Ovine maternal and fetal renal vasopressin receptor response to maternal dehydration Linda K. Kullama, PhD, Michael G. Ross, MD, Robert Lam, Rosemary D. Leake, MD, M. Gore Ervin, PhD, and Delbert A. Fisher, MD Torrance, California OBJECTIVE: Arginine vasopressin secretion increases in response to increased plasma osmolality or hypovolemia. Dehydration-induced increases in plasma arginine vasopressin levels have been shown to down-regulate arginine vasopressin V2 receptors in adult rat kidneys. Our study determined ovine maternal and fetal renal arginine vasopressin receptor characteristics and receptor response to maternal dehydration. STUDY DESIGN: Eight pregnant ewes (113 ± 1 days) were dehydrated for 72 hours; eight animals served as controls. Renal medullary tissue was isolated from maternal and fetal kidneys, and arginine vasopressin receptor characteristics determined with saturation and competition assays using tritiated arginine vasopressin, arginine vasopressin, and arginine vasopressin analogs. RESULTS: Euhydrated maternal and fetal renal medullary arginine vasopressin receptor dissociation constant (3.0 ± 0.3 and 1.9 ± 0.3 nmol/L) and maximal binding capacity (149 ± 15 and 111 ± 33 fmol/mg protein) values were similar. Pharmacologic profiles with selective agonists indicated a predominance of V2 receptors. Dehydration significantly increased maternal and fetal plasma osmolalities (304 ± 2 to 320 ± 2; 296 ± 1 to 319 ± 3 mOsm/kg water, respectively) and arginine vasopressin levels (3.8 ± 1.4 to 29.3 ± 4.6; 4.4 ± 1.0 to 16.9 ± 5.0 pg/ml, respectively) but had no effect on arginine vasopressin receptor binding. CONCLUSION: Specific, saturable, single-site tritiated arginine vasopressin binding is present in ovine maternal and fetal renal medullary membranes. Ovine maternal and fetal renal arginine vasopressin receptors do not down-regulate in response to dehydration-induced elevations in plasma arginine vasopressin levels. (AM J OBSTET GVNECOL 1992;167;1717-22.)
Key words: Ovine, fetal, renal, arginine vasopressin receptor, dehydration, down-regulation Body fluid homeostasis is critically dependent on the capacity of the kidneys to regulate free water excretion. The primary determinant of renal water reabsorption is circulating arginine vasopressin. The primary stimuli for arginine vasopressin secretion are increased plasma osmolality or decreased plasma volume, although several other physiologic events also contribute. I The antidiuretic effect of arginine vasopressin on renal collecting tubules is mediated via adenylate cyclase-linked V2 receptors! This hormone-receptor interaction may in itself be a regulatory mechanism. For instance, with its associated effects on plasma osmolality and arginine vasopressin levels, dehydration has been shown to down-regulate V2 receptors in adult rat kidneys.3
From the Departments of Obstetrics and Gynecology and Pediatrics, Harbor-UCLA Medical Center, UCLA School of Medicine. Supported in part l!y grants HL 40899 from the National Institute of Heart, Lung and Blood and HD 06335 from the National Institute of Child, Health and Human Development. Presented in part at the Thirty-ninth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18-21, 1992. Reprint requests: Michael C. Ross, MD, Harbor-UCLA Medical Center, 1124 West Carson St., RB-l, Torrance, CA 90502. 6/6/42192
The ovine maternal-fetal animal model is used extensively in fluid balance studies and has been very useful in characterizing the ontogeny of fetal renal responses to arginine vasopressin'" 5 To date, however, there is limited information available describing the maturation of ovine renal arginine vasopressin receptors 6 and no information concerning receptor responsiveness to physiologic alterations in plasma arginine vasopressin levels. In our study we compared ovine maternal and fetal renal medullary membrane arginine vasopressin receptor characteristics in control (euhydrated) animals and in animals after 72 hours of maternal dehydration.
Material and methods Animals and experimental. Eight time-dated pregnant ewes and fetuses (113 ± 1 days' gestation) were studied in the dehydration protocol; renal tissue was obtained from eight maternal and six fetal animals. For control renal values tissues were obtained from eight euhydrated pregnant ewes and four fetuses (122 ± 4 days) because the other four fetuses had been studied in protocols that may have compromised renal parameters. All animals were housed indoors in individual steel study cages and were acclimated to a 12-hour-light/12hour-dark cycle. They had free access to food (alfalfa
