Expression of the amiloride-blockable Na+ channel by RNA from control versus aldosterone-stimulated tissue.

The amiloride-blockable Na+ channel was expressed in Xenopus oocytes injected with total RNA isolated from the toad urinary bladder. This system was used to investigate mechanisms that mediate the natriferic action of aldosterone. Incubation of the epithelium with aldosterone for 3 h doubled its channel activity but did not increase the ability of isolated RNA to express functional channels in oocytes. A 20-h incubation with the hormone produced an additional increase of Na+ transport across the intact epithelium and also augmented the channel activity expressed in oocytes by nearly 10-fold. The data are in agreement with our model that aldosterone enhances the apical Na+ permeability of tight epithelia by a short term activation of pre-existing channels, followed by chronic induction of new channel protein. Blocking methyl transfer reactions, previously shown to inhibit the natriferic action of aldosterone in tight epithelia, did not alter the basal or aldosterone-induced response in oocytes.     

A search for a defect of proximal transport in denervated kidneys of conscious dogs

The influence of renal nerves on proximal Na+ reabsorption was studied in clearance experiments with unilaterally renal-denervated conscious dogs prepared by surgical bladder division. Two types of experiments were made : A. maximal water diuresis, and B. Total blockade of distal NaCl reabsorption with ethacrynic acid and chlorothiazide. In maximal water diuresis CH2O + CNa was used as a measure of fluid delivery to the distal nephron. At similar GFR on both sides, the proximal reabsorption estimated as GFR--(CH2O + CNa) was 38.4 +/- 5.6 ml/min for the intact and 35.9 +/- 4.2 ml/min for the denervated kidney (n = 6, difference NS). After distal tubular blockade, proximal Na+ reabsorption calculated as filtered load minus urinary excretion was 3.84 +/- 0.43 mmol/min for the intact and 3.91 +/- 0.36 mmol/min for the denervated kidney (n = 6, difference NS). The fractional reabsorption of NA+ was 64.9 +/- 1.0% for the intact and 66.9 +/- 1.1% for the denervated kidney (difference NS). In contrast to data from renal denervation studies with anaesthetized animals, the present experiments did not show any difference in proximal reabsorption between the innervated- and denervated kidney. We conclude that in absence of anaesthesia renal efferent nerves have no major effect on NaCl transport in dog proximal tubule.     

Role of antidiuretic hormone in hyponatremia in patients with isolated adrenocorticotropic hormone deficiency.

We found symptomatic hyponatremia in four elderly patients in which serum sodium (Na) levels ranged from 101 to 122 mEq/l. All 4 patients had low levels of plasma adrenocorticotropic hormone (ACTH), serum cortisol, and urinary excretion of 17-OHCS, and poor responses of ACTH to exogenous insulin and antidiuretic hormone (ADH). Other pituitary hormones were all normal. They were therefore diagnosed as having isolated ACTH deficiency. Plasma ADH was relatively high despite hypoosmolality which was associated with the hyponatremia. Water loading test revealed impaired water excretion and poor suppression of plasma ADH. Replacement with 20-30 mg hydrocortisone completely restored the serum Na level and restored the plasma ADH level to the normal range in all 4 patients. Other factors such as decreased glomerular filtration, enhanced urinary Na loss and decreased Na intake were also included. These results indicate that there is marked hyponatremia and that in the presence of hypoosmolality the sustained secretion of ADH is the key factor in causing the impaired water excretion and hyponatremia in isolated ACTH deficiency.     

Is hypercalcemic diet a possible antidote to oral contraceptive-induced hypertension?

