Acta Physiol Scand 1992, 145, 267-274
Effects on renal sodium and potassium excretion of vasopressin and oxytocin in conscious dogs S. E. A N D E R S E N , T. E N G S T R 0 M and P. B I E Department of Medical Physiology C, Panum Institute, University of Copenhagen, DK2200 Copenhagen N, Denmark ANDERSEN, s. E., ENGSTRBM, T. & BIE,P. 1992. Effects on renal sodium and potassium excretion of vasopressin and oxytocin in conscious dogs. Acta Physiol Scand 145, 267-273. Received 5 April 1991, accepted 7 November 1991. ISSN 0001-6772. Department of Medical Physiology C, University of Copenhagen, Denmark. Renal effects of arginine vasopressin and oxytocin were studied in conscious dogs, made water-diuretic by a waterload equivalent to 2% of body weight. Body water and content of sodium were maintained by separate servo-controlled infusions. Peptides were infused for 60 min at rates of 50 pg kg-' rnin-' (arginine vasopressin) or 1 ng kg-' min-' (oxytocin), either separately or combined. Infusions increased plasma arginine vasopressin to 1.9 f0.2 (arginine vasopressin alone) and 1.8f0.3 pg kg-' (arginine vasopressin plus oxytocin and plasma oxytocin to 7 2 f 5 (oxytocin alone) and 77 8 pg ml-' (oxytocin plus arginine vasopressin). Arginine vasopressin or arginine vasopressin plus oxytocin increased urine osmolality similarly by a factor of 13, decreased urine flow to between 5 and 7% of control and decreased free water clearance. Oxytocin reduced urine flow and free water clearance and increased urine osmolality by a factor of 2. Oxytocin and arginine vasopressin separately increased excretion of sodium from 4 f2 to 15 & 6 pmol min-' and from 7 f4 to 25 f 13 pmol min-', respectively. Arginine vasopressin plus oxytocin led to a pronounced natriuresis (13 f 4 to 101f27 pmol min-I). Arginine vasopressin and arginine vasopressin plus oxytocin increased the excretion of potassium by a factor of 2.5. Oxytocin and arginine vasopressin plus oxytocin increased urinary Na+/K+ ratio by a factor of 3.7. It is concluded, that oxytocin at plasma concentrations of 70-80 pg ml-' has modest antidiuretic and natriuretic effects and that the combined action of arginine vasopressin oxytocin may elicit supra-additive natriuretic effects. Key words : Neurohypophysial peptides, renal effects.
T h e role of vasopressin in the control of renal excretion of water and electrolytes in the conscious waterdiuretic dog is relatively well quantified (e.g. Baerwolf & Bie 1988, Bie et al. 1984). Oxytocin ( O T ) and arginine vasopressin (AVP) show extensive homologies (two amino acids [3-ILE] and [&LEU] distinguish OT from AVP), and both seem to be released in response to an increment in plasma osmolality (Weitzman et al. 1978). However, very few experiments on the renal effects of OT seem to have been Correspondence : Peter Bie, Department of Medical Physiology C, Panum Institutet, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark.
conducted in the dog (Ali 1958, Brooks 8~ Pickford 1958). These results indicated that OT may increase the rate of excretion of sodium at low urine flows and may act as an antidiuretic agent in water diuretic dogs. T h e observations were later confirmed by Chan & Sawyer (1961), who also found that OT in rather high doses could evoke natriuresis during water diuresis. Experiments in other species have yielded highly variable results, depending on species and the state of water and salt balance. Experiments in neurohypophysectomized and acutely hypophysectomized rats have demonstrated interactions between AVP and OT on renal functions (Balment et al. 1986). I t was
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observed that doses of OT, which by t h e m s e h e s had n o measurable renal effect, potentiate the narriuretic action of .-\I-P and that concurrent administration of both hormones largely reversed the renal sodium retention normally seen in neurohypophysectomized rats. T h c present stud!- was conducted in order to unveil a possible interaction of .41'P and OT on the renal excretion of sodium and potassium in the conscious dog under precisely defined experimental conditions with regard to body content of sodium and body w t .