1718 Kullama et al.
pellets) and water except for the withholding of food for 24 hours before surgery and the withholding of water as will be described later. The protocol was approved by the Harbor-UClA Animal Use Committee and was in accordance with American Association for Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. Animals in the dehydration protocol were prepared with chronically implanted maternal and fetal intravascular catheters, a fetal bladder catheter, and an amniotic catheter as described previously.' All catheters were exteriorized to the maternal flank and placed in a cloth pouch. Animals were allowed ~ 5 days for postoperative recovery, which included catheter maintenance and antibiotic administration.' On study day 1 the fetal bladder catheter was drained to gravity and an intravenous infusion of tritiated inulin (10 fLCilhr) in 0.15 moVL sodium chloride (0.05 mVkg/min) was initiated for determination of glomerular filtration rate. Animals were monitored for cardiovascular parameters (arterial blood pressure, heart rate) during a I-hour control period during which two maternal and two fetal arterial blood samples (4 cm 3 ) were withdrawn. Fetal urinary parameters (osmolality, electrolytes, urine flow per 10 minutes) were also assessed. Water was then removed from the cage. Animals were monitored daily. On study day 4 after 72 hours of maternal dehydration, maternal and fetal cardiovascular parameters were again assessed and two arterial blood samples withdrawn before euthanization and tissue harvest. On study days 1 and 3 (48 hours of dehydration) fetuses were used in an atrial natriuretic factor infusion protocol (data reported separately). Fetal blood samples were replaced with an equivalent volume of heparinized maternal blood withdrawn before the study, and maternal blood samples were replaced with an equivalent volume of 0.15 moVL saline solution. Blood aliquots were assessed for hematocrit, pH, P0 2 , Pco 2 , plasma osmolality and electrolyte (sodium, chloride, and potassium) concentrations, and plasma arginine vasopressin. Blood P0 2 , Pco 2 , and pH were measured at 39° C with a blood gas analyzer system (Radiometer BMS 33 MK2-PHM 73 GMA2, Radiometer, Copenhagen). Plasma and urine osmolality was measured by freezing point depression on an osmometer (Advanced Digimatic, model 3MO, Advanced Instruments, Needham Heights, Mass.), and plasma and urine sodium, potassium, and chloride concentrations were determined (NOVA 5 electrolyte analyzer, NOVA Biomedical, Waltham, Mass). Aliquots (100 fLl) of fetal plasma and urine were taken for measurements of tritiated inulin levels for determination of glomerular filtration rate. Plasma arginine vasopressin levels were measured by radioimmunoassay of 1 ml plasma samples extracted on
December 1992 Am J Obstet Gynecol
Sep Pak (Waters Associates, Milford, Mass.) columns as described previously.s The arginine vasopressin radioimmunoassay used by our laboratory is sensitive to 0.8 pg/ml plasma; intraassay and interassay coefficients of variation were 6% and 9%, respectively. Preparation of renal medullary membranes. Maternal ewes were killed by intravenous pentobarbital (60 mg/kg) infusion. Fetuses were delivered from the uterus and killed by intraumbilical pentobarbital (60 mg/kg) infusion. Kidneys were removed and placed in iced homogenization buffer (10 mmoVL Tris-hydrochloride, 0.10 mmoVL phenylmethyl sulfonyl fluoride, 1 mmoVL ethylenediaminetetraacetic acid, pH 7.4). Kidneys were decapsulated and cut into 1 cm horizontal slices. The renal cortex was removed by blunt dissection, and the medullopapillary region was minced and weighed. Tissue was homogenized in homogenization buffer by using a motor-driven Teflon glass homogenizer, filtered through cloth (Nitex Monoscreen, Tetko Corp., Elmsford, N.Y.), and centrifuged at 600g for 10 minutes at 4° C to remove debris. The resulting supernatant was centrifuged (40,000g for 20 minutes at 4° C), and the pellet was resuspended and homogenized in receptor buffer (100 mmoVL Tris-hydrochloride; 10 mmoVL magnesium chloride; 0.5 mg/ml bacitracin, and 100 IU/ml aprotinin, pH 7.4). This suspension was recentrifuged at 40,000g for 20 minutes at 4° C, the pellet was resuspended and homogenized in a volume of receptor buffer equal to two times the initial tissue wet weight, and an aliquot was taken for protein measurement by the Bradford method. 9 The remainder of the membrane suspension was frozen at -70° C. Tritiated arginine vasopressin saturation studies. Saturation assays included duplicate tubes containing increasing tritiated arginine vasopressin concentrations (0.5 to 15 nmoVL; 54 to 68 Cilmmol) in the absence (total binding) or presence (nonspecific binding) of 0.5 fLmoVL unlabeled arginine vasopressin and 400 to 500 fLg of membrane protein in a total volume of 500 fLl. Samples were incubated at 25° C for 60 minutes, and bound and free radioactivity was separated by rapid filtration through GF/C (Fisher Scientific, Pittsburgh; 1.2 fLm) filters presoaked with receptor buffer. Filters were suspended and vortexed in 10 ml CytoScint (WestChem Products, San Diego), and radioactivity was counted in a liquid scintillation counter (Beckman LS 350, Beckman Instruments, Irvine, Calif.). Specific binding (total binding - nonspecific) represented 30% to 70% of total binding (specific binding at 3 nmoVL averaged 50% to 60% for both maternal and fetal membrane preparations). Tritiated arginine vasopressin displacement studies. Displacement of tritiated arginine vasopressin binding by a series of arginine vasopressin analogs, including: V2 agonist desmopressin, selective V I agonist
Volume 167 Number 6
Ovine maternal and fetal renal arginine vasopressin receptors
(Phe 2 , He', Orn 8 -vp), and oxytocin was assessed in duplicate 500 ILl incubation volumes with a fixed concentration of tritiated arginine vasopressin (1 nmoVL) and analog concentrations ranging from 10- 12 to 10- 4 .5 mo1!L. Incubations were carried out as described earlier (400 to 500 ILg of membrane protein, 25° C for 60 minutes). Materials. Tritiated arginine vasopressin was purchased from Dupont New England Nuclear, Boston; unlabeled arginine vasopressin, V2 agonist desmopressin, and oxytoxin from Bachem California, Torrance, Calif.; and Phe 2 , He', Orn8 -VP was obtained from Peninsula Laboratories, Inc., Belmont, Calif. Data analysis. Data were analyzed with the iterative nonlinear model-fitting program LIGAND. IO Significance of differences among experimental values was determined by t test or by analysis of variance, and Bonferroni's method was used for individual comparisons. The two control period blood samples and cardiovascular parameter measurements (determined at 30-minute intervals) were averaged, and the mean value for study days 1 and 4 is reported. Values of p < 0.05 were considered significant. Data are expressed as mean ± SEM.
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Control. Specific tritiated arginine vasopressin binding to ovine renal medullary membranes was linear with increasing amounts of membrane protein (Fig. 1). Binding was saturable and best fit a single-site model. The euhydrated maternal equilibrium dissociation constant was 3.0 ± 0.4 nmoVL, and the maximal binding capacity was 149 ± 15 fmoVmg protein. Euhydrated fetal dissociation constant (1.9 ± 0.3 nmoVL) and maximal binding capacity (111 ± 33 fmoVmg protein) values were similar to maternal values. Displacement of tritiated arginine vasopressin by arginine vasopressin analogs allowed estimation of dissociation constant values; the rank order of potency was arginine vasopressin (3.5 nmo1!L) ~ V2 agonist desmopressin (16 nmoVL) > > VI agonist (> 1500 nmoVL) > > oxytocin (> 1 mmoVL) (Fig. 2). Membranes from both euhydrated and dehydrated maternal kidneys were used for displacement studies because no differences in dissociation constant or maximal binding capacity values were observed with dehydration. Dehydration Plasma parameters. Maternal arterial blood values and plasma arginine vasopressin levels before dehydration and on day 4 (after 72 hours of dehydration) are shown in Table I. Data are presented for six ewes; for two animals, blood samples were not obtained on day 4 before tissue harvest. In response to dehydration maternal hematocrit, plasma osmolality, and sodium, chloride, and arginine vasopressin levels increased and maternal arterial pH and P0 2 decreased. There were no
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changes in other measured maternal variables. Plasma samples collected during daily monitoring indicated that maternal plasma arginine vasopressin values were significantly increased by 48 hours of dehydration (24 hours: 12.3 pg/ml; 48 hours: 24.1 pg/ml, p < 0.05). Fetal arterial blood values and plasma arginine vasopressin levels before maternal dehydration and on day 4 are shown in Table II. In response to maternal dehydration, fetal plasma osmolality and sodium, chloride, and arginine vasopressin concentrations increased and fetal arterial pH decreased; other measured fetal values did not change. Fetal plasma arginine vaso-
