Proc. Nati. Acad. Sci. USA Vol. 85, pp. 2407-2411, April 1988 Physiological Sciences Desensitization of rat renal thick ascending limb cells to vasopressin (Brattleboro rats/urine concentration/glucagon/calcitonin) JEAN-MARC ELALOUF*, DAOUDI CHABANE SARI, NICOLE ROINEL, AND CHRISTIAN DE ROUFFIGNAC Service de Biologie Cellulaire, Centre d'Etudes Nucleaires de Saclay, 91191 Gif-sur-Yvette Cedex, France Communicated by Gerhard Giebisch, December 8, 1987 ABSTRACT Previous studies from this laboratory have collecting duct but also enhances Cl, Na, K, Mg, and Ca demonstrated that vasopressin stimulates K, Mg, Ca, Cl, and reabsorption in the thick ascending limb of Henle's loop Na reabsorption by the thick ascending limb of Henle's loop (TALH) of the rat kidney (2-4). In addition, three hor- (TALH) of the rat kidney. Micropuncture of superficial neph- mones-glucagon, calcitonin, and parathyroid hormone rons and clearance experiments were performed to determine (PTH)-act on the same pool of adenylate cyclase as vaso- whether desensitization of the TALH to vasopressin may be pressin in the rat TALH (5) and also strongly stimulate K, demonstrated in vivo and whether such desensitization is Mg, and Ca reabsorption in this nephron segment (6, 7). The specific for the effects of vasopressin (i.e., homologous) or also present study was undertaken to determine whether (i) the alters the response to the other hormones acting on the same biological response of the TALH to vasopressin can be pool of adenylate cyclase in this nephron segment. Brattleboro desensitized in vivo, (ii) this desensitization is homologous or rats, with hereditary hypothalamic diabetes insipidus (DI), heterologous, and (iii) the TALH and the collecting ducts can were given i.m. injections of 1-desamino-8-D-arginine-vaso- be independently desensitized. Confirmation of iii would pressin {des-l-amino-[DArg8JVP (herein designated dDAVP); 2 provide further evidence that vasopressin can affect the two ,ug/day} for 3 days. The effects of maximal physiological doses segments independently; it was previously demonstrated that of arginine-8-vasopressin {[Arg8]VP (herein designated AVP); a higher concentration of vasopressin is required to induce a 20 pg/min per 100 g of body weight} were studied 2 days after biological response (8) or to stimulate the cyclase (9) in the the cessation of treatment, when the animals had returned to TALH than in the collecting duct. DI. The K, Mg, Ca, and, to a lesser extent, Cl and Na The present experiments were performed in vivo. The concentrations in the fluid leaving the TALH of superficial TALH functions were studied by micropuncture of superfi- nephrons were higher in dDAVP-treated than in untreated rats cial nephrons. Our attention was focused on the effects of given similar amounts of AVP during the experiments. A arginine-8-vasopressin {[Arg8]VP (herein designated AVP)} 50-60% desensitization of the TALH to AVP was still appar- on K, Mg, and Ca reabsorption in the loop of Henle since ent 2 days after stopping the dDAVP injections. Desensitization these effects are easier to detect than those on Cl and Na (3). is homologous, as judged from normal responses to physiolog- The Mg excretion rate in urine closely depends on the ical doses of glucagon and calcitonin, two hormones acting on reabsorption of Mg in the TALH (3, 6, 8, 10) and was the same cyclase pool as AVP in the rat TALH. The AVP- therefore also measured to assess TALH function. With dependent increase of urine osmolality, however, indicated regard to the collecting duct system, the response to AVP that its effects on the permeability to water of the collecting was studied by measuring the urine osmolality. The effects duct were scarcely affected in dDAVP-treated rats. It is of glucagon and calcitonin, which act on the same cyclase concluded that (i) AVP induces homologous desensitization in pool as vasopressin in the cortical and medullary TALH (5), the rat TALH and (ii) the TALH can be markedly desensitized were studied to determine whether the desensitization of the to AVP when the collecting duct response to this hormone is TALH was homologous or heterologous. To avoid undesir- poorly affected or even fully maintained. able interference from endogenous vasopressin, glucagon, calcitonin, and PTH, this study was performed on hormone- It is well documented that, for a variety of hormones acting deprived rats (3, 6-8, 10)-i.e., Brattleboro rats {lacking by way of membrane-bound receptors, the number of such AVP production (11)} that were acutely thyroparathyroidec- cell-surface receptors and the magnitude of the biological tomized (thus suppressing calcitonin and PTH) and given response induced by an agonist can be depressed by acute or somatostatin during the experiments to inhibit glucagon chronic increases of the concentration of the agonist in the secretion. blood (1). This phenomenon is called