Administration of oral contraceptive (OC) has been associated with body fluid retention and in high doses over a long period, promotes hypertension. This present investigation tests the hypothesis that the dietary calcium supplementation increases salt and water excretion in OC (norgestre/ethinylestradiol) treated 32 female albino rats randomly distributed into four (1-4) groups of 8 rats each: Control, OC-treated, OC-treated+ Calcium diet fed and Calcium diet fed only respectively. OC was administered to the appropriate groups by gavage. Experimental diet contained 2.5% calcium supplement. Plasma and urinary [Na+] [K+] were evaluated after 8 weeks of experimentation by flame photometry and plasma [Ca2+] by colorimetric method. OC-treatment induced a significant fall in urinary [Na+]. Water excretion was significantly reduced in these animals (control, 3.1±0.56 Vs OC-treated rats, 1.47±0.16). OC-treated rats had significantly higher plasma [K+] compared to control rats. Calcium supplementation induced increases in plasma [Na+], [K+] and augmented urinary Na+ excretion (OC-treated + Ca2+ diet Vs OC-treated only). Compared with the control rats, high Ca2+ diet fed rats exhibited significant increases in plasma [Na+] and [K+] accompanied by significant decreases in urinary H20 excretion. These results strongly suggest that high dietary Ca2+ supplementation increases salt and water excretion in OC-treated rats and potentially moderates fluid retention and blood pressure in these animals, and may be of clinical significance in OC-induced abnormal fluid retention and perhaps OC-induced hypertension.Keywords: Hypercalcemic-diet, Oral contraceptive, Plasma electrolytes, Hypertension, Female-albino-rats.     

Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder: Effects of bradykinin,kallidin, eledoisin,and physalaemin

Summary It has been shown by means of Bentley'sin vitro preparation of the isolated urinary bladder of the toad,Bufo marinus paracnemis Lutz, that bradykinin reversibly inhibited the increase brought about by vasopressin on the permeability to water of the toad bladder. The increased hydro-osmotic response of the bladder to oxytocin was also inhibited by the kinin. The effect on water permeability was observed when bradykinin was added either to the serosal Ringer's solution or to the mucosal solution. The addition of bradykinin alone did not alter the basal osmotic water transfer across the bladder. In this context, bradykinin acted as a competitive antagonist of vasopressin (and oxytocin). Although lacking intrinsic activity, bradykinin exhibited affinity for receptor sites that are also common to the neurohypophysial hormones, causing a parallel shift of the log-dose/response curve for vasopressin without changing the maximal responses. The effects of other kinins (namely kallidin, eledoisin and physalaemin) on the toad bladder were also tested. Each of these drugs alone did not change the basal water flux across the bladder wall. Like bradykinin, these peptides inhibited the increase in water permeability evoked by vasopressin and oxytocin in the bladder. In view of the importance of neurohypophysial hormones and their target tissues to the osmotic homeostasis of amphibians, and the observation of antagonism between the kinins and the pituitary hormones coupled to the abundance of kinins in the amphibian organism, particularly in the skin and urinary bladder, teleological reasoning predicts a physiological role for the kinins, possibly functioning to dampen excesses and oscillations in membrane permeability that could occur in face of a constant and variable secretion of neurohypophysial hormone, thus adding to the homeostatic response of the amphibian organism.     

Effect of oleamide on water and sodium ion transport in osmoregulatory organs of vertebrates

In the frog Rana temporaria L., oleamide solution (10 μmole/L) applied to the isolated basal surface of the skin augmented the short-circuit current (SCC) from 59.8 ± 2.5 to 78.2 ± 1.4 μA/cm². When applied to the serous membrane of the urinary bladder, oleamide (1 μmole/L) induced more than a 30-fold increase in osmotic water permeability. The addition of argininevasotocin against the background of oleamide further increased SCC across the skin and osmotic water permeability in the bladder. In Wistar rats, intraperitoneal injection of oleamide (0.1 μmole/L per 100 g of body weight) to non-anesthetized animals after water load reduced diuresis by 22% and increased solute-free water reabsorption and urinary sodium excretion by 31% and 55%, respectively, but did not affect urinary potassium excretion. These findings provide evidence of the similarity between the effects of oleamide and nonapeptide neurohypophyseal hormones on water and ion transport in epithelial cells of osmoregulatory organs in vertebrates.     