ZIATERI.ILS A N D M E T H O D S .4ninra/s. Experiments were performed on six trained, conscious female Beagle dogs weighing 10.5-13.5 kg. They \\ere fed once dail! with commercial food (Latz Komplett mised with Latz canned food) usually at 1400 hrs, and had free access to tap water. The intake of sodium and potassium was determined regularly and a\eraged 3.5k0.3 and 4.1 k 1.3mmol d q - ' kg bod! \ \ t I . respectively. The dogs were trained to accept percutaneous catheterization, catheterization of the hladder and to stand quietly for several hours supported by a canvas sling. The common carotid arteries had been placed in skin loops prior to the experiments. Prepuration. Each dog was used for all types of experiments with intervals of at least a week. .4 sterile catheter (Intracath'?) was placed in a saphenous vein and another in or near the right atrium via an external jugular yein. third catheter (Angiocath^) was placed in a carotid artery. .I modified silicone Foley catheter was inserted into the bladder. The venous catheters were used for blood sampling and infusion, while the arterial one was used to measure the arterial blood pressure (Sratham P50). Heart rate was measured hy registration of the electrocardiogram. Values of arterial blood pressure and heart rate were collected automatically every min using a Dialog 2000 monitor (Danica) interrogated bj- a DEC pVAX computer. Data were stored on disc and averaged over 15 min. Protocd. T h e dogs were hydrated via a gastric tube with a load of water at 37 "C equivalent to 2 O , of bodv wt. Thereafter, the body wt was kept within 102.0_+ 0.2O, of prehydration value by use of a seryomechanism (Bie 1976) infusing a hqpotonic solution of glucose and urea (G/U-solution), 40 and 25 mM, respectively. After hydration a 20 ml bolus injection of inulin, 40 g I-', was given, and a continuous infusion was started at a rate of 0.33 ml min-' in order to measure inulin clearance. During the experiment the quantity of sodium excreted per unit time was continuously replaced by a sodium servo-system
infusing a 400 rnhl solution of sodium chloride into the jugular catheter (Andersen & Bie 1990). Ninety min after h!-dration, when steady state water diuresis had been achieved, sampling of urine was started (time = 0). .-\fter a control period of 15 min, a continuous infusion of hormones lasting 60 min was carried out. ,4VP and OT were infused at rates of 50 pg kg-' min-' and 1 ng kg-' min-', respectively, either separately or in combination. In the control series only the vehicle was infused. Pure synthetic peptides (Ferring, Malmo, Sweden) were dissolved in a solution of acetic acid adjusted to p H = 4.5 and stored at - 18 "C. Immediately prior to infusion aliquats were thawed and diluted with G/U-solution and Haemaccels (Behringwerke, Marburg, Germany) in a ratio of 9 to 1. HaemacceF was added to avoid adsorption of peptide to tubing and glassware. Observations were continued in a 90min postinfusional phase. Urine was sampled at intervals of 15 min. Blood samples of 4.0ml (at 37.5, 97.5 and 157.5 min) and 16 ml (at 7.5,67.5 and 127.5 min) were obtained from the saphenous vein. After sampling the blood was centrifuged immediately at 4 "C. The erythrocytes were resuspended in isotonic sodium chloride and reinfused. Plasma samples were immediately frozen and stored at - 18 "C. Analyses. Na' and K' were measured on an IL 243 LED flame photometer. Osmolalities on an osmometer (Digimatic* osmometer model 3DI1, Advanced Instruments, MA, USA). Inulin was measured by the method described by Steele (1969) with minor modifications. Radioimmunoassay for AVP on extracted dog plasma was performed by the method described by Sakurai et al. (1986) with minor modifications. Extraction recovery averaged 7 2 yo.Specific and nonspecific binding was 39 and 2.2%, respectively. Intraassay coefficient of variation was about S.O"& All samples were run in one assay. Sensitivity of the assay was 0.15 pg tube-'. Half maximal binding (ED-50 value) occurred at 1.47 k 0.05 pg tube-'. Radioimmunoassay for oxytocin on infusates and unextracted dog plasma was performed by the method described by Engstrem et al. (1990). Specific and nonspecific binding was 35 and 2.0y0,respectively. Intraand interassay coefficients of variation were 7.894 and 7.700at 5 pg tube-'and 5.6%and 2.506at 20 pg tube. Sensitivity of the assay was 2 pg tube. Half maximal binding (ED-50 value) occurred at 14.5 Ifr 0.3 pg. Statistics. Results are given as mean+SE. Data were subjected to a one-way analysis of variance for repeated measures (Winer 1971). Because of variance inhomogeneity, as indicated by Bartlett's test, the data for sodium excretion and urine osmolality were transformed logarithmically prior to analysis of variance (Winer 1971). I n case of significantly large Fvalues all possible differences were evaluated systematically by Newman-Keuls' test (Winer 1971).
