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Home , Calcitonin, Vasopressin

Proc. Nadl. Acad. Sci. USA Vol. 83, pp. 2276-2280, April 1986 Physiological Sciences Sensitivities of rat thick ascending limbs and collecting ducts to in vivo (Brattleboro rats/urinary concentration) JEAN-MARC ELALOUF, ANTONIO Di STEFANO, AND CHRISTIAN DE ROUFFIGNAC Service de Biologie Cellulaire, DIpartement de Biologie, Centre d'Etudes Nuc1daires de Saclay, 91191 Gif-sur-Yvette Cedex, France Communicated by Gerhard Giebisch, October 28, 1985

ABSTRACT experiments were performed to act in the same way as [Arg8]vasopressin on the permeability characterize the sensitivity to vasopressin ofthe thick ascending of the cortical collecting duct to water (11, 12). To avoid limbs and ofthe rat kidney. The response undesirable interference from these , the experi- of the thick ascending limbs was evaluated by measuring the ments were performed with -deprived rats (4); i.e., Mg2+ rate in the , since the [-8]vas- Brattleboro D. I. rats (lacking [Arg8]vasopressin) that were opressin-mediated effects on Mg2+ excretion are the direct acutely thyroparathyroidectomized (thus suppressing calci- result of a stimulation of Mg2' in this tonin and PTH levels) and given to inhibit segment, and the response ofthe collecting ducts was evaluated secretion (6-11). by changes in urine flow. To avoid the effects of , glucagon, and , which stimulate Mg2+ reabsorption in the thick ascending limb and distal tubule, and METHODS of calcitonin, which increases the permeability of the cortical Experiments were performed on 38 male rats with hereditary collecting ducts to water, experiments were performed on hypothalamic (Brattleboro D. I. rats) bred Brattleboro D. I. rats (with hereditary diabetes insipidus, due in our laboratory. Laboratory chow of constant composition to a lack of [Arg8]vasopressin) acutely deprived of endogenous (UAR, Villemoisson-sur-Orge, France; composition: P = parathyroid hormone, calcitonin, and glucagon. Vasopressin 6.6, Ca = 7.5, K = 9.3, Na = 3, Mg = 1.2 g/kg) was available infused at rates up to 5 pg/min did not reduce the Mg2+ to all rats until 17 hr before the experiments. The animals fractional excretion rate, whereas at 5 pg/min water excretion were then placed in metabolic cages to verify by their urine was decreased by 50%. The half-maximal reduction of Mg2+ osmolality (<250 milliosmoles/kg) that they were homozy- excretion occurred at vasopressin infusion rates 4-6 times gous. Free access to distilled water was available until higher than those necessary to diminish the water excretion rate anesthesia. The animals were prepared in order to reduce the to the same extent. We conclude that in vivo, two segments plasma concentration of calcitonin, PTH, and glucagon involved in the production ofconcentrated urine have different (6-11). They are called hormone-deprived animals through- sensitivities to vasopressin and that this difference in sensitivity out this paper. is very similar for the biological response in vivo and the After anesthesia (Inactin, 10 mg/100 g ofbody weight), the adenylate cyclase activation in vitro. We suggest that both the animals, placed on a heated table to maintain a body magnitude and the nature ofthe effects of [Arg8]vasopressin on temperature of 370C, underwent tracheotomy after removal the kidney may vary according to the required of the thyroparathyroid glands. For the perfusion of solu- response. tions, one polyethylene (PE50) catheter (Becton Dickinson) was inserted into the right jugular vein and another into the Vasopressin (antidiuretic hormone) increases the water per- right femoral vein. A PE50 catheter was inserted into the right meability of the collecting ducts and stimulates reabsorption femoral artery for arterial blood sampling and arterial blood of , such as NaCl (1-3), Mg2+, Ca2+, and K+ (4), pressure recording and two such catheters were inserted into by the thick ascending limb of Henle's loop. Imbert-Teboul the left and right ureters for urine collection. During surgery, et al. (5) showed that the apparent Km value of adenylate estimated fluid losses were replaced by intravenous admin- cyclase activation by [arginine-8]vasopressin was much high- istration of a 0.9% NaCl solution (1.5 ml/100 g of body er in the thick ascending limb than in the collecting tubule, weight). For the changes in urinary flow rate to reflect as indicating that the two segments have different sensitivities to closely as possible the [Arg8]vasopressin-mediated alteration of the permeability of the collecting ducts to water, it is this hormone. crucial to maintain the water balance equilibrium throughout We undertook the present study to test whether vasopres- the clearance studies (i.e., to adjust the water infusion rates sin, at a concentration higher than that necessary to increase to the water losses). It was therefore necessary to perform a the permeability of the collecting duct to water, elicits a preliminary experimental series, to establish the time course physiological response in the thick ascending limb in vivo. and degree of urine-flow reduction during sustained [Arg8]- The biological effects on the thick ascending limb were vasopressin infusion over the range of doses to be studied in evaluated by measuring the Mg2' excretion rate, since the the second experimental series. In the second series of effects on the loop are directly responsible for the hormone- experiments, the magnitudes of the physiological responses mediated decrease of Mg2+ excretion in urine (4, 6). The of the thick ascending limbs and collecting ducts were response ofthe collecting ducts was evaluated by the changes measured at each ofthe doses of vasopressin used in the first in urine flow. Calcitonin, parathyroid hormone (PTH), and series. glucagon may act on Mg2+ reabsorption in the distal tubule [Arg8]Vasopressin-Dependent Reduction of (7, 8) and the thick ascending limb (9, 10), and calcitonin may (Series I). These experiments were performed to determine the appropriate administration rate of water in the second The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: PTH, parathyroid hormone; dDAVP, 1-desamino-[D- in accordance with 18 U.S.C. §1734 solely to indicate this fact. Arg8]vasopressin. 2276 Downloaded by guest on September 25, 2021 Physiological Sciences: Elalouf et al. Proc. Natl. Acad. Sci. USA 83 (1986) 2277 series. Throughout the experiments, the water losses through extrarenal pathways were taken to be constant, whereas the urinary losses were variable, depending on the administration rate of the antidiuretic hormone. During clearance experi- ments on anesthetized 200-g rats, it is generally accepted (24) 75 that the extrarenal losses are well compensated by a water infusion rate of 20 A.l/min. Thus, in these experiments, a r. 4 continuous infusion of 0.4% NaCl solution at 20 ,ul/min was started through the jugular vein catheter immediately after so surgery. This solution also contained [3H]inulin (10 ,uCi/ml; Lo. New England Nuclear; 1 Ci = 37 GBq), bovine serum albumin (1 mg/ml; fraction V), somatostatin (7 Ag/ml; Clin-Midy, catalog no. 93537), and, when necessary, syn- thetic [Arg]vasopressin (Sigma Grade VIII, 400 units/mg). The administration rate of somatostatin (140 ng/min) was r_ chosen to ensure inhibition ofglucagon secretion. Bailly etal. 25 h (10) showed that Brattleboro rats receiving such a dose of somatostatin had a plasma glucagon concentration undetect- able by radioimmunoassay (<7 pM). At time zero after surgery, the rats were also infused, through the femoral vein catheter, with a 0.4% NaCl solution at 60 Aul/min (4), in order to establish a mild water . One hour after the -30 0 30 60 90 120 beginning of perfusions, the urinary flow rate from both Time, min kidneys was determined over a 10-min collection period. The rate at which the 0.4% NaCl solution was infused through the FIG. 1. Water excretion rate in hormone-deprived rats during femoral vein catheter was then adjusted to compensate for control periods (e) and during administration (indicated by solid bar) renal water losses (this adjustment being made after each of vasopressin at 5 (o), 10 (c), 20 (a), 40 (o), or 80 (E) pg/min. After urine collection the each urine collection, the perfusion rate was adjusted to replace throughout experiment). Two 10-min water losses adequately, in order not to infuse excess water. The control periods of urine collection were then carried out. urinary flow rate is that of the two kidneys. Each point