Pathways for movement of ions and water across toad urinary bladder

Hypertonicity of the mucosal bathing medium increases the electrical conductance of toad urinary bladder by osmotic distension of the epithelial "tight" or limiting junctions. However, toad urine is not normally hypertonic to plasma. In this study, the transmural osmotic gradient was varied strictly within the physiologic range; initially hypotonic mucosal bathing media were made isotonic by addition of a variety of solutes. Mucosal NaCl increased tissue conductance substantially. This phenomenon could not have reflected soley an altered conductance of the transcellular active transport pathway since mucosal KCl also increased tissue conductance, whether or not Na+ was present in the bathing media. The effect of mucosal NaCl could not have been mediated solely by a parallel transepithelial pathway formed by damaged tissue since mucosal addition of certain nonelectrolytes also increased tissue conductance. Finally, the osmotically-induced increase in conductance could not have occurred soley in transcellular transepithelial channels in parallel with the active pathway for Na+, since the permeability to 22Na from serosa to mucosa (s to m) was also increased by mucosal addition of NaCl; a number of lines of evidence suggest that s-to-m movement of Na+ proceeds largely through paracellular transepithelial pathways. The results thus establish that the permeability of the limiting junctions is physiologically dependent on the magnitude of the transmural osmotic gradient. A major role is proposed for this mechanism, serving to conserve the body stores of NaCl from excessive urinary excretion.     

Response of rainbow trout gill Na+-ATPpase to T(3) and NaCl administration

The effect of the administration of commercial diets supplemented with 9 mg kg(-1) 3,5,3'-triiodo-l-thyronine (T(3)) or 10% (w/w) NaCl was evaluated on the ouabain-insensitive Na+-ATPase activity in rainbow trout gill microsomes. The trial, carried out following the seasonal trend from March to mid-May, included a treatment phase in freshwater and a subsequent transfer to brackish water (22 per thousand salinity) where trout were not treated. pH dependence, apparent Km values for Mg(2+) and Na+, and Hill coefficients evaluated throughout the trial for Na+-ATPase were generally not affected by the treatments and habitat change. In comparison with the control group, in both treated groups, Na+-ATPase activity was lower during the freshwater phase and higher after brackish-water transfer. As compared with untreated trout, gill (Na++K+)-ATPase activity during the freshwater phase was stimulated by NaCl treatment and also by T(3) treatment after transfer to brackish water. The results indicate that NaCl and T(3) administration act differently on the two ATPase activities involved in Na+ regulation and suggest a prevalent role of Na+-ATPase activity in hypoosmotic conditions.     

Water ingestion by rats fed a high-salt diet may be mediated, in part, by visceral osmoreceptors

After surgical removal of all salivary secretions ("desalivation"), rats increase their consumption of water while eating dry laboratory chow. In the present experiments, desalivated rats drank even more water while they ate "powdered" high-salt food (i.e., <15-mg food particles). The Na+ concentration of systemic plasma in these animals was not elevated during or immediately after the meal, which suggests that cerebral osmoreceptors were not involved in mediating the increased water intake. A presystemic osmoregulatory signal likely stimulated thirst because the Na+ and water contents of the gastric chyme computed to a solution approximately 150 mM NaCl. In contrast, desalivated rats drank much smaller volumes of water while eating "pulverized" high-salt food (i.e., 60-140-mg food particles), and the fluid mixture in the gastric chyme computed to approximately 280 mM NaCl solution. These and other findings suggest that the NaCl ingested in the powdered high-salt diet was dissolved in the gastric fluid and that duodenal osmoreceptors (or Na+-receptors) detected when the concentration of fluid leaving the stomach was elevated after each feeding bout, and promptly stimulated thirst, whereupon rats drank water until the gastric fluid was diluted back to isotonicity. However, when rats ate the pulverized high-salt diet, much of the NaCl ingested may have been embedded in the gastric chyme and therefore was not accessible to visceral osmoreceptors once it emptied from the stomach. Consistent with that hypothesis, fluid intakes were increased considerably when desalivated rats drank 0.10 M NaCl instead of water while eating either powdered or pulverized high-salt food.     

Amiloride-inhibited Na+ uptake into toad bladder microsomes is Na+-H+ exchange

Amiloride-inhibited Na+ transport into toad urinary bladder microsomes is sensitive to a pH gradient across the vesicular membrane. The magnitude of the gradient was measured directly with acridine orange. Also Na+ could stimulate amiloride-sensitive proton efflux from the microsomes. These results indicated that the transport process was Na+-H+ exchange.     