desensitization or METHODS hormone-induced refractoriness. For those cell types in which several hormones act through a common mechanism, Experiments were performed on 72 male rats (body weight, desensitization is considered of the homologous type if the 160-200 g) with hereditary diabetes insipidus (DI) (Brattle- alteration of the cellular response is selective for one ago- boro strain). The experimental procedures have been de- nist-i.e., the one that is responsible for the desensitization scribed in detail (3, 6, 8, 10) and will only be recalled briefly. process. Conversely, desensitization is referred to as heter- The animals were fed ad libitum until 17 hr before the ologous or nonspecific when one hormone induces cell experiments. Free access to water was allowed until anes- refractoriness to several hormones. thesia (inactin, 10 mg/100 g of body weight). The animals In the mammalian kidney vasopressin is known to act on were prepared to reduce the plasma concentration of endog- several different cell types, with distinct functional proper- enous calcitonin, PTH, and glucagon (3). For this purpose, a ties. This hormone increases the permeability to water of the Abbreviations: TALH, thick ascending limb of Henle's loop; AVP, arginine-8-vasopressin; dDAVP, 1-desamino-8-D-arginine-vasopres- The publication costs of this article were defrayed in part by page charge sin; DI, diabetes insipidus; PTH, parathyroid hormone; HD, hor- payment. This article must therefore be hereby marked "advertisement" mone-deprived; HDD, HD densensitized; HCT, human calcitonin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. Downloaded by guest on September 26, 2021 2407 2408 Physiological Sciences: Elalouf et al. Proc. Natl. Acad. Sci. USA 85 (1988) thyroparathyroidectomy was performed immediately after filtrate samples were obtained by centrifuging plasma sam- anesthesia (i.e., 3 hr before the onset of clearance measure- ples on Amicon cuprophan membranes (19). ments), and somatostatin (Clin Midy Laboratory, Montpel- During experiments, the diuretic animals (i.e., the rats not lier, France) was administered i.v. at a rate of 70 ng/min per infused with AVP) were given NaCl solution (4 g/liter) 100 g of body weight from the end of surgery up to the end through the left femoral vein at a rate of 85 ILI/min to of the experiment to inhibit glucagon secretion (6). compensate for urinary losses (8). Another NaCl solution (4 Desensitization was achieved by i.m. injections of 1- g/liter) containing [3H]inulin, as a glomerular indicator, as desamino-8-D-arginine-vasopressin {des-1-amino-[DArg8]VP well as somatostatin and, where necessary, glucagon or (herein designated dDAVP); Ferring Pharmaceuticals, calcitonin was given at a constant rate of 20 Al/min. The Malmo, Sweden}. The peptide, dissolved in isotonic saline, nondiuretic animals (those given AVP during the experi- was administered once a day for 3 days at a dose of 1 ,ug/100 ment) were only infused at 20 Aul/min with a NaCl solution (9 g of body weight. It was shown previously that i.m. admin- g/liter) containing [3H]inulin and the required hormones. istration of such a pharmacological dose of dDAVP markedly However, when the urine flow rate from both kidneys decreases the number of [3H]vasopressin binding sites (12) exceeded 15 ,ul/min, these animals were also given a NaCl and the adenylate cyclase responsiveness to vasopressin in solution (4 g/liter) through the left femoral vein at a rate membranes prepared from the rat kidney medulla (12, 13). equaling the urinary losses (8). The peptide was given i.m. in saline to ensure a sudden and During the micropuncture experiments, the tubular fluid dramatic rise of the plasma dDAVP concentration. Bouby et samples were collected from late proximal and early acces- al. (14) have demonstrated that chronic administration of sible distal convolutions belonging to the same superficial much lower doses (200-500 ng/day) of dDAVP delivered nephrons. One hundred and thirteen nephrons were micro- progressively (with implantable osmotic minipumps or by punctured in these experiments (20-25 nephrons in each of s.c. injection in a peanut oil emulsion) promotes a hypertro- the five groups of rats studied). phy of the medullary TALH cells in the DI Brattleboro rat. Analytical Procedures. The [3H]inulin contents and the In this model, the TALH response to dDAVP is maintained osmolality and electrolyte concentration in the plasma and (15). In the present study, the higher dose and more rapid urine samples were measured as described (8). diffusion of dDAVP (due to i.m. administration) ensure that The electrolyte concentration in the tubular fluid and the maximal concentration of peptide reached in the blood plasma ultrafiltrate samples was determined with an electron should be at least 10-100 times higher than in the model of probe analyzer (Cameca, Camebax; Courbevoie, France) Bouby et al.