is a mean of After these two periods, AVP was added to the solution the means obtained from 3 or 4 rats, except for the control periods, infused through thejugular vein, at concentrations chosen to for which the data from all animals were pooled. ensure an administration rate of either 5 (n = 3 rats), 10 (n = 3), or 40 (n = 4) pg/min. Five minutes after the beginning of (5-10% of the filtered load) is rather limited when compared [Arg8]vasopressin infusion, the urine flow rate from both with that carried out by the (60-85% of this kidneys was measured during five 5-min periods and there- load, depending on the hormonal status of the animal). Thus after during two 20-min collection periods. At the end of the determination of Mg2" urinary excretion is a satisfactory second 20-min period, the administration rate of [Arg8]vas- quantitative measure of Mg2+ transport by the loop. opressin was doubled for rats previously receiving 10 or 40 The hormone-deprived model requires a depletion of the pg/min. The results of these experiments are depicted in Fig. four hormones that act on the adenylate cyclase present in the 1. After [Arg8]vasopressin administration, the urine flow thick ascending limb. The Brattleboro rat, which lacks rapidly decreased and then reached a steady state after about [Arg8]vasopressin, was chosen for establishing the model. In 20 min, irrespective of the dose used. After 70 min of this rat, the ofthe thick ascending limb is reduced perfusion, rats receiving 5, 10, and 40 pg/minhad mean urine in length and thickness (14). The threshold of the cyclase flows of54, 28, and 11 ,u/min, respectively. [Arg8]Vasopres- response ofthis epithelium to vasopressin is nevertheless the sin at 20 pg/min (subsequent to 70 min of perfusion at 10 same as that of either Wistar (5) or Long-Evans rats (15), pg/min) induced further reduction of the urinary flow rate, lying between 10 and 100 pM. In addition, the epithelium of which stabilized at 20 ,ul/min. [Arg8]Vasopressin at 80 the thick ascending limb of the Brattleboro rat given dDAVP pg/min(subsequent to 70 minperfusion at 40 pg/min) didnot daily hypertrophied after 6 weeks of treatment (14). The alter the urine flow rate, indicating that the maximal reduction of Mg2' excretion that accompanied this treatment antidiuretic response was reached at 40 pg/min. was nevertheless very similar to that observed in Brattleboro Evaluation of Hormone Action on the Thick Ascending rats receiving an acute infusion of vasopressin (16). More- Limb. The action of [Arg8]vasopressin on the thick ascending over, when the treatment was interrupted, the Mg2+ frac- limb was evaluated by the -mediated decrease of tional excretion rose to 20-30% ofthe filtered load within 2-3 urinary Mg2+ excretion. Several lines of evidence support days, despite the persistence of the epithelial hypertrophy this protocol. (i) Mg transport was shown to be unchanged by (16). This indicates that the high Mg2+ excretion rate in the 1-desamino-[D-Arg8]vasopressin (dDAVP), a synthetic ana- Brattleboro rat results from a lack of antidiuretic hormone, log of [Arg8]vasopressin, both in the proximal (4) and in the which normally maintains a high rate ofMg2+ reabsorption in distal tubule (6). (ii) In these previous studies (4, 6), the the loop, and not from an impaired capacity of the thick amount ofMg2+ excreted in urine, in the presence or absence ascending limb to reabsorb Mg2+. ofdDAVP, was directly correlated to the amount delivered to Effects of Various Doses of [Arg8]Vasopressin on Kidney the early distal tubule (r = 0.83, P < 0.001) and, therefore, to Function (Series II). These experiments were carried out on the ability of the loop of Henle to reabsorb Mg2+. (iii) We seven groups of four rats. The first group consisted of found (6, 7), in agreement with others (13), that in the hormone-deprived rats. The other six consisted of hormone- superficial distal tubule, Mg2+ reabsorption is load-depen- deprived rats infused with [Arg8]vasopressin at rates of 3, 5, dent. However, within the normal range of Mg2+ load 10, 20, 40, or 80 pg/min. The animals were infused with the delivered to this nephron segment (i.e., tubular concentration same solutions as those used in experimental series I, except <2 mM), the amount