Na+-H+ exchange and Na+ entry across the apical membrane of Necturus gallbladder

The role of Na+-H+ exchange in Na+ transport across the apical membrane was evaluated in Necturus gallbladder epithelium by means of intracellular Na+ activity (aNai) and 22Na+ uptake measurements. Under control conditions, complete replacement of Na+ in the mucosal solution with tetramethylammonium reduced aNai from 14.0 to 6.9 mM in 2 min (P less than 0.001). Mucosal addition of the Na+-H+ exchange inhibitor amiloride (10(-3) M) reduced aNai from 15.0 to 13.3 mM (P less than 0.001), whereas bumetanide (10(-5) and 10(-4) M) had no effect. Na+ influx across the apical membrane was studied by treating the tissues with ouabain, bathing them in Na-free solutions, and suddenly replacing the mucosal solution with an Na-containing solution. When the mucosal solution was replaced with Na-Ringer's, aNai increased at approximately 11 mM/min. This increase was inhibited by 54% by amiloride (10(-3) M, P less than 0.001) and was unaffected by bumetanide (10(-5) M). Amiloride-inhibitable Na+ fluxes across the apical membrane were also induced by the imposition of pH gradients. Na+ influx was also examined in tissues that had not been treated with ouabain. Under control conditions, 22Na+ influx from the mucosal solution into the epithelium was linear over the first 60 s and was inhibited by 40% by amiloride (10(-3) M, P less than 0.001) and by 19% by bumetanide (10(-5) M, P less than 0.025). We conclude that Na+-H+ exchange is a major pathway for Na+ entry in Necturus gallbladder, which accounts for at least half of apical Na+ influx both under transporting conditions and during exposure to ouabain. Bumetanide-inhibitable Na+ entry mechanisms may account for only a smaller fraction of Na+ influx under transporting conditions, and cannot explain influx in ouabain-treated tissues. These results support the hypothesis that NaCl entry results primarily from the operation of parallel Na+-H+ and Cl--HCO-3 exchangers, and not from a bumetanide-inhibitable NaCl cotransporter.     

Hyponatremia-induced brain edema in guinea pigs is reduced by treatment with the novel anion transport inhibitor L-644,711

Cerebral edema in various disease states may result from astroglial swelling due to increased NaCl uptake mediated by enhanced Cl-HC03 exchange. We evaluated this mechanism in the pathogenesis of cerebral edema in acute hyponatremia by administering L-644,711, a fluorenyloxyacetate derivative that functions as an anion exchange inhibitor, to guinea pigs with severe reductions in serum Na+ concentration. Acute hyponatremia was induced for 54 hr by daily injections of arginine vasopressin (10 U/day) and 5% dextrose in water (7.5% body wt/day). Experimental animals received L-644,711, 20 mg/kg/day, while controls were given an equal volume of the diluent. This regimen lowered the serum Na from normal levels to 108 +/- 3 and 109 +/- 4 mM in experimental and control animals, respectively. Drug treatment resulted in less cerebral edema characterized by a reduction in brain total tissue water 432 +/- 4 vs 466 +/- 8 ml/100 g dry wt experimental vs control, P less than 0.005. This difference was composed mainly of less expansion of the intracellular water space, 287 +/- 11 vs 323 +/- 9 ml/100 g dry wt experimental vs control, p less than 0.005. The cerebral cortical Na+ +Cl content was reduced from 55.5 +/- 1.3 (control) to 39.5 +/- 1.1 mEq/100 g dry wt (experimental), p less than 0.01. These results indicate that treatment of guinea pigs with L-644,711 decreases brain NaCl content and attenuates cerebral edema during severe acute hyponatremia without normalizing the serum Na+ concentration.     

Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase

The kidney plays an essential role in regulating potassium and acid balance. A major site for these regulations is in the collecting tubule. In the present study, we report the primary sequence of a novel alpha subunit of the P-ATPase gene family, which we isolated from the urinary bladder epithelium of the toad Bufo marinus, the amphibian equivalent of the mammalian collecting tubule. The cDNA encodes a protein of 1,042 amino acids which shares approximately 67% identity with the alpha 1 subunit of the ouabain-inhibitable Na,K-ATPase and approximately 69% identity with the alpha subunit of the SCH28080- inhibitable gastric H,K-ATPase. When coexpressed in Xenopus oocytes with a beta subunit isolated from the same cDNA library, the ATPase is able to transport rubidium (a potassium surrogate) inward, and hydrogen outward, leading to alkalization of the intracellular compartment and acidification of the external medium. The novel ATPase has a unique pharmacological profile showing intermediate sensitivity to both ouabain and SCH28080. Our findings indicate that the bladder ATPase is a member of a new ion motive P-ATPase subfamily. The bladder ATPase is expressed in the urinary tract but not in the stomach or the colon. This H,K-ATPase may be one of the molecules involved in H+ and K+ homeostasis, mediating the transport of these ions across urinary epithelia and therefore regulating their urinary excretion.     

The identity of the current carriers in canine lingual epithelium in vitro

Ion transport across the lingual epithelium has been implicated as an early event in gustatory transduction. The fluxes of isotopically labelled Na+ and Cl- were measured across isolated canine dorsal lingual epithelium under short-circuit conditions. The epithelium actively absorbs Na+ and to a lesser extent actively secretes Cl-. Under symmetrical conditions with Krebs-Henseleit buffer on both sides, (1) Na+ absorption accounts for 46% of the short-circuit current (Isc); (2) there are two transcellular Na+ pathways, one amiloride-sensitive and one amiloride-insensitive; (3) ouabain, added to the serosal solution, inhibits both Isc and active Na+ absorption. When hyperosmotic (0.25 M) NaCl is placed in the mucosal bath, both Isc and Na+ absorption increase; net Na+ absorption is at least as much as Isc. Ion substitution studies indicate that the tissue may transport a variety of larger ions, though not as effectively as Na+ and Cl-. Thus we have shown that the lingual epithelium, like other epithelia of the gastrointestinal tract, actively transports ions. However, it is unusual both in its response to hyperosmotic solutions and in the variety of ions that support a transepithelial short-circuit current. Since sodium ion transport under hyperosmotic conditions has been shown to correlate well with the gustatory neural response, the variety of ions transported may likewise indicate a wider role for transport in taste transduction.     

On the mechanism of sodium-proton exchange in crayfish

In salt depleted crayfish net sodium and proton fluxes were coupled 1:1 as required by the frog skin-turtle bladder model. In addition, three proton pump inhibitors produced equal reductions of both fluxes. It is concluded that the model operates in these animals. Net Na+ and H+ fluxes were very small in tap water adapted animals, but regression analysis clearly showed that they were coupled, though perhaps not 1:1. Proton pump inhibitors, at concentrations that suppressed fluxes in salt-depleted crayfish, had no measureable effect on proton movement in tap water-adapted animals. Two of them (dicyclocarbodiimide and N-ethyl maleimide), caused a small reduction in Na+ influx without affecting proton efflux. These experiments provide no support for operation of the frog-turtle system in adult crayfish adapted to tap water. A 2Na+-H+ exchanger is considered from an energetic point of view. Such a system might be able to couple Na+ and H+ fluxes in dilute, near neutral solutions ([Na+] approximately 1-2 mM; pH 7).     

Cation-selective channels in amphibian epithelia: electrophysiological properties and activation

1. Na+ as well as Li+ move across the apical membrane through amiloride-sensitive ionic channels. 2. K+ movements across the apical membrane occur through Ba2+- and Cs+-sensitive channels which do not allow the passage of Na+ or Li+. 3. A third pathway in the apical membrane is permeable for Na+, K+, Cs+, Rb+, NH+4 and Ti+. The currents carried by these monovalent cations are blocked by Ca2+ and divalent cations as well as La3+. 4. In the urinary bladder, the Ca2+-sensitive currents are stimulated by oxytocin, activators of cytosolic cAMP and cAMP analogues. Also the oxytocin activated currents are blocked by divalent cations and La3+. 5. Nanomolar concentrations of mucosal Ag+ activate the third channel and open the pathway for movements of Ca2+, Ba2+ and Mg2+, which are known to permeate through Ca2+ channels in excitable tissues.     