1720 Kullama et al.
December 1992 Am J Obstet Gynecol
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Table II. Fetal arterial blood values and plasma arginine vasopressin levels before (euhydrated) and after (dehydrated) 72 hours of maternal dehydration
Table I. Maternal arterial blood values and plasma arginine vasopressin levels before (euhydrated) and after (dehydrated) 72 hours of maternal dehydration Euhydration
n=6 (mean ± SEM)
Hematocrit (%) 28.9 ± 0.8 pH 7.51 ± 0.01 P0 2 (mm Hg) 86 ± 3 Pco 2 (mm Hg) 30 ± 1 Blood pressure (mm Hg) Systolic 101 ± 5 Diastolic 69 ± 4 Heart rate (beats/min) 110 ± 3 Osmolality (mOsm/kg 304 ± 2 water) 148.7 ± 0.8 Sodium (mEqlL) Chloride (mEqlL) 112.6 ± 0.8 Potassium (mEqlL) 4.4 ± 0.1 3.8 ± 1.4 Arginine vasopressin (pg/ml)
Euhydration (mean ± SEM)
Dehydration
n=6 (mean ± SEM)
31.6 7.45 78 30
± 1.2* ± 0.02*
99 71 107 320
± 4
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± 0.8* ± 1.3* ± 0.1 ± 4.6*
*Significant difference (p < 0.05) between euhydrated and dehydrated values.
pressin levels did not significantly increase until 72 hours of dehydration (24 hours: 5.8 pg/ml; 48 hours: 12.4 pg/ml). Urinary parameters. Dehydration had no effect on fetal urine flow rates (0.25 ± 0.06 to 0.21 ± 0.07 mVmin) or glomerular filtration rate (2.2 ± 0.2 to 1.9 ± 0.5 mVmin). Urine osmolality significantly increased (172 ± 13 to 300 ± 27 mOsmlkg water), and free water clearance significantly decreased (0.12 ± 0.04 to 0.01 ± 0.01 mVmin). Arginine vasopressin binding characteristics. Dehydration
Hematocrit (%; n = 5) pH P0 2 (mm Hg) Pco2 (mm Hg) Blood pressure (mm Hg; Systolic Diastolic Heart rate (beats/min; n = 4) Osmolality (mOsm/kg water) Sodium (mEqlL) Chloride (mEqlL) Potassium (mEqlL) Arginine vasopressin (pgiml)
34.4 7.41 22 42
± 1.2
± 0.01 ± 2 ± 1
n = 4) 49 ± 2 32 ± 1 190 ± 4
Dehydration (mean ± SEM)
32.6 7.38 21 42
± 0.9 ± 0.01* ± 1
± 2
55 ± 2 35 ± 2 198 ± 5
296.2 ± 1.1
319.0 ± 2.9*
139.6 106.4 4.0 4.4
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± 1.0
± 0.8 ± 0.2 ± 1.0
± 1.4*
± 2.2" 0.1 5.0*
± ±
Except as noted, n = 6. *Significant difference (p < 0.05) between euhydrated and dehydrated values.
had no effect on maternal or fetal dissociation constant values when compared with euhydrated values (maternal control: 3.0 ± 0.3 vs dehydrated: 2.7 ± 0.3 nmoVL; fetal control: 1.9 ± 0.3 vs dehydrated: 1.2 ± 0.3 nmoVL). Neither maternal nor fetal maximal binding capacity values (maternal control: 149 ± 15 vs dehydrated, 128 ± 10 fmoVmg protein; fetal control: 111 ± 33 vs dehydrated, 122 ± 22 fmoVmg protein) changed in response to dehydration. The rank order of
Volume 167 Number 6
Ovine maternal and fetal renal arginine vasopressin receptors
potency for analog displacement of tritiated argmme vasopressin binding was not affected by dehydration.
Comment Ovine fetal renal responsiveness to arginine vasopressin has been examined in numerous studies'" 5. s. II Although the magnitude of the response is limited, ovine fetal kidneys respond appropriately to changes in plasma arginine vasopressin levels. The relatively limited fetal renal concentrating capability has been variously attributed to a decreased number of arginine vasopressin binding sites,5. 12 to immaturity of postreceptor coupling,13 or to a limited ability to form an osmotic gradient in the inner medulla of the fetal and neonatal kidney.14 In the current study arginine vasopressin dissociation constant values in the nanomolar range and receptor densities of llO to 150 fmoVmg protein are comparable with reported adult rat kidney values." 15 Our results demonstrate arginine vasopressin binding to ovine renal medullary membranes by 113 days' gestation, with binding characteristics not significantly different from adult maternal values. Thus the obtunded fetal renal response is not the result of the developmental immaturity of arginine vasopressin receptor binding. Species differences between rats and rabbits, both altricial species, and the precocious sheep fetus may partially explain the earlier maturation of ovine arginine vasopressin receptor binding. Increased urine osmolality in the absence of a significant antidiuretic effect has been reported previously." However, arginine vasopressin-induced increases in free water reabsorption have been demonstrated in fetuses after 130 days' gestation. 14 One explanation may be that even though arginine vasopressin increases fetal free water reabsorption, associated arginine vasopressin effects to alter blood pressure or efferent arteriolar tone (increasing glomerular filtration rate and/or filtration fraction), combined with a limited potential of the fetal kidney to reabsorb sodium, may result in a mild osmotic diuresis, thereby limiting antidiuretic potential. Our demonstration of the presence of functional arginine vasopressin receptors in fetuses as young as 113 days' gestation suggests that limited ability to form an osmotic gradient, and not arginine vasopressin-receptor immaturity, is the primary explanation for the limited ability of the fetal kidney to form a concentrated urine. Adult rat renal V2 receptors down-regulate in response to 72 hours of dehydration.' Maternal dehydration sufficient to increase both maternal and fetal plasma osmolalities and arginine vasopressin levels had no effect on ovine maternal or fetal renal arginine vasopressin receptor binding capacity. In our study maternal plasma arginine vasopressin levels significantly increased after 48 hours of dehydration, and fetal levels were not significantly increased until 72 hours.
