The role of dehydration and antidiuretic hormone on water exchange in Rana pipiens

• 1.1. Experiments were performed to evaluate the effects of 8-arginine vasotocin on the effective water potential and on the hydraulic conductance of leopard frogs (Rana pipiens) during water uptake through their ventral integument.
• 2.2. Vasotocin apparently decreases the effective water potential of intact frogs, thus bringing the effective water potential into close correspondence with the osmotic potential of extracellular fluids.
• 3.3. Thus, well-hydrated frogs, which release little AVT, have effective water potentials considerably higher (more positive) than the osmotic potentials of their plasma, and therefore, demonstrate a diminished capacity to absorb water.
• 4.4. Dehydrated frogs release AVT which causes their effective water potential to become essentially identical to the osmotic potential of their plasma.
• 5.5. We hypothesize that the action of AVT is to mobilize water from the site of absorption by the frog, thereby resulting in maintenance of a high water potential gradient between the environment and the frog. and in increased rates of water uptake.

     

Ultracytochemical localization of Ca++-ATPase activity in pituicytes of the neurohypophysis of the guinea pig

Summary Ca⁺⁺-ATPase activity (cf. Ando et al. 1981) was examined both light- and electron-microscopically in the neurohypophysis of the guinea pig. Apart from a strong activity within the walls of the blood vessels, in the parenchyma of the neurohypophysis the reaction product of the Ca⁺⁺-ATPase activity was restricted to the plasmalemma of the pituicytes. This reaction was completely dependent upon Ca⁺⁺ and the substrate, ATP; the reaction was inhibited by 0.1 mM quercetin, an inhibitor of Ca⁺⁺-ATPase. A reduction of the enzyme activity occurred by 1) adding Mg⁺⁺ to the standard incubation medium, and 2) substituting Ca⁺⁺ with Mg⁺⁺ at varing concentrations. In all experiments the neurosecretory fibers were devoid of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity in the plasmalemma of the pituicytes is discussed in connection with the regulation of the extracellular Ca⁺⁺ concentration, which seems to be important with respect to the discharge of secretory material from the neurosecretory fibers.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany.     

Evidence for calcium inactivation during hormone release in the rat neurohypophysis.

1. A study has been made of the relationship between 45Ca uptake into and hormone release from isolated rat neurohypophyses incubated in vitro. 2. Hormone secretion is triggered by high-K (56 mM) but long exposure to the stimulus does not generate a maintained release of hormone. 3. When hormone release began to wane, addition of Ba of La increased hormone output which suggests that the decline in output did not result from depletion of the neurosecretory granules at the nerve terminals. 4. 45Ca uptake is enhanced in the presence of high-K concentration, but the initial high rate declines during long exposure to the potassium stimulus with a time constant similar to that of the decline in hormone release. 5. After a period of incubation in a K-rich, calcium-free medium, addition of calcium to the medium induced hormone release. The magnitude of this release was dependent on the time of exposure to excess potassium. 6. After inactivation of secretion, mobilization of internal calcium by means of a calcium ionophore increased hormone release.     

The role of microtubules and microfilaments in the hydrosmotic response to antidiuretic hormone

To test the effects of colchicine and cytochalasin B on the ADH-induced response, unidirectional and net water fluxes were measured at one or two minutes intervals in frog urinary bladder. The action of these agents on the appearance of intramembrane particles aggregates in the luminal membrane of target cells under oxytocin stimulation and the changes in the tissue ultrastructure induced by cytochalasin B were also studied. It was observed that: the time-course of the response to oxytocin was strongly slowed by colchicine while the washout was not affected; the time-course of the 'on and off' of the response to oxytocin was not modified by cytochalasin B; cytochalasin B pretreatment proportionally reduced unidirectional and net water fluxes measured after glutaraldehyde fixation; the combined action of colchicine and cytochalasin B proportionally reduced the net water flux and the number of intramembrane particles aggregates, observed in freeze-fracture studies; after cytochalasin B action the dilation of intercellular spaces classically observed under oxytocin stimulation is strongly reduced. It is concluded that: microtubules probably play an important role in the water channels plug-in, but not in their removal; microfilaments integrity is necessary for the mechanisms inducing intercellular space dilation and the observed results confirm that water permeability is controlled by the number of permeation units present in the luminal border of granular cells and probably represented by the intramembrane particle aggregates.     