reabsorbed increases linearly with the that [Arg8]vasopressin was added to the solution exactly at load delivered (r = 0.90, P < 0.001); the fraction of this load time zero and perfused throughout the equilibration and reabsorbed is therefore constant (6, 7) and unaltered by clearance periods, and that the infusion rates to compensate dDAVP (6). (iv) Mg2' reabsorption by the distal tubule for urinary water losses were therefore chosen for urine flows Downloaded by guest on September 25, 2021 2278 Physiological Sciences: Elalouf et al. Proc. Natl. Acad. Sci. USA 83 (1986) deduced from experimental series I. In the hormone-deprived (control) rats and in those receiving [Arg8jvasopressin at 3 pg/min, this infusion rate was set at 60 Al/min. The clearance 10 periods began after a 60-min equilibration period (that is, 150-180 min after thyroparathyroidectomy) during which the urine from both kidneys was collected. The experiments

consisted of five 30-min clearance periods. After each blood 5 collection, samples were centrifuged, the plasma was sepa- rated, and the erythrocytes were resuspended for reinjection through the femoral vein catheter. The urine flow rate ofboth kidneys was determined after each urine collection. The phosphate concentration of the plasma and urine of each rat 0 was determined in order to verify that the fractional excretion of phosphate was low (3.2 ± 0.4% of the filtered load, n = 7 groups) and, thus, that parathyroidectomy had been success- ful. At the end of each experiment, the left kidney was weighed after dissection of the perirenal fat. 30 Analytical Procedures. For determination of the [3H]inulin known volumes of urine and plasma samples concentration, T 1I were dissolved in Picofluor scintillation solution (Packard Instrument, Downers Grove, IL) and the radioactivity was 20 measured by liquid scintillation counting (Intertechnique SL Lk+ 4000). The Mg2+ concentrations in the plasma and urine a samples were determined with an atomic fluorescence spec- trometer (Baird), and the phosphate concentrations by the P-,- method of Chen et al. (17). The urinary Mg2' fractional 10 excretion rate was calculated using an ultrafiltrable fraction of 0.70 (4, 6). Osmolality was determined with a 5100c vapor pressure osmometer (Wescor, Logan, VT). Only the urine I from the left kidney was analyzed for inulin and concentrations. The values for each parameter in the plasma 0 60 90 120 150 180 210 and urine samples were averaged and considered as one Time, min datum for each animal. Statistical comparisons between excretion (FE, groups were performed by variance analysis and, when FiG. 2. Time course of water and Mg2" fractional as % of the filtered load) in hormone-deprived rats (o) and in by the F value, Tukey's test was applied (18). The allowed hormone-deprived rats receiving vasopressin at 5 (o) or 40 (o) data are presented as means ± SEM. pg/min. Each point is a mean value obtained from four rats studied by the clearance technique. RESULTS The mean arterial was very similar in all seven opressin inducing a maximal antidiuretic response (40 groups (Table 1). Neither did the plasma Mg2+ concentration pg/min)]. The effects of [Arg8]vasopressin on water and differ between groups, except for that receiving the highest Mg2+ reabsorption by the kidney are dissociated according to dose of hormone. Table 1 also shows that the urine/plasma the dose employed. A maximal dose of [Arg8]vasopressin osmolality ratio gradually increases with the infusion rate of reduced the fractional excretion rate of both H20 and Mg2+, [Arg8]vasopressin; the response was maximal when [Arg8]- as originally observed with the synthetic analog dDAVP (4, vasopressin was given at 40 pg/min. The glomerular 6). In rats given [Arg8]vasopressin at 5 pg/min, the Mg2+ rate remained constant, irrespective of the dose. fractional excretion did not differ statistically from the The time course for water and Mg2+ fractional excretion control values, whereas the water fractional excretion rate during experiments is depicted in Fig. 2 for three groups of was already reduced by a factor of 2. rats [controls, rats receiving a submaximal dose of [Arg8]vas- The relationships between the amounts of water or Mg2` opressin (5 pg/min), and rats receiving a dose of [Arg8]vas- excreted by the kidney and the administration rate of