1721
Thus it is possible that longer exposure to elevated plasma arginine vasopressin levels might cause receptor down-regulation. Down-regulation of rat renal receptors' occurred in response to a tenfold increase in arginine vasopressin levels; however, Steiner and Phillips note, the rats' arginine vasopressin values were unusually low (0.2 to 2.4 pg/ml after 72 hours of dehydration). In addition, these authors did not measure daily plasma arginine vasopressin levels. In another study of the effects of 72 hours of dehydration on rat plasma arginine vasopressin levels, 16 basal levels (2.8 pg/ml) were significantly elevated after 24 hours (13.9 pg/ml) and attained levels of 31.3 pg/ml after 72 hours. These absolute values and the time course of their increase are very similar to those reported in our study for maternal sheep. Thus the difference in renal receptor response to dehydration was apparently not caused by differences in plasma arginine vasopressin concentrations or time of exposure to the agonist. An alternative explanation would be a species difference, with sheep being more resistant to dehydration-induced renal effects. Ovine renal concentrating ability is over twofold the capacity of humans. I? The lack of downregulation of renal arginine vasopressin receptors may be advantageous in a species adapted to an arid environment. The development of differentially selective arginine vasopressin V I and V2 receptor agonists and antagonists lS permits the characterization of arginine vasopressin receptor subtypes on the basis of relative analog potency in displacement assays. The pharmacologic profile of maternal renal arginine vasopressin receptors (arginine vasopressin = V2 agonist desmopressin> > VI> > oxytocin) observed in our study indicates a predominance of V2 receptors; both arginine vasopressin and V2 agonist desmopressin (a V2 agonist) were significantly more potent than the selective V I agonist in displacing tritiated arginine vasopressin binding. However, the affinity of V2 agonist desmopressin was consistently, though not significantly, less than that of arginine vasopressin. Although the V2 receptor population predominates in the kidney, V I-specific arginine vasopressin binding sites have been localized autoradiographically to the rat kidney medulla. I9 The difference in affinity of arginine vasopressin for V I and V2 receptors is slight; V2 agonist desmopressin is highly selective for V2' and the V I agonist used is highly selective for V I receptors. Thus our studies were not designed to differentiate the small proportion of V I receptors. Future studies in which recently developed iodinated V I receptor analogs are used may enable determination of the V I receptor proportion and of whether changes occur during renal maturation. In summary, specific, saturable, single-site tritiated arginine vasopressin binding has been demonstrated in ovine maternal and fetal renal medullary membranes
1722 Kullama et al.
from 2: 113 days' gestation. Pharmacologic profiles indicate a predominance of V2 receptors. Whereas dehydration-induced increases in plasma arginine vasopressin levels have been shown to down-regulate arginine vasopressin receptors in rat kidney; neither maternal nor fetal ovine renal arginine vasopressin receptors down-regulate in response to increases in plasma arginine vasopressin levels induced by 72 hours of maternal dehydration. REFERENCES 1. Schrier RW, Berl T, Anderson RJ. Osmotic and nonosmotic control of vasopressin release. Am j Physiol 1979; 236:F321-2. 2. jard S. Volume 18: vasopressin isoreceptors in mammals: relation to cyclic AMP dependent and cyclic AMP independent transduction mechanisms. In: Kleinzeller A, Martin BR, eds. Current topics in membranes and transport. New York: Academic, 1983:255-85. 3. Steiner M, Phillips MI. Renal tubular vasopressin receptors down-regulated by dehydration. Am j Physiol 1988; 254:C404-10. 4. Robillard jE, Weitzman RE. Developmental aspects of the fetal renal response to exogenous arginine vasopressin. Am j Physiol 1980;238:F407-14. 5. Wintour EM, Congiu M, Hardy Kj, Hennessy DP. Regulation of urine osmolality in fetal sheep. Q j Exp Physiol 1982;67:427-35. 6. Ervin MG, Miller Sj, Ramseyer Lj, Ross MG, Leake RD, Fisher DA. Renal arginine vasopressin receptors in newborn and adult sheep. Clin Res 1990;38:170A. 7. Ervin MG, Castro R, Sherman Dj, et al. Ovine fetal renal and hormonal responses to changes in plasma epinephrine. Am j Physiol 1991;260:R82-9. 8. Ross MG, Sherman Dj, Ervin MG, Castro R, Humme J. Maternal dehydration-rehydration: fetal plasma and urinary responses. Am j Physiol 1988;255:E674-9.
December 1992 Am J Obstet Gynecol
9. Bradford MM. A rapid and sensItive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-54. 10. Munson Pj, Rodbard D. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 1980;107:220-39. 11. Ervin MG, Ross MG, Youssef A, Leake RD, Fisher DA. Renal effects of ovine fetal arginine vasopressin secretion in response to maternal hyperosmolality. AM j OBSTET GYNECOL 1986;155:1341-7. 12. Rajerison RM, Butlen D, jard S. Ontogenic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation. Mol Cell Endocrinol 1976;4:271-85. 13. Schlondorff D, Weber H, Trizna H, Fine LG. Vasopressin responsiveness of renal adenylate cyclase in newborn rats and rabbits. Amj PhysioI1978;234:F16-21. 14. Robillard jE, Matson jR, Sessions C, Smith FG jr. Developmental aspects of renal tubular reabsorption of water in the lamb fetus. Pediatr Res 1979; 13: 1172-6. 15. Dorsa DM, Majumdar LA, Petracca FM, Baskin DG, Cornett LE. Characterization and localization of [tritiated]arginine 8 -vasopressin binding to rat kidney and brain tissue. Peptides 1983;4:699-706. 16. Woods RL, johnston CI. Contribution of vasopressin to the maintenance of blood pressure during dehydration. Amj PhysioI1983;245:F615-21. 17. Madarlane WV, Morris RjH, Howard B, McDonald j, Budtz-Olsen OE. Water and electrolyte changes in tropical Merino sheep exposed to dehydration during summer. Austj Agric Res 1961;12:889-912. 18. Manning M, Lowbridgej, Haldar j, SawyerWH. Design of neurohypophyseal peptides that exhibit selective agonistic and antagonistic properties. Fed Proc 1977;36: 1848-52. 19. Gerstberger R, Fahrenholz F. Autoradiographic localization of VI vasopressin binding sites in rat brain and kidney. Eur j Pharmacol 1989;167:105-16.
