Rabary M. Rajerison

Ontogenic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation.

The development of adenylate cyclase responsiveness to vasopressin and parathyroid hormone was studied using membrane fractions prepared from medullo-papillary and cortical portions of kidneys of 2-46-day-old rats. The development of vasopressin binding capacity was followed on the same preparations, using [3H]vasopressin. The characteristics of medullo-papillary adenylate cyclase response to vasopressin were identical in young and adult control animals as regards apparent Km values for [Lys8]vasopressin (3 X 10(-8) M), specificity towards the natural neurohypophysial peptides and the effects of Mg2+. However, the magnitude of maximal enzyme activation by vasopressin was much lower in very young than adult animals. Accordingly vasopressin responsiveness increased sharply between the 10th and 25th days but the magnitude of the maximal response only reached the adult value between the 30th and 45th days after birth. During both periods basal adenylate cyclase activity was almost independent of age. Specific vasopressin binding sites were detected on kidney medullo-papillary membranes from young animals. Vasopressin binding capacity and adenylate cyclase responsiveness to the hormone followed similar development patterns. However, the appearance of specific binding sites slightly preceded the onset of adenylate cyclase responsiveness. Basal cortical adenylate cyclase activity/mg protein was 12 times higher in 2-day-old rats than in the adult controls. It dropped with age but only fell to the adult value between the 25th and the 35th days after birth. For the youngest animals tested (2 days old), the increase in activity due to parathyroid hormone was about half the increase measured in adults, and gradually rose to about 75% of the adult response between the 2nd and 46th days after birth. Apparent Km values for parathyroid hormone were identical in young and adult animals (3.2 and 3.0 U/ml, respectively).

The isolated frog skin epithelium: Presence of α and β adrenergic receptors regulating active sodium transport and water permeability

SummaryThe effects of stimulation of α and β adrenergic receptors on short circuit current (S.C.C.), Na+ and Cl− fluxes and osmotic water permeability were studied on isolated frog skin epithelial layers separated from the dermis.Low norepinephrine doses (final concentrations in the incubation medium ranging from 5×10−9 to 10−8 M) produced increased water permeability and S.C.C. The latter was entirely accounted for by an increase in the active Na+ influx. Na+ outflux and Cl− fluxes were not modified. Both these effects disappeared after treatment with the β blocking agent, Propranolol. Higher norepinephrine doses (final concentrations: 10−7 to 10−6 M) produced: 1. an increase in water permeability lower than that produced by low doses, the highest doses failing to increase water permeability, and 2. a triphasic change in S.C.C.: after an initial increase, S.C.C. dropped to its resting value and then rose again to a sustained value. Na+ and Cl− flux measurements showed that the variation in S.C.C. reflected variations in active Na+ transport. When the same high norepinephrine doses were applied after treatment with the α blocking agent Phentolamine, the effects observed were identical to those obtained with low doses.On β blocked preparations, large doses of norepinephrine inhibited the water permeability and sodium transport increases induced by theophylline or oxytocin but did not modify those induced by 3′5′-cyclic AMP. The inhibition was suppressed after blocking α receptors.From the foregoing, it was concluded that both α and β adrenergic receptors are present in frog skin epithelial cells and are involved in the regulation of water and sodium permeability.It is suggested that the inhibitory effect of α stimulation resulted from the inhibition of cyclic-AMP generating system, the activity of which is under the positive control effect of oxytocin and β stimulation.

The isolated frog skin epithelium: Permeability characteristics and responsiveness to oxytocin, cyclic AMP and theophylline

SummaryThe combined treatment of the frog skin with collagenase and hydrostatic pressure enables complete separation of the epithelial layer from the supporting dermis. The separation entirely preserves the epitheliums passive permeability and active sodium transport capacity. The short circuit current, d.c. resistance, unidirectional fluxes of Na+ and Cl− ions, and osmotic water permeability were found identical on series of isolated epitheliums and intact skins obtained from the same groups of animals. Furthermore, the isolated epithelium is fully responsive to oxytocin, cyclic AMP and theophylline—three agents known to enhance active sodium transport and water permeability of the intact skin.

Effects of temperature, ouabain and diuretics on the cell sodium and potassium contents of isolated rat kidney tubules

Cell Na+ and K+ contents were measured by flame photometry in single pieces of rat medullary thick ascending limb (MAL) and medullary collecting tubules (MCT) after an overnight incubation at various temperatures. Below 8°C, MCT samples were no more able to sustain high-K+, low-Na+ cell concentrations, and sodium progressively replaced cell potassium as the temperature decreased. The loss of potassium and the accompanying accumulation of sodium by MCT cells occurred at lower or higher temperature when amiloride (20 μmol/l) or ouabain (1 mmol/l) was present respectively in the incubation medium compared to that observed on control non-treated MCT. In contrast, MAL samples maintained normal cation gradients across their membrane at all temperatures, including 0°C, even in the presence of ouabain. However, MAL cells lost nearly all their potassium which was replaced by sodium when they were incubated in K+-free solution. These Na+-loaded, K+-depleted MAL cells restored high-K+ and low-Na+ contents similar to those of control samples when they were further incubated for 1 h at 0°C in presence of 5 mmol/l external potassium. Even in the presence of 1 mmol/l ouabain and at 0°C, a restoration of almost normal cation gradients occured provided that Na+-loaded MAL were incubated for 24 h after potassium addition to the external medium. The results are discussed in relation to the respective effects of low temperatures on the passive and active components of the cation balance in the cells of the two nephron segments.