The single file hypothesis and the water channels induced by antidiuretic hormone

Unidirectional and net water movements were determined at minute intervals in frog urinary bladders. The changes in both parameters were followed, during the action of antidiuretic hormone (ADH), at different temperatures and stirring conditions. After correction for external unstirred layer effects, the ratio of the osmotic (Pf) and diffusional (Pd) permeability coefficients was remarkably constant, at different times and in different experimental conditions. In the presence of ADH the delta Pf/delta Pd ratio in the mucosal border was probably greater than 9. On the other hand, in nonstimulated preparations the ratio was smaller, and probably not different from 1. These results, together with previous observations indicating that other small molecules (like urea) are excluded from the ADH-induced channel, might indicate that single-file water movement can occur through this structure. Alternatively, the delta Pf/delta Pd ratio could result from a complex geometric arrangement in series with the aqueous pore.     

Circulating antidiuretic hormone in the X-irradiated rat

Male rats exposed to 500 R of whole-body x-irradiation were allowed food and water ad libitum and housed in metabolism cages; water and food intake and urinary and fecal excretion were recorded daily. Urine output increased 200% during the first 24 hours after irradiation. No significant changes occurred in daily sodium, potassium, urea, or total solute excretion, although calcium excretion approximately doubled after irradiation. The marked increase in free water excretion implicates antidiuretic hormone (ADH) in this phenomenon. Application of a sensitive bioassay for ADH permitted measurement of plasma ADH concentrations in undisturbed, unanesthetized rats before and after irradiation. ADH levels were lower and frequently not detectable 24 hours after exposure. High ADH levels, however, could be provoked in irradiated rats by hemorrhage, indicating that the receptor cells and secretory ability of the posterior pituitary remained intact. Furthermore, irradiated rats responded normally to small intravenous injections (4 to 8 microU) of exogenous ADH. Rats with congenital diabetes insipidus given daily injections of Pitressin showed no postirradiation diuresis. Lastly, increased urinary calcium excretion may result from hypercalcemia which is known to induce diuresis through calcium-vasopressin antagonism. These results further suggest that the diuretic response is due to decreased circulating ADH.     

Fusion images and intramembrane particle aggregates during the action of antidiuretic hormone

Summary Antidiuretic hormone (ADH) causes the appearance of water-conducting particle aggregates in the luminal membrane of receptor cells in amphibian bladder and skin, and in the mammalian collecting duct. The aggregates originate from cytoplasmic tubules that fuse with the luminal membrane during ADH stimulation. We have studied the process of fusion and the structure of the particle aggregates by a rapid-freeze technique that renders chemical fixation and glycerol protection unnecessary. Our findings differ in some important respects from previously published work. Aggregate particles, in our study, partition equally between the external (EF) and protoplasmic (PF) membrane leaflets, rather than remaining in the protoplasmic leaflet exlcusively. By including the entire population of fusion images in our survey, we have found that aggregate delivery in ADH-treated cells proceeds preferentially from small fusion images whose diameter is significantly less than the 0.12 m characteristic of the carrier tubules themselves. We have also found that, even in unstimulated preparations, fusion images are numerous, being mostly of small diameter. ADH stimulation produces a moderate increase in the number of fusion images and a significant increase in fusion-image diameter. These findings indicate that the individual particles are mobile within the membrane, lacking interparticle linkage. In addition, contact of cytoplasmic tubules with the luminal membrane may take place even in the absence of ADH, producing small fusion images which are not associated with aggregate delivery to the luminal membrane.Faculty Scholar, Josiah Macey Jr. Foundation     

Contributions of the kidneys and intestines to water conservation,and plasma levels of antidiuretic hormone,during dehydration in house sparrows (Passer domesticus)

Summary The contributions of the kidneys, the small intestine and the lower intestine (rectum plus cloaca) to water conservation during dehydration in unanaesthetized, unrestrained house sparrows (Passer domesticus) were assessed. Thirty hours of acute dehydration resulted in a 12% loss in body mass and a significant increase in plasma osmolality. Glomerular filtration rate declined by 55%, from 7.7 to 3.5 ml/h, and urine flow rate delined by more than 80%, from 0.2 to 0.03 ml/h. These changes are likely attributable to a large increase in plasma levels of arginine vasotocin during dehydration, from <26 pg/ml in hydrated birds to greater than 200 pg/ml after 30 h dehydration. Flow of water from the ileum to the lower intestine was reduced during dehydration, primarily because of a reduced flow of dry matter (with no significant reduction in water content). The rate of water loss in the excreta declined from 0.2 ml/h in hydrated birds to 0.04 ml/h in dehydrated birds. The rate of water reabsorption in the lower intestine (equal to the rate of water loss in the excreta minus the combined rates of inflow into the lower intestine from the urine and the ileal contents) slightly exceeded the rate of water flow from the ileum in both hydrated and dehydrated birds. We suggest that much of the water reabsorbed in the lower intestine of hydrated birds derives from the urine, but that primarily water from ileal contents is reabsorbed in dehydrated birds. That is, urine undergoes significant post-renal modification in hydrated but not dehydrated house sparrows.