, we measured urinary aquaporin-2 and vasopressin by radioimmunoassay in normonatremic or hyponatremic patients after cerebral infarction and in healthy controls. In the normonatremia group, urinary aquaporin-2 and plasma AVP levels were higher than in controls. In the hyponatremia group, plasma AVP was relatively high despite low plasma osmolality in each patient. However, urinary aquaporin-2 in hyponatremia was significantly increased when compared with the other two groups. In conclusion, AQP-2 increment does not directly reflect non-osmotic AVP secretion in a hyponatremic state. This result indicates that the urinary excretion of AQP-2 is not only AVP-dependent in hyponatremic states.     

Ultrastructural modifications of frog urinary bladder epithelium under the influence of hypertonic media

Summary The frog urinary bladder undergoes a marked increase in its water permeability when incubated in hypertonic media. Many similarities are found between this effect and the hydrosmotic action of antidiuretic hormone. The ultrastructural modifications of the epithelium observed under the influence of serosal hypertonicity (the intercellular spaces are dilated while the tight junctions remain closed) lead us to assume that the pathways of water movement across the epithelium could be the same in this case and in hydrosmotic response to the hormone. In contrast, when the mucosal medium is made hypertonic, the ultrastructure is differently altered: the intercellular spaces are closed, the tight junctions show small vesicles and numerous large vacuoles appearing in the cytoplasm.     

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.     

Effect of adrenalectomy on distal tubule water permeability of the Brattleboro rat

The mechanism of impaired water excretion in adrenalectomized mammals is unclear. Previous workers have suggested that one cause might be increased water permeability of the distal nephron, allowing back diffusion of water from tubular fluid diluted by the ascending limb. Evidence to support this mechanism in previous studies has been confounded by simultaneous changes in steroid and antidiuretic hormone levels. We compared osmotic and diffusional water permeability of the surface late distal tubule in vivo in intact and adrenalectomized Brattleboro rats, which are free of antidiuretic hormone. The adrenalectomized rats were demonstrated to have impaired diluting capacity in clearance studies. Adrenalectomized rats had a sixfold increase in osmotic permeability and a 1.5-fold increase in diffusional permeability over intact controls. Adrenal steroids have a specific action on water permeability of the distal nephron, independent of antidiuretic hormone.     

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.     

Apical material extracted from amphibian urinary bladder epithelium by enzymes and detergent treatment

The apical plasma membrane of epithelial cells of frog and toad urinary bladder is subject to large modifications during the induction of water permeability by the antidiuretic hormone. A better characterization of the apical membrane is necessary for a clear understanding of the mechanisms of hormone action. Towards this end, apical material was extracted by enzymatic treatment and by incubation with detergent. Proteolytic enzyme alone had little effect under our conditions. A pretreatment with several glycosidases (alpha-mannosidase or endo-beta-N-acetylglucosaminidase H) increased the hydrolytic action of papain, elastase, proteinase K or Staphylococcus aureus V8 protease and allowed the detection of a major 76 kD in SDS gel electrophoresis. The n-octyl-beta-D-glucopyranoside (0.2%) led to the extraction after 150 mn of 1 to 5 micrograms proteins per cm2 of amphibian urinary bladder apical surface. The extracted proteins migrated as several bands on SDS gels. One of them probably corresponds to the 76 kD fragment obtained after proteolysis. The absence of alteration of the water permeability after extraction and the good preservation of the ultrastructure are evidence for the localisation of the 76 kD at the apical membrane surface. This protein may be the best candidate as antigen to raise antibodies against the apical surface of amphibian urinary bladder epithelial cells.     

Hypoalbuminaemic hyponatraemia: a new syndrome?

Six patients with severe hyponatraemia had neurological features of hyponatraemia and pronounced hypoalbuminaemia. All had biochemical features typical of the syndrome of inappropriate secretion of antidiuretic hormone with low serum osmolality and an inappropriately high urinary osmolality. All were given infusions of whole plasma or albumin solution, or both, to restore their plasma albumin concentrations to normal, which led to a dramatic increase in plasma sodium concentrations and serum osmolality, with a concomitant fall in urinary osmolality in all patients. Neurological features were reversed in four patients. It is suggested that severe hypoalbuminaemia is an important cause of appreciable hyponatraemia; infusions of plasma and albumin in such patients may reverse the biochemical and clinical features and should form the basis of management.     

Molecular mechanisms of action of prostaglandin E2 in the regulation of water osmotic permeability

The functional role and molecular mechanisms of action of prostaglandin E2 (PGE2) in the regulation of water osmotic permeability in osmoregulatory epithelia (mammalian collecting tubules and amphibian urinary bladder) are considered. The paper describes the modern classification of PGE2 receptors, their distribution along a nephron and receptor-coupled intracellular second messenger systems. The mechanism of the inhibitory action of PGE2 on the antidiuretic hormone-induced enhancement of water osmotic permeability is analyzed. Special attention is given to the role of PGE2 as an auto- or paracrine regulator of water osmotic permeability in the phenomenon of ADH-independent increase of water permeability observed in an isolated amphibian urinary bladder in replacements of the surrounding serous solution. It is concluded that the osmoregulatory epithelium is not only a place of the maximum level of PGE2 synthesis in the kidney but is also characterized by a great diversity of PGE2 receptor subtypes: EP1, EP2, EP3 and EP4 have been revealed in the mammalian collecting tubules. Such a diversity of PGE2 receptors is in a good agreement with different functional effects of PGE2 in the osmoregulatory epithelium. The data considered suggest that PGE2 is not less important in the regulation of water and ion transport in the osmoregulatory epithelium than antidiuretic hormone.     

Changes in plasma cortisol,blood antidiuretic hormone and urinary catecholamines in high—altitude pulmonary oedema

In 10 subjects susceptible to high altitude pulmonary oedema (HAPO) plasma cortisol and antidiuretic hormone (ADH) and urinary catecholamines were estimated both at sea level and daily during their stay at 3, 500 m (Leh). At high altitude 4 of the subjects developed HAPO, 2 got acute mountain sickness (AMS) and 4 remained unaffected. Plasma cortisol showed a sharp rise on the first day at high altitude in all the subjects. Thereafter, it declined gradually in the unaffected subjects. In the HAPO patients there was a sharp fall in the plasma cortisol level combined with antidiuresis. Changes in plasma ADH and urinary catecholamines were not consistent. It appears that failure in the normal adrenocortical response to altitude stress in susceptible subjects is a factor in precipitating HAPO.