J. Crabbé

Relevance of sodium transport pool measurements in toad bladder tissue for the elucidation of the mechanism whereby hormones stimulate active sodium transport

SummaryThe size of toad bladder sodium transport pool, defined as that amount of sodium of apical origin and recovered in tissue at equilibrium, was compared with sodium transport rate, derived from short-circuit current read immediately before tissue analysis.Provided certain precautions be taken, the relationship between both variables can be described by a curve starting at the intersect ofX (pool, in μEq) andY (transport, in μEq/hr) axes, with a tendency forX to increase faster thanY. Assuming sodium transport pool forms one compartment, its calculated half-life averages 2–3 min.sodium transport pool measurements are thought to shed light on mechanism of sodium transport by toad bladder because pool size was large with respect to transport rate when tissue was exposed to several inhibitors of sodium transport. Conversely, upon stimulation of activity of (substrate — depleted) preparations by glucose, a relative reduction of pool size was observed.Aldosterone, vasopressin (and adenosine 3′,5′-phosphate) increased sodium pool size and transport rate, proportionately; insulin stimulated sodium transport more than it increased pool size. Thus, insulin presumably exerts its effect at the sodium “pump” while such a site of action need not be postulated for aldosterone and vasopressin: these 2 hormones would instead induce, permeability changes faciliting sodium movement at the apical border of toad bladder epithelial cells.

The role of nucleic acids in the sodium-retaining action of aldosterone☆

Abstract 1. The stimulating effect of aldosterone on active sodium transport in vivo and in vitro by the ventral skin and urinary bladder of the toad, Bufo marinus , can be blocked by actinomycin D. No safe conclusion can be drawn from experiments with puromycin, since this antibiotic depressed the baseline activity of the preparations. 2. Large amounts of actinomycin D or puromycin added to bladders already reacting to aldosterone in vitro , rapidly eliminate the effect of the latter, thus permitting an evaluation of the half-life of the intermediates synthesized under influence of the hormone. 3. In the presence of aldosterone, [ 3 H]uridine incorporation into bladder tissue RNA increases over control values, whether or not sodium ions are available for active transport; it is concluded that stimulation of RNA synthesis is not secondary to intracellular sodium concentration changes.

Action of aldosterone and vasopressin on the active transport of sodium by the isolated toad bladder.

Summaries – Résumés Action of aldosterone and vasopressin on the active transport of sodium by the isolated toad bladder By Crabbé, J. and Weer, P. de: J. Physiol., Lond. 180: 560-568 (1965). The isolated urinary bladder of the toad, Bufo marinus, was incubated in the presence, on its mucosal side, of 24Na and 14C-carboxyl inulin added to sodium-Ringer’s diluted to 1i5 with choline-Ringer’s. The serosal surface was exposed to straight sodium-Ringer’s. Active sodium transport by the tissue was stimulated upon removal of sodium from the animal environment, by treatment of the toad with aldosterone, or by addition to the incubation solution of vasopressin. The short-circuit current, taken as a reflection of active sodium transport in the experimental conditions selected, agreed closely with the flux of 24Na from the mucosal to the serosal surface of the membranes. The amount of radioactive sodium in toad-bladder tissue was found, after correction for sodium present in the inulin space on the mucosal side, to be directly proportional to the short-circuit current in the case of untreated bladders. The results obtained with membranes stimulated by the above treatments conformed with the same current-tissue sodium relation. It is concluded that aldosterone and vasopressin alter the diffusion barrier at the mucosal surface of the cells to allow more sodium to reach the sodium ‘pump’ at the serosal surface. Author’s address: Dr. J. Crabbé. Section of Endocrinology, Laboratory of Experimental Surgery, University Clinics St. Pierre, L·ouvain (Belgium). Effects of spinal section and renal denervation on the renal response to blood volume expansion By Pearce, J.W. and Sonnenberg, H.: Canadian J. Physiol. Pharmacol. 43: 211 (1965). Expansion of the blood volume with ‘artificial blood’ infusion was previously shown to cause diuresis and natriuresis in anaesthetized dogs (1). As the magnitude of response was related to the state of prehydration as reflected in some parameter other than the initial or expanded blood volume, the possibility of extravascular fluid volume receptors was considered. In the present work, artificial blood infusion failed to produce increased renal excretion in chronic high-spinal dogs. This observation was considered best explained as the result of interruption of nervous pathways essential to a reflex mechanism. In other dogs, unilateral renal denervation did not significantly alter the time course of response or the magnitude of the diuresis, but did result in a reduced natriuresis. As there was no qualitative difference between the responses of the innervated and denervated kidneys in each animal, it is concluded that renal motor nerve supply is not involved in the effector limb of the reflex, which must then be humoral. Bilateral renal denervation led to a marked reduction in natriuretic response to infusion and, while not reducing the diuretic response, significantly delayed the maximum increase in urine flow. These effects, like those of spinal section, are attributed to the loss of sensory pathways. The hypothesis is advanced that receptors of extracellular fluid volume with spinal 64 Summaries – Resumes