Key words: Ovine, fetal, renal, arginine vasopressin receptor, dehydration, down-regulation Body fluid homeostasis is critically dependent on the capacity of the kidneys to regulate free water excretion. The primary determinant of renal water reabsorption is circulating arginine vasopressin. The primary stimuli for arginine vasopressin secretion are increased plasma osmolality or decreased plasma volume, although several other physiologic events also contribute. I The antidiuretic effect of arginine vasopressin on renal collecting tubules is mediated via adenylate cyclase-linked V2 receptors! This hormone-receptor interaction may in itself be a regulatory mechanism. For instance, with its associated effects on plasma osmolality and arginine vasopressin levels, dehydration has been shown to down-regulate V2 receptors in adult rat kidneys.3
From the Departments of Obstetrics and Gynecology and Pediatrics, Harbor-UCLA Medical Center, UCLA School of Medicine. Supported in part l!y grants HL 40899 from the National Institute of Heart, Lung and Blood and HD 06335 from the National Institute of Child, Health and Human Development. Presented in part at the Thirty-ninth Annual Meeting of the Society for Gynecologic Investigation, San Antonio, Texas, March 18-21, 1992. Reprint requests: Michael C. Ross, MD, Harbor-UCLA Medical Center, 1124 West Carson St., RB-l, Torrance, CA 90502. 6/6/42192
The ovine maternal-fetal animal model is used extensively in fluid balance studies and has been very useful in characterizing the ontogeny of fetal renal responses to arginine vasopressin'" 5 To date, however, there is limited information available describing the maturation of ovine renal arginine vasopressin receptors 6 and no information concerning receptor responsiveness to physiologic alterations in plasma arginine vasopressin levels. In our study we compared ovine maternal and fetal renal medullary membrane arginine vasopressin receptor characteristics in control (euhydrated) animals and in animals after 72 hours of maternal dehydration.
Material and methods Animals and experimental. Eight time-dated pregnant ewes and fetuses (113 ± 1 days' gestation) were studied in the dehydration protocol; renal tissue was obtained from eight maternal and six fetal animals. For control renal values tissues were obtained from eight euhydrated pregnant ewes and four fetuses (122 ± 4 days) because the other four fetuses had been studied in protocols that may have compromised renal parameters. All animals were housed indoors in individual steel study cages and were acclimated to a 12-hour-light/12hour-dark cycle. They had free access to food (alfalfa
1718 Kullama et al.
pellets) and water except for the withholding of food for 24 hours before surgery and the withholding of water as will be described later. The protocol was approved by the Harbor-UClA Animal Use Committee and was in accordance with American Association for Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. Animals in the dehydration protocol were prepared with chronically implanted maternal and fetal intravascular catheters, a fetal bladder catheter, and an amniotic catheter as described previously.' All catheters were exteriorized to the maternal flank and placed in a cloth pouch. Animals were allowed ~ 5 days for postoperative recovery, which included catheter maintenance and antibiotic administration.' On study day 1 the fetal bladder catheter was drained to gravity and an intravenous infusion of tritiated inulin (10 fLCilhr) in 0.15 moVL sodium chloride (0.05 mVkg/min) was initiated for determination of glomerular filtration rate. Animals were monitored for cardiovascular parameters (arterial blood pressure, heart rate) during a I-hour control period during which two maternal and two fetal arterial blood samples (4 cm 3 ) were withdrawn. Fetal urinary parameters (osmolality, electrolytes, urine flow per 10 minutes) were also assessed. Water was then removed from the cage. Animals were monitored daily. On study day 4 after 72 hours of maternal dehydration, maternal and fetal cardiovascular parameters were again assessed and two arterial blood samples withdrawn before euthanization and tissue harvest. On study days 1 and 3 (48 hours of dehydration) fetuses were used in an atrial natriuretic factor infusion protocol (data reported separately). Fetal blood samples were replaced with an equivalent volume of heparinized maternal blood withdrawn before the study, and maternal blood samples were replaced with an equivalent volume of 0.15 moVL saline solution. Blood aliquots were assessed for hematocrit, pH, P0 2 , Pco 2 , plasma osmolality and electrolyte (sodium, chloride, and potassium) concentrations, and plasma arginine vasopressin. Blood P0 2 , Pco 2 , and pH were measured at 39° C with a blood gas analyzer system (Radiometer BMS 33 MK2-PHM 73 GMA2, Radiometer, Copenhagen). Plasma and urine osmolality was measured by freezing point depression on an osmometer (Advanced Digimatic, model 3MO, Advanced Instruments, Needham Heights, Mass.), and plasma and urine sodium, potassium, and chloride concentrations were determined (NOVA 5 electrolyte analyzer, NOVA Biomedical, Waltham, Mass). Aliquots (100 fLl) of fetal plasma and urine were taken for measurements of tritiated inulin levels for determination of glomerular filtration rate. Plasma arginine vasopressin levels were measured by radioimmunoassay of 1 ml plasma samples extracted on
December 1992 Am J Obstet Gynecol