Table 1. Body weight, kidney weight, arterial blood pressure, plasma magnesium concentration, , urine/plasma osmolality ratio, and glomerular filtration rate (GFR) for rats receiving various doses of vasopressin Kidney Plasma Urine/plasma [Arg8]Vasopressin, Body weight, weight, pressure, Mg2+, osmolality, osmolality GFR, pg/min g mg mm Hg* mM milliosmoles/kg ratio yIA/mint 0 178 ± 6 675 ± 25 84 ± 4 0.52 ± 0.02 313 ± 3 0.51 ± 0.03 975 ± 53 3 187 ± 6 761 ± 51 93 ± 5 0.54 ± 0.01 316 ± 5 0.75 ± 0.05 850 ± 29 5 170 ± 1 652 ± 28 83 ± 7 0.47 ± 0.02 307 ± 2 0.92 ± 0.06$ 922 ± 96 10 174 ± 8 633 ± 25 85 ± 5 0.47 ± 0.01 297 ± 7 1.42 ± 0.19t 985 ± 86 20 182 ± 7 744 ± 66 85 ± 5 0.54 ± 0.02 308 ± 3 2.79 ± 0.19t 796 ± 48 40 177 ± 3 687 ± 37 91 7 0.53 ± 0.01 304 ± 6 3.25 ± 0.33* 934 ± 90 80 181 ± 4 629 ± 20 88 3 0.68 ± 0.06t 317 ± 8 2.56 ± 0.04* 964 ± 63 Values are means ± SEM (n = 4 rats for each dose). *1 mm Hg = 133.3 Pa. tPer gram of kidney weight. *Significant difference (P < 0.05) as compared with rats not receiving vasopressin. Downloaded by guest on September 25, 2021 Physiological Sciences: Elalouf et al. Proc. Natl. Acad. Sci. USA 83 (1986) 2279

[Arg8]vasopressin are summarized in Fig. 3. Mg2" excretion agreement with our data) and that concomitantly the plasma was maximally reduced when [Arg8]vasopressin was given at [Arg8]vasopressin was 2.3 pg/ml. In addition, these authors 40 pg/min and was decreased 50% for an [Arg8]vasopressin found that the plasma concentration of [Arg8]vasopressin infusion rate of 20 pg/min. The water excretion rate showed increased linearly with the administration rate of the hor- a different response pattern. The maximal antidiuretic effect mone: for an administration rate of 100 pg/min, the was obtained at 20 pg/min, and a half-maximal reduction of [Arg8]vasopressin concentration was 8.0 pg/ml. From these diuresis was found at rates of 3-5 pg/min. There was, data, we calculate that in our experiments the [Arg8]vaso- therefore, a 4- to 6-fold difference between the doses giving pressin concentration varied between 0 and 6 pg/ml, values the half-maximal response of these two renal parameters. that lie within the physiological range. In addition, Fig. 3 shows that [Arg8]vasopressin infusion The present study indicates that at a low plasma [Arg8]vas- rates between 0 and 10 pg/min caused the steepest reduction opressin concentration ([Arg8]vasopressin infusion rates 3-5 ofwater excretion by the kidney, whereas the Mg2+ excretion pg/min) the antidiuretic response mainly involves an action was scarcely affected. Comparison of Table 1 and Fig. 3 on the collecting duct system, since the diuresis was reduced shows that at the half-maximal response, the urine 50%, whereas the Mg2+ excretion rate was unaltered, indi- osmolality value was <300 milliosmoles/kg. cating that the thick ascending limb had not yet been stimulated. A 50% decrease of the diuresis would result in a DISCUSSION urine osmolality of 250-300 milliosmoles/kg, as indeed was recorded. Concomitantly, the osmolality ofthe fluid reaching This study shows that two biological effects of [Arg8]vaso- the hairpin turn ofthe long-looped could not be less pressin on the kidney, namely the stimulation ofreabsorption than 350 milliosmoles/kg, which is a limit value measured in of water and Mg2+, exhibit different sensitivities to the the hormone-deprived animal (i.e., in the absence of the four hormone. To increase Mg2+ reabsorption requires higher hormones, including [Arg8]vasopressin, that stimulate elec- doses of [Arg8]vasopressin than to decrease urine flow. The trolyte transport in the thick ascending limb) (11). Thus, the maximal antidiuretic response, however, was not registered incomplete osmotic equilibration between the urine and the until Mg2+ reabsorption had clearly been stimulated. The fluid of Henle's loop confirms that at this dose, [Arg8]vaso- half-maximal reduction of Mg2+ excretion occurred at infu- pressin does not exert its maximal effect on the water sion rates 4- to 6-fold higher than those necessary to decrease permeability of the collecting duct, as deduced from the data the urinary flow to the same extent. presented in Fig. 3. It is therefore