Effects of AVP andlor O T on renal Na' and K+
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Infusion of -4i-P at -iO pg lip-' m i n - ' increased plasma concentration of -41P (p.11.P) from 0.9 f0 . 1 to I .9 f0 . 2 pg mI '. After cessation of infusion p.\VP had returned to 0.9 0.1 pg ml-'. Urine flov decreased from 3.8k0.4 to 0 . 2 i 0.1 ml min I ( P < O.01, Table I ) . J.ihenise free \$att'r clearance (CH,,,) decreased fimm 3.0 i0.3 to -0..7&(J.2ml min-' (P < 0.01, Fig. I ) . This \$as accompanied b!- a 13-fold increase in urine osmolalit! (uOsm), from 0 2 i 3 to 821 1X4 mOsm kg ( P < 0.01, 'Table 1). T h e rate of excrction of sodium showed an increase of ( 7 k 4 to 25 f 13 pmol min-.'; 18 pmoI min Fig. 2) as did the rate of excretion of potassium ( 1 3 iL t o 31 6 pmol min~-'). \lean arterial blood pressure (MABP) (mean 1 I3 & 1 mmHg), heart rate (FIR), plasma osmolality (pOsm), plasma concentrations of sodium (pNa') and potassium (PIC') remained constant throughout the experiment. Infusion ( f 0 T
Infusion of 07' at 1 ng kg-' min-' increased plasm;i concentration of OT ( p O T ) from below
1
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limit of detecting to 72 f5 pg ml-'. After cessation of infusion p O T returned to undetectable IeL-els. Urine flow (Table 1) and CH,O (Fig. 1) decreased from 4.2 i0.5 to 3.0 f 0 . 7 mi min-' and from 3.3f0.4 to 2.2+0.6ml min-', respectively, and Uosm doubled (Table 1). After the termination of hormone infusion both urine flow and CH,O increased to levels significantly higher than values observed in control and AVP experiments. T h e rate of excretion of sodium showed a persistent increment averaging 11 pmol min-' and did not return to control level (Fig. 2). T h e rate of excretion of potassium tended to increase, but the rate was not significantl!- higher than that during the preinfusional period. At the end of the experiment, though, the rate of excretion was significantly higher than in control and AVP experiments. Plasma K' decreased from 4.1 kO.1 to 3.7+ 0.1 mmol I-' during the infusion. Administration of OT did not affect MARP (mean 108f 1 mmHg), HR, pOsm, pAVP, or pNA+.