Increased Chloride Permeability of Amphibian Epithelia Treated With Aldosterone

The transepithelial flux of chloride was increased by aldosterone treatment of amphibian skin and bladder and this was reflected by increased “shunt” conductance. The hormonal effect depended on the presence of chloride on the epithelial side of the preparation. These changes in tissue conductance and chloride permeability appear to be a direct effect of aldosterone as they did not occur when sodium transport was stimulated with vasopressin or hypotonicity.Chloride efflux was reduced in magnitude by indacrinone and DIDS, as well as after removal of chloride from the solution on the epithelial side of the preparations. These results suggest that, rather than merely diffusing along (a) paracellular pathway(s), chloride flows through (a) cellular structure(s), notably mitochondria-rich cells. These cells can therefore be considered as targets for aldosterone.

Decreased sensitivity to amiloride of amphibian epithelia treated with aldosterone

The reversible inhibition of transepithelial sodium transport achieved with amiloride (and triamterene) was evaluated in amphibian preparations stimulated with aldosterone so as to provide further information regarding a possible influence of this hormone on the apical border of target cells.When aldosterone secretion was enhanced by withdrawal of sodium from toad (Bufo marinus) habitat, sensitivity of abdominal skin to amiloride decreased; the same occurred in skin and bladder preparations incubated with aldosterone for several hours.Amiloride proved a less efficient blocker of sodium transport by toad skin exposed to vasopressin and to ouabain; both substances are capable or raising cell sodium content. It is therefore proposed that the decrease in sensitivity to amiloride of amphibian epithelia treated with aldosterone results from an increase in target cell sodium, itself due to a hormone-induced increase in sodium conductance at the apical cell border.Glucose, which enhanced markedly the rate of sodium transport in preparations treated with aldosterone for several hours, failed to decrease any further the response to amiloride; this is taken as an argument for an additional (? secondary) influence of aldosterone on the cells metabolic machinery connected with the operation of the sodium ‘pump’.

Stimulation by aldosterone of a rapidly labelled RNA fraction in toad bladder tissue

Abstract The mechanism of action of a steroid hormone, aldosterone, has been further studied by examining its influence on nucleic acid metabolism in a responsive tissue, the toad bladder. Nucleic acids from a single toad bladder have been extracted and analyzed by ultracentrifugation or fractionated by chromatography on methylated serum albumin-kieselguhr columns. Incubation of toad bladder tissue in the presence of a labelled precursor has made it possible to characterize a rapidly labelled RNA fraction which could, at least in part, represent messenger RNA. Isotope incorporation in this fraction is increased after incubation of the tissue with aldosterone.

Changes in Corticosteroid Secretory Pattern Induced By Prolonged Corticotropin Treatment in the Rabbit

The effects of corticotropin (ACTH) on adrenocortical steroidogenesis were studied on rabbit adrenocortical cells harvested from control animals and from ACTH-treated rabbits. Treatment (200 μg ACTH1–24 s.c. daily for 12 days) led to an increase in the maximal secretory capacity of a given number of adrenocortical cells in response to ACTH as well as to dibutyryl c-AMP in vitro. This increase in the steroidogenic activity of ACTH was associated with a clearcut reduction in the sensitivity to the peptide, as much higher concentrations of ACTH were needed to elicit a half-maximal increase in steroidogenesis with cells from ACTH-treated animals. Moreover, the ACTH-dependent generation of c-AMP was significantly reduced as a result of in vivo treatment with ACTH. The production of aldosterone in response to ACTH or dibutyryl c-AMP was also much lower with cells from ACTH-treated rabbits. In addition to quantitative changes in steroidogenesis, prolonged treatment with ACTH led to increased generation of 17-hydroxylated steroids, with increased adrenocortical production and plasma levels of cortisol. cortisone and 11-deoxycortisol; concomitantly synthesis and circulating levels of corticosterone were reduced. It was therefore concluded that besides the well-known prolonged trophic influence of ACTH on adrenal growth this peptide influences in a lasting way the function of adrenocortical cells. The latter effect is characterized in the rabbit by an increased capacity but decreased sensitivity to ACTH, and by a stimulation of 17α-hydroxylase activity with ensuing shift from corticosterone to cortisol production.