Effects of external potassium concentrations on the cell sodium and potassium contents of isolated rat kidney tubules

The effects of 20 μmol/l amiloride, 10 μmol/l furosemide and 1 mmol/l ouabain on cell Na and K concentrations were investigated by flame microphotometry in isolated rat medullary collecting tubules and medullary thick ascending limbs (MCT and MAL) as a function of the external potassium concentration [Ke]. The results are expressed as Na and K concentrations per liter cell volume ([Nac] and [Kc], mmol/l) and relative sodium content, [Nac]/([Nac]+[Kc]). From the experimental curves, [Ke]1/2 is defined as the [Ke] value corresponding to half maximal exchange of K against Na in cells. When [Ke] was 5 mmol/l, the relative Na content was less than 15% in control and amiloride-treated MCT as well as in control and furosemide-treated MAL, and about 24% in ouabain-treated MCT and MAL. In MCT, relative cell Na content increased up to 90% or more when [Ke] was reduced from 2.5 to 0.25 mmol/l. [Ke]1/2 was 0.55, 0.45 and 1.25 mmol/l for control, amiloride-treated and ouabain-treated MCT respectively. In MAL, similar increases in relative Na content were observed when [Ke] was reduced from 0.5 to 0.05 mmol/l. [Ke]1/2 was 0.25, 0.10 and 1.75 mmol/l for control, furosemide-treated and ouabain-treated MAL respectively. When [Ke] was reduced from 5 to 1 mmol/l, [Nac] dropped from 16.4 to 8.4 mmol/l (P<0.01) in control MAL. When [Ke] was 5 mmol/l, [Nac] was lower in furosemide-treated MAL (7.8 mmol/l) than control MAL (P<0.01). At 1 mmol/l [Ke], [Nac] was similar in both groups.These results are discussed in terms of the balance between the active and passive components of Na and K fluxes across apical and basolateral cell membranes. They indicate that a K-dependent passive Na entry process exists in the membranes of MAL cells but not of MCT cells. This process was proportionally more inhibited than the active Na pump when [Ke] was reduced from 5 to 1 mmol/l. In addition, it was found sensitive to furosemide. It presumably corresponds to the luminal 1 Na-1 K-2 Cl contransport mechanism known to exist in the thick ascending limb.

Renal adenylate cyclase activation by amino acylated vasopressin and oxytocin

Two series of neurohypophysial peptide amino-acylated derivatives were tested for their ability to activate plasma membrane adenylate cyclase prepared from pig or rat kidney. They were firstly [8-lysine]-vasopressin-related derivatives (Na-[Glycyl-Cys]1-[8-Lysine]-vasopressin and Na-[Glycyl-Glycyl-Cys51-[8-Lysine]-vasopressin) and secondly oxytocin-related derivatives (Na-[Glycyl-Cys-a1)-oxytocin, Na-[Leucyl-Glycyl-Glycyl--Cys]-oxytocin, and Na-[Glycyl-Cys]-[2-0methyl tyrosine]-oxtocin). The maximal adenylate cyclase activation induced by these peptides was lower than that induced by their respective parent hormones. After incubation of these analogues with plasma membranes obtained from the renal medulla, no significant release of parent hormones occurred. Good qualitative correlations were observed between relative antidiuretic activities measured in vivo and relative potencies in activating adenylate cyclase. It was concluded that direct action of peptides tested on the kidney is at least partly responsible for their antidiuretic activity in vivo.

Relationship between cell volume and cation content in thick ascending limb of rat kidney