safe to conclude that the Vasopressin, apart from its effects on the permeability of half-maximal reduction of the diuresis corresponds to the the collecting tubule to water, stimulates electrolyte reab- half-maximal response of the collecting ducts to vasopressin. sorption by the thick ascending limb (1-3). In rats deprived [Arg8]Vasopressin must induce its biological effects on the of endogenous [Arg8]vasopressin, calcitonin, PTH, and collecting duct and the thick ascending limb by generating glucagon, dDAVP enhances Na+, Cl-, Mg2+, Ca2+, and K+ intracellular cyclic AMP, since the hormone stimulates the reabsorption (4). As far as Mg2+ is concerned, its urinary adenylate cyclase activity in both segments (5) and its effects excretion rate has been considered as an indicator of an can be reproduced with cyclic AMP or cyclic AMP analogs action ofthe peptide on the thick ascending limb. The present (1, 2, 21). Imbert-Teboul et al. (5) established dose-response study, which shows distinct dose-response relationships for curves for the [Arg8]vasopressin-dependent adenylate cy- [Arg8]vasopressin action on Mg2+ and water excretion, clase activity in the medullary thick ascending limbs and strongly argues in favor of different sensitivities to [Arg8]vas- collecting tubules. As generally observed for any tubular opressin of two target sites, the thick ascending limb and the segment, the doses required to induce an effect on the cyclase collecting tubule system. Such a specific concentration in vitro are higher than those necessary to elicit a biological dependency of each target site is likely to be ofphysiological response in vivo. The reasons for this difference are both relevance. In the normally hydrated rat, the plasma concen- methodological and biological, and a discussion of them is tration of vasopressin is 2-3 pg/ml and increases to 15 pg/ml beyond the scope ofthis paper. Nevertheless, Imbert-Teboul after a 24-hr water-deprivation (19). Gellai et al. (20) found et al. (5) reported that a half-maximal response ofthe cyclase that [Arg8]vasopressin administered at 25 pg/min to Brat- occurred in the medullary thick ascending limb at an tleboro D. I. rats elicited a maximal antidiuretic response (in [Arg8]vasopressin concentration 5 times greater than that required for half-maximal response in the medullary collect- ing tubule; the same shift was noted between the respective responses ofthe cortical thick ascending limb and the cortical loo1 0 CF collecting tubule (M. Imbert-Teboul, personal communica- 0 tion). The receptors for [Arg8]vasopressin located in the two 4) S. E segments must be similar, because several vasopressin ana- u 751 X z 0 logs gave Kmvalues of were 4) E adenylate cyclase activation that C4-. x systematically higher in the thick ascending limb than in the 0 CO 0 r. E 501 collecting tubule (5). Thus, the -0 [Arg8]vasopressin-dependent u , 0 adenylate cyclase activity exhibited similar dose-response z 10 relationships in the cortical and medullary thick ascending Q 25 -1 04 limb, and both segments were found less sensitive than the cortical and medullary collecting tubules. That the concen- oL tration of [Arg8]vasopressin required to induce 50% of the biological response and 50% of the cyclase response must be 0 3 5 10 20 40 80 5 times higher for the thick ascending limb than for the collecting tubule shows that the difference in cyclase sensi- Vasopressin, pg/min tivity among cell populations may be interpreted in physio- FIG. 3. Dose-dependent effects of [Arg8]vasopressin on the logical terms. In this connection, it is relevant to note that the reduction of absolute water (o) and Mg2" (e) excretion rates by the amplitude of the effects induced by [Arg8]vasopressin on the kidney in hormone-deprived rats, expressed as % of the maximal adenylate cyclase activity in the thick ascending limb in reduction. Each point is the mean of the means obtained from each different species of is correlated with urinary of the four rats studied at each vasopressin-infusion rate. concentrating ability (22). Downloaded by guest on September 25, 2021 2280 Physiological Sciences: Elalouf et al. Proc. Natl. Acad. Sci. USA 83 (1986) Electrolyte reabsorption by the thick ascending limbs and 1. Hall, D. A. & Varney, D. M. (1980) J. Clin. Invest. 