Sep Pak (Waters Associates, Milford, Mass.) columns as described previously.s The arginine vasopressin radioimmunoassay used by our laboratory is sensitive to 0.8 pg/ml plasma; intraassay and interassay coefficients of variation were 6% and 9%, respectively. Preparation of renal medullary membranes. Maternal ewes were killed by intravenous pentobarbital (60 mg/kg) infusion. Fetuses were delivered from the uterus and killed by intraumbilical pentobarbital (60 mg/kg) infusion. Kidneys were removed and placed in iced homogenization buffer (10 mmoVL Tris-hydrochloride, 0.10 mmoVL phenylmethyl sulfonyl fluoride, 1 mmoVL ethylenediaminetetraacetic acid, pH 7.4). Kidneys were decapsulated and cut into 1 cm horizontal slices. The renal cortex was removed by blunt dissection, and the medullopapillary region was minced and weighed. Tissue was homogenized in homogenization buffer by using a motor-driven Teflon glass homogenizer, filtered through cloth (Nitex Monoscreen, Tetko Corp., Elmsford, N.Y.), and centrifuged at 600g for 10 minutes at 4° C to remove debris. The resulting supernatant was centrifuged (40,000g for 20 minutes at 4° C), and the pellet was resuspended and homogenized in receptor buffer (100 mmoVL Tris-hydrochloride; 10 mmoVL magnesium chloride; 0.5 mg/ml bacitracin, and 100 IU/ml aprotinin, pH 7.4). This suspension was recentrifuged at 40,000g for 20 minutes at 4° C, the pellet was resuspended and homogenized in a volume of receptor buffer equal to two times the initial tissue wet weight, and an aliquot was taken for protein measurement by the Bradford method. 9 The remainder of the membrane suspension was frozen at -70° C. Tritiated arginine vasopressin saturation studies. Saturation assays included duplicate tubes containing increasing tritiated arginine vasopressin concentrations (0.5 to 15 nmoVL; 54 to 68 Cilmmol) in the absence (total binding) or presence (nonspecific binding) of 0.5 fLmoVL unlabeled arginine vasopressin and 400 to 500 fLg of membrane protein in a total volume of 500 fLl. Samples were incubated at 25° C for 60 minutes, and bound and free radioactivity was separated by rapid filtration through GF/C (Fisher Scientific, Pittsburgh; 1.2 fLm) filters presoaked with receptor buffer. Filters were suspended and vortexed in 10 ml CytoScint (WestChem Products, San Diego), and radioactivity was counted in a liquid scintillation counter (Beckman LS 350, Beckman Instruments, Irvine, Calif.). Specific binding (total binding - nonspecific) represented 30% to 70% of total binding (specific binding at 3 nmoVL averaged 50% to 60% for both maternal and fetal membrane preparations). Tritiated arginine vasopressin displacement studies. Displacement of tritiated arginine vasopressin binding by a series of arginine vasopressin analogs, including: V2 agonist desmopressin, selective V I agonist
Volume 167 Number 6
Ovine maternal and fetal renal arginine vasopressin receptors
(Phe 2 , He', Orn 8 -vp), and oxytocin was assessed in duplicate 500 ILl incubation volumes with a fixed concentration of tritiated arginine vasopressin (1 nmoVL) and analog concentrations ranging from 10- 12 to 10- 4 .5 mo1!L. Incubations were carried out as described earlier (400 to 500 ILg of membrane protein, 25° C for 60 minutes). Materials. Tritiated arginine vasopressin was purchased from Dupont New England Nuclear, Boston; unlabeled arginine vasopressin, V2 agonist desmopressin, and oxytoxin from Bachem California, Torrance, Calif.; and Phe 2 , He', Orn8 -VP was obtained from Peninsula Laboratories, Inc., Belmont, Calif. Data analysis. Data were analyzed with the iterative nonlinear model-fitting program LIGAND. IO Significance of differences among experimental values was determined by t test or by analysis of variance, and Bonferroni's method was used for individual comparisons. The two control period blood samples and cardiovascular parameter measurements (determined at 30-minute intervals) were averaged, and the mean value for study days 1 and 4 is reported. Values of p < 0.05 were considered significant. Data are expressed as mean ± SEM.
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Control. Specific tritiated arginine vasopressin binding to ovine renal medullary membranes was linear with increasing amounts of membrane protein (Fig. 1). Binding was saturable and best fit a single-site model. The euhydrated maternal equilibrium dissociation constant was 3.0 ± 0.4 nmoVL, and the maximal binding capacity was 149 ± 15 fmoVmg protein. Euhydrated fetal dissociation constant (1.9 ± 0.3 nmoVL) and maximal binding capacity (111 ± 33 fmoVmg protein) values were similar to maternal values. Displacement of tritiated arginine vasopressin by arginine vasopressin analogs allowed estimation of dissociation constant values; the rank order of potency was arginine vasopressin (3.5 nmo1!L) ~ V2 agonist desmopressin (16 nmoVL) > > VI agonist (> 1500 nmoVL) > > oxytocin (> 1 mmoVL) (Fig. 2). Membranes from both euhydrated and dehydrated maternal kidneys were used for displacement studies because no differences in dissociation constant or maximal binding capacity values were observed with dehydration. Dehydration Plasma parameters. Maternal arterial blood values and plasma arginine vasopressin levels before dehydration and on day 4 (after 72 hours of dehydration) are shown in Table I. Data are presented for six ewes; for two animals, blood samples were not obtained on day 4 before tissue harvest. In response to dehydration maternal hematocrit, plasma osmolality, and sodium, chloride, and arginine vasopressin levels increased and maternal arterial pH and P0 2 decreased. There were no
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Fig. 1. Specific tritiated arginine vasopressin tH AVP) binding of a single maternal euhydrated renal medullary membrane preparation with increasing protein concentration (top panel), with increasing radioligand concentration (middle panel), Scatchard plot (bottom panel)._
changes in other measured maternal variables. Plasma samples collected during daily monitoring indicated that maternal plasma arginine vasopressin values were significantly increased by 48 hours of dehydration (24 hours: 12.3 pg/ml; 48 hours: 24.1 pg/ml, p < 0.05). Fetal arterial blood values and plasma arginine vasopressin levels before maternal dehydration and on day 4 are shown in Table II. In response to maternal dehydration, fetal plasma osmolality and sodium, chloride, and arginine vasopressin concentrations increased and fetal arterial pH decreased; other measured fetal values did not change. Fetal plasma arginine vaso-