Conihinivi infirsion o f .4 VP und 0 T Combined infusion of AVP and OT 5 0 pg kg-' rnin and 1 ng kg-' min-', respectively, produced plasma concentrations of hormones similar to those found after single infusions (pAVP = 1.8i0.3 pg ml-' ( n = 4) and POT = 77 f 8 pg ml.'). T h e effects on urine flow (Table l), uOsm (Table 1) and C,.,,, (Fig. 1) h~eresimilar to the changes seen in dogs during single infusion of -1L-P. How-ever, during recovery urine flow increased to a level significantly higher than seen in control and .4\)-P experiments. T h e rate of excretion of sodium, however, increasing by 89 pmol min-' from 13 f4 to 101 +27 pmol min-.' (P< 0.01), greatly exceeded the sum of the excretion rates (18f 11 pmol min-') measured in the single hormone series (Fig. 2). T h e masimum was reached shortly after termination of infusion at a time when urine flow was still reduced. T h e pronounced natriuresis was accompanied by an increase in the rate of excretion of potassium (13 f3 to 35 f7 pmol min-') similar to that seen with AVP alone. T h e effect of combined infusion on the rate of excretion of potassium was prolonged, and at the end of the experiment the rate of excretion was signilicantl!- higher compared to control and -4L-Pesperiments. A trend towards a decrease in
Efects o f AVP andlor OT on renal Na' and Ki
271
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Time (rnin) Fig. 2. Rate of excretion of sodium (Pmol min-'). Hormone or vehicle were infused from min 15 to min 75. = controls, 0 = AVP 50 pg kg-' min-',
A = OT 1 ng kg-' min-', 4 = AVP SO pg kg-' min-' and OT 1 ng kg-' min-'. Dotted line indicates sum of single hormone effects. Values significantly different from preinfusion values (" = 0.05 and * = 0.01) of the same series are indicated. Values are means SEM ; n = 6 dogs.
Fig. 3. Urinary Na'/K+ ratio. Hormone or vehicle were infused from min 15 to min 75. =controls, = AVP 50 pg kg-' min-', A = OT 1 ng kg-' min-', 4 = AVP 50 pg kg-' min-' and OT 1 ng kg-' min-'. Values significantly different from preinfusion values (" = 0.05 and * = 0.01) of the Same series are indicated. Values are means+SEM; n = 6 dogs.
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ttx pK+, statistically insignificant but of similar magnitude to the decrease found in OT single hormone series, was observed. pOsm showed a decrease from 292+ 1 to 289+ 1 mosm kg-l ( P < 0.01). T h e concurrent administration of hormones did not affect HR, MABP (mean 117 2 1 mmHg) or pNa+ measurably.
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Fig. 4. Fractional excretion of sodium (0J. Hormone During the control series pOsm, pNa+ and p K + = or vehicle were infused from min 15 to min 75. did not change significantly. Likewise, detectable controls, = AVP 50 pg kg-' min-', A= changes did not occur in HR, urine flow, CHZO OT 1 ng kg-' min-', 4 = AVP 50 pg kg-' min-' and (Fig. l), uOsm (Table I), rates of excretion of OT 1 ng kg-' min '. Values significantly different sodium (Fig. 2) and potassium or pAVP. p O T from preinfusion values (" = 0.05 and * = 0.01) ofthe was below the detection limit of the assay same series are indicated. Values are means SEM ; throughout the experiment. Towards the end of n = 6 dogs. the post-infusion phase MABP increased slightly maximum obtained during combined hormone from look5 to 1 0 9 + 3 mmHg. During infusion of OT and combined infusion infusion was significantly higher than in control of AVP and OT the urinary sodium/potassium and AVP experiments. T h e urinary sodium/ ratio increased from 0 . 4 5 0 . 2 to 1.7 F0.8 and potassium ratio did not change during control 1.1k0.3 to 4.lf 1.3, respectively (Fig. 3 ) . T h e and AVP experiments. 13-2
272
S . E. Anderseii et al.
I h t a f’or inuiin clearance are g i w n in ‘Table 1 For certain periods in time it wa5 mident that the urine flou- data did not represent a steady state. T h e values for these periods h a w been omitted. I l o w e x r , assuming only minor changes in GFK, calculations of the fractional escretions of electrol!-tes remain d i d . During the control, .%W,and (IT series fractional escretion of sodium did not change significantly (Fig. 4). T h e combined infusion of A1.P and OT, hon e\-er, increased fractional sodium escretion significanti!- from 0 . 2 6 ~ 0 . 1 0 t o1.21 i O . 3 7 , i . e . almost h! a fhctor of 5.