We examined the relationship between the cell volume and cation concentration ([Nai] and [Ki]) of isolated segments of rat medullary thick ascending limb (MAL) after incubation at 30°C in various isotonic solutions. When the tubules were incubated in a normal NaCl solution containing 5 mmol/l K+, addition of 1 mmol/l of ouabain increased [Nai] and decreased [Ki] but did not change the total ([Nai]+[Ki]) concentration (about 90 mEq/l) or tubular volume. After incubation in various K+-free solutions, the tubules were almost fully K+-depleted; their volume per unit of length was similar in the three solutions, although the choline Cl-treated tubules had a very low sodium content compared to the NaCl-and Na2SO4-treated tubules (8 vs. 97 and 95 mEq/l respectively). Ouabain altered neither volume nor [Nai] of tubules incubated in choline Cl or Na2SO4 solution. Transfer of tubules from K+-free Na2SO4 or K+-free choline Cl solution into K+-free NaCl solution resulted in an increase in [Nai] (by 29 and 97 mEq/l respectively) without much increase in tubular volume. A marked swelling of the tubules was only observed when the K+-free NaCl solution contained also ouabain. Under this condition, [Nai] was comparable to the Na+ concentration of the incubation medium. After washing and incubation in a normal NaCl solution containing K+, the swollen tubules recovered their initial volume and restored Na+ and K+ concentration gradients across the cell membranes. The ([Nai]+[Ki]) concentration centration measured in the tubules preincubated in choline Cl solution was always smaller than that of the tubules preincubated either in NaCl or Na2SO4 solutions, an observation suggesting that choline ions enter rat MAL cells. Barium (3 mmol/l) prevented tubular swelling. This inhibition corresponded to a smaller increase in [Nai] than that observed in control tubules. Furosemide or bumetanide (even at 0.1 mmol/l) did not alter the increases in tubular volume and in Na+ content induced by ouabain. The data provide additional evidence that the isoosmotic swelling of MAL cells requires an almost full inhibition of Na+-pump activity and involves coupled net fluxes of Na+ and Cl− ions.

Involvement of Na and Cl in ouabain-induced cell swelling in thick ascending limb of rat kidney

Changes in the volume of isolated segments of rat medullary thick ascending limb (MAL) were studied by a photographic technique, after tubule incubation in isotonic solutions in the absence or presence of ouabain and/or K. When segments were incubated at 30°C in NaCl solution, their volume increased by 75% after removal of external K, and by 170% after removal of external K plus addition of 1 mmol/l ouabain. At steady state, tubular volume was a function of the external K concentration. Resting volume was obtained with external K concentrations higher than 0.1 and 1.0 mmol/l in the absence and presence of ouabain respectively. When MAL samples were incubated in isotonic K-free Na2SO4 or K-free choline Cl solution, their volume per unit of length was similar to that determined in NaCl medium, but there was no swelling after the addition of ouabain. The ouabain-induced swelling was shown to depend on both the Na and Cl concentrations in the incubate (apparentKm of 87 and 80 mmol/l for Na and Cl respectively). Swollen tubules recovered their resting volume when ouabain, Na or Cl was removed from the incubation medium. Recovery of resting volume was also observed after addition of K into the incubation medium. These observations indicate that rat MAL cell volume is the result of coupled passive net fluxes of Na and Cl, which depend on the respective electrochemical gradients for Na or Cl across the cell membranes and the Na-pump activity which continuously extrudes Na.

Hormone Binding to Receptors: Vasopressin

It is now clearly established that the primary action of antidiuretic hormone on the mammalian kidney consists in the activation of a membrane-bound adenylate cyclase present in the tubular cells from the responding parts of the nephron (1, 2). Membrane fractions prepared from the kidney medullary portion contain a vasopressin- sensitive adenylate cyclase (3–8); they exhibit the expected high sensitivity to the hormone and marked stereospecificity towards the natural neurohypophysial peptides and artificial structural analogues (7, 9–11). These acellular preparations thus appear to be a convenient biological material for a tentative characterization of the hormonal receptors involved in the response of the mammalian kidney to vasopressin. The purpose of the present paper is to discuss some of the methodological and technical problems encountered during the course of recent studies by this group, on the pig kidney lysine-vasopressin receptor (4, 9–12).

Inhibition des effects de l'ocytocine sur la peau et la vessie de la grenouille par l'[2-O-ethyltyrosine]-ocytocine

Abstract Hydrosmotic and natriferic activities of [O-ethyl-Tyr2]-oxytocin in the frog (Rana esculenta) are studied. Hydrosmotic action is measured by the increase in water permeability of the isolated bladder and the natriferic action by the stimulation of active sodium transport by the isolated skin. The affinity of [O-ethyl-Tyr2]-oxytocin for these two receptors is much lower than that of oxytocin, used as the reference standard: it is 28 times lower on the skin and 109 times lower on the bladder. The intrinsic hydrosmotic and atriferic activities of this analogue, as defined by the maximal response, are 35% and 18%, respectively, of that of oxytocin. [O-Ethyl-Tyr2]-oxytocin partially inhibits hydrosmotic and natriferic responses of small doses of oxytocin but does not modify responses of the same magnitude evoked by3′,5′-AMP on the bladder and by 3′,5′-AMP or norepinephrine on the skin. The inhibition can be reversed by increasing amounts of oxytocin. The responses of supramaximal doses of oxytocin are identical in the presence or in the absence of [O-ethyl-Tyr2]-oxytocin. It is concluded that this analogue behaves like a competitive inhibitor of oxytocin on both the frog skin and bladder.