66, water removal from the collecting tubules are two processes 792-802. primarily involved in the elaboration of concentrated urine. 2. Sasaki, S. & Imai, M. (1980) Pflugers Archiv. 383, 215-221. 3. Hebert, S., Culpepper, M. & Andreoli, T. (1981) Am. J. The reabsorption of electrolytes, in excess of water, by the Physiol. 241, F412-F431. thick ascending limb participates in the constitution of a 4. de Rouffignac, C., Corman, B. & Roinel, N. (1983) Am. J. corticomedullary concentration gradient. When [Arg8]vaso- Physiol. 244, F156-F164. pressin is present, the fluid entering the collecting duct 5. Imbert-Teboul, M., Chabardes, D., Montegut, M., Clique, A. equilibrates its concentration with that of the surrounding & Morel, F. (1978) Endocrinology 102, 1254-1261. medium by osmotic water withdrawal. That these two pro- 6. Elalouf, J. M., Roinel, N. & de Rouffignac, C. (1984) Pflugers cesses (electrolyte and water reabsorptions) exhibit different Archiv. 401, 167-173. dose-response relationships may be of 7. Elalouf, J. M., Roinel, N. & de Rouffignac, C. (1983) Pflugers [Arg8]vasopressin Archiv. 399, 111-118. physiological interest. The present data show that maximal 8. Bailly, C., Roinel, N. & Amiel, C. (1985) Pflugers Archiv. 403, urine concentration occurred when [Arg8]vasopressin acted 28-34. not only on the collecting ducts but also on the thick 9. Elalouf, J. M., Roinel, N. & de Rouffignac, C. (1984) Am. J. ascending limb. Both the magnitude and the nature of the Physiol. 246, F213-F220. effects of[Arg8]vasopressin on the kidney may therefore vary 10. Bailly, C., Roinel, N. & Amiel, C. (1984) Am. J. Physiol. 246, to the required antidiuretic response. This idea was F205-F212. according 11. de Rouffignac, C. & Elalouf, J. M. (1983) Am. J. Physiol. 245, advanced several years ago by Atherton et al. (23) who F506-F511. proposed, from work on the medullary content of water and 12. Morel, F., Chabardes, D., Imbert-Teboul, M., Le Bouffant, solutes as a function of antidiuretic hormone perfusion rates, F., Hus-Citharel, A. & Montegut, M. (1982) Kidney Int. 21, that high-affinity sites are implicated in the control of water 55-62. permeability and low-affinity sites are implicated in the 13. Quamme, G. A. & Dirks, J. H. (1980) Am. J. Physiol. 238, of in the medulla. It is now clear that F187-F198. accumulation 14. Kriz, W. & Bankir, L. (1982) Ann. N.Y. Acad. Sci. 394, the low-affinity sites suggested by these authors correspond 424-434. to the thick ascending parts of Henle's loop. 15. Imbert-Teboul, M., Chabardes, D., Montegut, M., Clique, A. In conclusion, our work shows that, in rats, the vasopres- & Morel, F. (1978) Renal Physiol. 1, 3-10. sin-dependent reduction of water and Mg2' excretion each 16. Bouby, N., Trinh-Trang-Tan, M. M. & Bankir, L. (1984) exhibits a specific dose-response relationship. The hormonal Pflugers Archiv. 402, 458-464. to elicit a response in 17. Chen, P. S., Toribara, T. Y. & Warner, H. (1956) Anal. Chem. concentrations required physiological 28, 1756-1758. two target segments, the thick ascending limb and the 18. Snedecor, G. W. (1956) Statistical Methods (Iowa State Univ. collecting tubule, are therefore different in vivo. A low Press, Ames, IA), 5th Ed. concentration of vasopressin increases the permeability of 19. Dunn, F. L., Brennan, T. J., Nelson, A. E. & Robertson, the nephron terminal segments to water, and only at higher G. L. (1973) J. Clin. Invest. 52, 3212-3219. concentrations does the hormone also stimulate electrolyte 20. Gellai, M., Silverstein, J. H., Hwang, J. C., La Rochelle, the thick limb. The difference in F. T. & Valtin, H. (1984) Am. J. Physiol. 246, F819-F827. reabsorption by ascending 21. Grantham, J. J. & Burg, M. B. (1966) Am. J. Physiol. 211, sensitivity to vasopressin of these two processes may allow 255-259. the hormone to control the renal concentrating ability with 22. Morel, F. (1981) Am. J. Physiol. 240, F159-F164. greater efficiency. 23. Atherton, J. C., Green, R. & Thomas, S. (1971) J. Physiol. 213, 291-309. We gratefully acknowledge the technical assistance ofP. Phillippe, 24. Andreucci, V. E. (1978) Manual of Renal Micropuncture N. Soyeux, and A. Zimmerman. (Idelson, Naples, Italy). Downloaded by guest on September 25, 2021