Influence of glucocorticoids on the secretion of pancreatic juice in the rat

The influence of adrenalectomy and hydrocortisone treatment on the exocrine pancreatic secretion has been studied in anaesthetized rats. In the adrenalectomized animals Na+ administered in the saline solution provided for drinking was able to maintain standard sodium levels in serum. In these animals an increase of Na+ secretion in pancreatic juice was observed. Furthermore, the osmotic effect created by the increase in Na+ would account for the increase in pancreatic flow. In these adrenalectomized rats, an increase in K+ output is observed, which can be explained by the high K+ concentrations in serum. Likewise adrenalectomy decreased pancreatic enzyme secretion and produced a loss in weight of the organ that is accounted for by a lack of glucocorticoids. Hydrocortisone administration did not affect neither the secretion nor the weight of the pancreas of the control rats but the hormone proved to be effective in adrenalectomized rats producing a pancreatic secretion close to normal, balancing the secretory rate of water, Na+ and K+, completely restoring total protein secretion and the weight of the pancreas but amylase secretion in part only. It is therefore concluded that the weight of the pancreas and its exocrine secretion are clearly influenced by adrenalectomy and by substitution therapy with hydrocortisone. The administration of this hormone (25 mg.kg-1.day-1 along 6 days) did not affect intact animals.     

Aldosterone stimulates Na+ transport without affecting citrate synthase activity in cultured cells

Aldosterone increases citrate synthase activity in toad urinary bladder and mammalian kidney. It has been suggested that this action is important to aldosterone stimulation of Na+ transport, and it has been used as a marker of those epithelia which are stimulated by aldosterone. We describe three continuous lines of cultured cells derived from toad urinary bladder and toad kidney in which aldosterone increases active Na+ transport but does not increase the activity of citrate synthase. Therefore, in cultured cells at least, citrate synthase is not a critical enzyme for, or a suitable marker of, aldosterone stimulation of Na+ transport.     

Serotonergic mechanisms of the lateral parabrachial nucleus in renal and hormonal responses to isotonic blood volume expansion

This study investigated the involvement of serotonergic mechanisms of the lateral parabrachial nucleus (LPBN) in the control of sodium (Na+) excretion, potassium (K+) excretion, and urinary volume in unanesthetized rats subjected to acute isotonic blood volume expansion (0.15 M NaCl, 2 ml/100 g of body wt over 1 min) or control rats. Plasma oxytocin (OT), vasopressin (VP), and atrial natriuretic peptide (ANP) levels were also determined in the same protocol. Male Wistar rats with stainless steel cannulas implanted bilaterally into the LPBN were used. In rats treated with vehicle in the LPBN, blood volume expansion increased urinary volume, Na+ and K+ excretion, and also plasma ANP and OT. Bilateral injections of serotonergic receptor antagonist methysergide (1 or 4 microg/200 etal) into the LPBN reduced the effects of blood volume expansion on increased Na+ and K+ excretion and urinary volume, while LPBN injections of serotonergic 5-HT(2a)/HT(2c) receptor agonist, 2.5-dimetoxi-4-iodoamphetamine hydrobromide (DOI; 1 or 5 microg/200 etal) enhanced the effects of blood volume expansion on Na+ and K+ excretion and urinary volume. Methysergide (4 microg) into the LPBN decreased the effects of blood volume expansion on plasma ANP and OT, while DOI (5 microg) increased them. The present results suggest the involvement of LPBN serotonergic mechanisms in the regulation of urinary sodium, potassium and water excretion, and hormonal responses to acute isotonic blood volume expansion.     

Frog skin epithelium: electrolyte transport and chytridiomycosis

One unique physiological characteristic of frogs is that their main route for intake of water is across the skin. In these animals, the skin acts in concert with the kidney and urinary bladder to maintain electrolyte homeostasis. Water absorption across the skin is driven by the osmotic gradient that develops as a consequence of solute transport. Our recent study demonstrated that chytridiomycosis, an infection of amphibian skin by the fungal pathogen, Batrachochytrium dendrobatidis, inhibits epithelial Na(+) channels, attenuating Na(+) absorption through the skin. In frogs that become severely affected by this fungus, systemic depletion of Na(+), K(+) and Cl(-) is thought to cause deterioration of cardiac electrical function, leading to cardiac arrest. Here we review the ion transport mechanisms of frog skin, and discuss the effect of chytridiomycosis on these mechanisms.