1720 Kullama et al.
December 1992 Am J Obstet Gynecol
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Table II. Fetal arterial blood values and plasma arginine vasopressin levels before (euhydrated) and after (dehydrated) 72 hours of maternal dehydration
Table I. Maternal arterial blood values and plasma arginine vasopressin levels before (euhydrated) and after (dehydrated) 72 hours of maternal dehydration Euhydration
n=6 (mean ± SEM)
Hematocrit (%) 28.9 ± 0.8 pH 7.51 ± 0.01 P0 2 (mm Hg) 86 ± 3 Pco 2 (mm Hg) 30 ± 1 Blood pressure (mm Hg) Systolic 101 ± 5 Diastolic 69 ± 4 Heart rate (beats/min) 110 ± 3 Osmolality (mOsm/kg 304 ± 2 water) 148.7 ± 0.8 Sodium (mEqlL) Chloride (mEqlL) 112.6 ± 0.8 Potassium (mEqlL) 4.4 ± 0.1 3.8 ± 1.4 Arginine vasopressin (pg/ml)
Euhydration (mean ± SEM)
Dehydration
n=6 (mean ± SEM)
31.6 7.45 78 30
± 1.2* ± 0.02*
99 71 107 320
± 4
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± 0.8* ± 1.3* ± 0.1 ± 4.6*
*Significant difference (p < 0.05) between euhydrated and dehydrated values.
pressin levels did not significantly increase until 72 hours of dehydration (24 hours: 5.8 pg/ml; 48 hours: 12.4 pg/ml). Urinary parameters. Dehydration had no effect on fetal urine flow rates (0.25 ± 0.06 to 0.21 ± 0.07 mVmin) or glomerular filtration rate (2.2 ± 0.2 to 1.9 ± 0.5 mVmin). Urine osmolality significantly increased (172 ± 13 to 300 ± 27 mOsmlkg water), and free water clearance significantly decreased (0.12 ± 0.04 to 0.01 ± 0.01 mVmin). Arginine vasopressin binding characteristics. Dehydration
Hematocrit (%; n = 5) pH P0 2 (mm Hg) Pco2 (mm Hg) Blood pressure (mm Hg; Systolic Diastolic Heart rate (beats/min; n = 4) Osmolality (mOsm/kg water) Sodium (mEqlL) Chloride (mEqlL) Potassium (mEqlL) Arginine vasopressin (pgiml)
34.4 7.41 22 42
± 1.2
± 0.01 ± 2 ± 1
n = 4) 49 ± 2 32 ± 1 190 ± 4
Dehydration (mean ± SEM)
32.6 7.38 21 42
± 0.9 ± 0.01* ± 1
± 2
55 ± 2 35 ± 2 198 ± 5
296.2 ± 1.1
319.0 ± 2.9*
139.6 106.4 4.0 4.4
150.8 113.1 4.4 16.9
± 1.0
± 0.8 ± 0.2 ± 1.0
± 1.4*
± 2.2" 0.1 5.0*
± ±
Except as noted, n = 6. *Significant difference (p < 0.05) between euhydrated and dehydrated values.
had no effect on maternal or fetal dissociation constant values when compared with euhydrated values (maternal control: 3.0 ± 0.3 vs dehydrated: 2.7 ± 0.3 nmoVL; fetal control: 1.9 ± 0.3 vs dehydrated: 1.2 ± 0.3 nmoVL). Neither maternal nor fetal maximal binding capacity values (maternal control: 149 ± 15 vs dehydrated, 128 ± 10 fmoVmg protein; fetal control: 111 ± 33 vs dehydrated, 122 ± 22 fmoVmg protein) changed in response to dehydration. The rank order of
Volume 167 Number 6
Ovine maternal and fetal renal arginine vasopressin receptors
potency for analog displacement of tritiated argmme vasopressin binding was not affected by dehydration.
Comment Ovine fetal renal responsiveness to arginine vasopressin has been examined in numerous studies'" 5. s. II Although the magnitude of the response is limited, ovine fetal kidneys respond appropriately to changes in plasma arginine vasopressin levels. The relatively limited fetal renal concentrating capability has been variously attributed to a decreased number of arginine vasopressin binding sites,5. 12 to immaturity of postreceptor coupling,13 or to a limited ability to form an osmotic gradient in the inner medulla of the fetal and neonatal kidney.14 In the current study arginine vasopressin dissociation constant values in the nanomolar range and receptor densities of llO to 150 fmoVmg protein are comparable with reported adult rat kidney values." 15 Our results demonstrate arginine vasopressin binding to ovine renal medullary membranes by 113 days' gestation, with binding characteristics not significantly different from adult maternal values. Thus the obtunded fetal renal response is not the result of the developmental immaturity of arginine vasopressin receptor binding. Species differences between rats and rabbits, both altricial species, and the precocious sheep fetus may partially explain the earlier maturation of ovine arginine vasopressin receptor binding. Increased urine osmolality in the absence of a significant antidiuretic effect has been reported previously." However, arginine vasopressin-induced increases in free water reabsorption have been demonstrated in fetuses after 130 days' gestation. 14 One explanation may be that even though arginine vasopressin increases fetal free water reabsorption, associated arginine vasopressin effects to alter blood pressure or efferent arteriolar tone (increasing glomerular filtration rate and/or filtration fraction), combined with a limited potential of the fetal kidney to reabsorb sodium, may result in a mild osmotic diuresis, thereby limiting antidiuretic potential. Our demonstration of the presence of functional arginine vasopressin receptors in fetuses as young as 113 days' gestation suggests that limited ability to form an osmotic gradient, and not arginine vasopressin-receptor immaturity, is the primary explanation for the limited ability of the fetal kidney to form a concentrated urine. Adult rat renal V2 receptors down-regulate in response to 72 hours of dehydration.' Maternal dehydration sufficient to increase both maternal and fetal plasma osmolalities and arginine vasopressin levels had no effect on ovine maternal or fetal renal arginine vasopressin receptor binding capacity. In our study maternal plasma arginine vasopressin levels significantly increased after 48 hours of dehydration, and fetal levels were not significantly increased until 72 hours.