STIMULATION BY INSULIN OF TRANSEPITHELIAL SODIUM TRANSPORT

A decrease in plasma potassium concentration is mentioned in the first reports of insulin effects on the whole 0rganism.lThis obscrvation became amenable to interpretation much later, when Zierler 3, reported that the electrical potential difference across rat striated-muscle membrane increased when the tissue was exposed to insulin; glucose was not required for this hormonal effect. Moore,5 studying frog striated muscle, provided evidence for this hyperpolarization resulting from stimulation of sodium extrusion-and potassium uptake-by the tissue as a consequence of stimulation of the sodium “pump.” o* One could therefore raise the question of transcellular sodium transport being also influenced by insulin-since, for all we know, a characteristic of cells forming epithelia with this property is that while they allow passive sodium entry at their apical pole, they. expel it towards the milieu inth-ieur at their basal-lateral borders by means of this sodium “pump.” In fact, Herrera reported a stimulating effect of insulin on transepithelial sodium transport by frog skin and toad bladder.v For the experiments described here, sodium transport was usually measured by the short-circuit current method devised by Ussing and Zerahn.’O In general, toad ( B u f o niarinus) epithelia-urinary bladder, colon, abdominal skin-were treated with mammalian crystallized insulin added to substrate-free Ringer’s fluid on the inner (serosal) side of the preparations.

Parotid-saliva Cortisol in Normal Subjects - Increase During Pregnancy

Cortisol concentration was measured by radioimmunoassay in parotid saliva: values for normal subjects averaged 15 nM at 8:00 and 4 nM at 18:00. Parotid saliva cortisol was shown to react promptly and in a clearcut fashion to factors leading to rise and fall in cortisol secretion. Cortisol levels in parotid saliva were uninfluenced in young women taken estrogen-containing drugs for contraceptive purposes; on the other hand, they increased significantly during the third trimester of pregnancy. This increase corresponds to the reported increase in ultrafilterable cortisol, itself being presumably the result of the hypersection of progesterone characterizing this physiological condition since progesterone competes with cortisol not only at the level of transcortin but also at that of cytoplasmic glucocorticoid receptors.

Subtypes of Madin-Darby canine kidney (MDCK) cells defined by immunocytochemistry: Further evidence for properties of renal collecting duct cells

The Madin-Darby canine kidney (MDCK) cell line has been proposed as a model for studying intercalated (IC) cells of the renal cortical collecting duct. The IC cells are characterized by peanut lectin (PNA) binding capacity, carbonic anhydrase (CA) activity and Cl-−HCO3-exchange mediated by a band 3-related protein. It has been suggested that these properties are also expressed in MDCK cells. So far however, the nature of the specific protein involved in Cl-−HCO3-exchange, the type of CA isozyme and the relationship between these two characteristics and PNA binding, have not been investigated in MDCK cells by immunocytochemical methods. Using two antibodies raised against human erythrocyte band 3 protein and two against human erythrocyte CA I and II isozymes, our study provides evidence that a protein related to band 3 is expressed in about 5% of cultured MDCK cells; these band 3-positive cells do not bind PNA and are not reactive for CAI or CAII. About 30% of the MDCK cells bind PNA, two-thirds of which are also CAII-positive. A majority (about 65%) of MDCK cells is not reactive for the three markers used; their density is increased after incubation with aldosterone. These data indicate (i) that the Cl-−HCO3-exchanger of the MDCK cells could be related to human erythrocyte band 3, (ii) that the CA activity of the MDCK cell line bears antigenic identity with the erythrocyte CA II isozyme and (iii) that the latter is always co-localized with PNA binding. These results provide immunocytochemical evidence for the heterogeneity of the MDCK cell line, which might reflect the cellular heterogeneity encountered in the renal cortical collecting duct. Our data also indicate that clonal selection will be required for future functional studies of the MDCK cells.