1721
Thus it is possible that longer exposure to elevated plasma arginine vasopressin levels might cause receptor down-regulation. Down-regulation of rat renal receptors' occurred in response to a tenfold increase in arginine vasopressin levels; however, Steiner and Phillips note, the rats' arginine vasopressin values were unusually low (0.2 to 2.4 pg/ml after 72 hours of dehydration). In addition, these authors did not measure daily plasma arginine vasopressin levels. In another study of the effects of 72 hours of dehydration on rat plasma arginine vasopressin levels, 16 basal levels (2.8 pg/ml) were significantly elevated after 24 hours (13.9 pg/ml) and attained levels of 31.3 pg/ml after 72 hours. These absolute values and the time course of their increase are very similar to those reported in our study for maternal sheep. Thus the difference in renal receptor response to dehydration was apparently not caused by differences in plasma arginine vasopressin concentrations or time of exposure to the agonist. An alternative explanation would be a species difference, with sheep being more resistant to dehydration-induced renal effects. Ovine renal concentrating ability is over twofold the capacity of humans. I? The lack of downregulation of renal arginine vasopressin receptors may be advantageous in a species adapted to an arid environment. The development of differentially selective arginine vasopressin V I and V2 receptor agonists and antagonists lS permits the characterization of arginine vasopressin receptor subtypes on the basis of relative analog potency in displacement assays. The pharmacologic profile of maternal renal arginine vasopressin receptors (arginine vasopressin = V2 agonist desmopressin> > VI> > oxytocin) observed in our study indicates a predominance of V2 receptors; both arginine vasopressin and V2 agonist desmopressin (a V2 agonist) were significantly more potent than the selective V I agonist in displacing tritiated arginine vasopressin binding. However, the affinity of V2 agonist desmopressin was consistently, though not significantly, less than that of arginine vasopressin. Although the V2 receptor population predominates in the kidney, V I-specific arginine vasopressin binding sites have been localized autoradiographically to the rat kidney medulla. I9 The difference in affinity of arginine vasopressin for V I and V2 receptors is slight; V2 agonist desmopressin is highly selective for V2' and the V I agonist used is highly selective for V I receptors. Thus our studies were not designed to differentiate the small proportion of V I receptors. Future studies in which recently developed iodinated V I receptor analogs are used may enable determination of the V I receptor proportion and of whether changes occur during renal maturation. In summary, specific, saturable, single-site tritiated arginine vasopressin binding has been demonstrated in ovine maternal and fetal renal medullary membranes
1722 Kullama et al.
from 2: 113 days' gestation. Pharmacologic profiles indicate a predominance of V2 receptors. Whereas dehydration-induced increases in plasma arginine vasopressin levels have been shown to down-regulate arginine vasopressin receptors in rat kidney; neither maternal nor fetal ovine renal arginine vasopressin receptors down-regulate in response to increases in plasma arginine vasopressin levels induced by 72 hours of maternal dehydration. REFERENCES 1. Schrier RW, Berl T, Anderson RJ. Osmotic and nonosmotic control of vasopressin release. Am j Physiol 1979; 236:F321-2. 2. jard S. Volume 18: vasopressin isoreceptors in mammals: relation to cyclic AMP dependent and cyclic AMP independent transduction mechanisms. In: Kleinzeller A, Martin BR, eds. Current topics in membranes and transport. New York: Academic, 1983:255-85. 3. Steiner M, Phillips MI. Renal tubular vasopressin receptors down-regulated by dehydration. Am j Physiol 1988; 254:C404-10. 4. Robillard jE, Weitzman RE. Developmental aspects of the fetal renal response to exogenous arginine vasopressin. Am j Physiol 1980;238:F407-14. 5. Wintour EM, Congiu M, Hardy Kj, Hennessy DP. Regulation of urine osmolality in fetal sheep. Q j Exp Physiol 1982;67:427-35. 6. Ervin MG, Miller Sj, Ramseyer Lj, Ross MG, Leake RD, Fisher DA. Renal arginine vasopressin receptors in newborn and adult sheep. Clin Res 1990;38:170A. 7. Ervin MG, Castro R, Sherman Dj, et al. Ovine fetal renal and hormonal responses to changes in plasma epinephrine. Am j Physiol 1991;260:R82-9. 8. Ross MG, Sherman Dj, Ervin MG, Castro R, Humme J. Maternal dehydration-rehydration: fetal plasma and urinary responses. Am j Physiol 1988;255:E674-9.
December 1992 Am J Obstet Gynecol
9. Bradford MM. A rapid and sensItive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-54. 10. Munson Pj, Rodbard D. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 1980;107:220-39. 11. Ervin MG, Ross MG, Youssef A, Leake RD, Fisher DA. Renal effects of ovine fetal arginine vasopressin secretion in response to maternal hyperosmolality. AM j OBSTET GYNECOL 1986;155:1341-7. 12. Rajerison RM, Butlen D, jard S. Ontogenic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation. Mol Cell Endocrinol 1976;4:271-85. 13. Schlondorff D, Weber H, Trizna H, Fine LG. Vasopressin responsiveness of renal adenylate cyclase in newborn rats and rabbits. Amj PhysioI1978;234:F16-21. 14. Robillard jE, Matson jR, Sessions C, Smith FG jr. Developmental aspects of renal tubular reabsorption of water in the lamb fetus. Pediatr Res 1979; 13: 1172-6. 15. Dorsa DM, Majumdar LA, Petracca FM, Baskin DG, Cornett LE. Characterization and localization of [tritiated]arginine 8 -vasopressin binding to rat kidney and brain tissue. Peptides 1983;4:699-706. 16. Woods RL, johnston CI. Contribution of vasopressin to the maintenance of blood pressure during dehydration. Amj PhysioI1983;245:F615-21. 17. Madarlane WV, Morris RjH, Howard B, McDonald j, Budtz-Olsen OE. Water and electrolyte changes in tropical Merino sheep exposed to dehydration during summer. Austj Agric Res 1961;12:889-912. 18. Manning M, Lowbridgej, Haldar j, SawyerWH. Design of neurohypophyseal peptides that exhibit selective agonistic and antagonistic properties. Fed Proc 1977;36: 1848-52. 19. Gerstberger R, Fahrenholz F. Autoradiographic localization of VI vasopressin binding sites in rat brain and kidney. Eur j Pharmacol 1989;167:105-16.