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T h e main findings of the stud!- were a modest antiduretic and natriuretic effect of OT infused alone, and supra-additive effects of -41-Pand OT on renal escretion of sodium. In the pattern of the response it should be noted that the changes in the rate of’ercretion of sodium are reflected in urinarl- X a - / I C ratio and in the fractional excretion rates and that the peak sodium excretion occurred a t the nadir of urine flo\\. ‘I’ahcn together this demonstrates that washout of‘ the dead space of the urinar!- drainage s!-stem can not account for our main findings. T o the contrar!, the dead space (1-olume of the lower urinary tract and external drainage tubing) is liliel! to hake caused deminuation and dela! of the ohserxeetl events. Calculation of inulin clearance requires stead!state conditions. ..lt times of rapid changes in urinc flon inulin clearances do not express GFR. In the present study the d u e s obtained prior to hormone infusions and at the end of the infusion and recover!- periods were all obtained a t stead!state urine flov and are, therefore, most likel!- to reflect GFR. Other clearance data should be interpreted with great caution. T h e rate of excretion of sodium in the control period of the ..lI-P+OT series was slightly greater than in the -41-Pand OT series (Fig. 2 ) . I i o a e ~ e rat , the beginning of the infusion period prior to the appearance of other effects of the hormones no significant differences between rates of excretion of sodium of the three series could be detected. I t seems thus 1-ery unliliel!-. that the obserwd supraadditive natriuretic effect was due to small initial differences in sodium excretion betlieen the series.
U-hen AVP $+as infused separately, the expected reduction in urine flow and C H L 0was accompanied by a minor increase in the rate of escretion of sodium. This result is in agreement with earlier experiments on the effect of’ overh!-dration during different AVP infusions (Bie t t a / . 1984). I n that study larger doses caused further elevation in sodium excretion. I n a subsequent study it w-as shown that infusions of -41.P a t the very low rates of 2 and 5 pg kg-’ min-’ reduced urine flow by 39 and 61 O o , respectivel!-, without measurable alterations in sodium excretion (Baerwolf & Bie 1988). These results together demonstrate a threshold for the natriuretic action of AVP which is rather low. None of these studies included measurements of hormone concentrations in plasma. Physiological rele1-ance of the supra-additive effect of AVP and OT on renal excretion of sodium requires simultaneous release of both hormones in response to physiological stimuli, e.g. increasing plasma osmolality. While the osmotic regulation of AVP secretion is well established (e.g. Robertson 1987), the role of plasma osmolalitl- in regulation of OT secretion is poorly elucidated also in dogs. I n conscious dogs plasma concentrations of both hormones increase in response to increments in plasma osmolalit>- from 3 0 4 5 1 mOsm kg-’ to 316f 1 mOsm kg-’ (Weitzman et 01. 1978). I n rats a strong positive correlation between plasma osmolality and plasma oxytocin concentration (Balment et ul. 1980) and osmotic sensitivity of OTand ri1-P-producing neurons has been observed (Rrimble 1977, Cheng & North 1986). Simultaneous release of both hormones in response to a varier>-of stimuli e.g. intravenous injection of hypertonic saline and isotonic hypovolemia has been demonstrated (Kasting 1988). T h e plasma concentrations of AVP measured during the present study are well u-ithin the normal physiological range. Increments in piasma OT, though, may be considered substantial or supraphysiological. However, POT of the same magnitude has been observed in lactating rhesus monkeys during nursing (Amico et t i / . 1990) and in Brattleboro rats in response to an increment in pOsm from 3 0 7 i 3 to 3 1 9 i 2 mosm kg-’ (Brimble et a / . 1990). I t might be questionable whether the demonstrated supra-additive effect of AVP and OT is part of physiological sodium homeostasis, but the phen-
Efects of AVP andlor OT on renal Na' and Kf omenon may obviously be of relevance to body fluid control during labour and nursing. T h e renal effect of normal as well as supranormal concentrations of OT is a topic of some controversy. I n the present study we observed a modest natriuretic and antidiuretic effect of OT (1 ng kg-' min-' giving 70 pg ml-' in plasma). This is discordant with results obtained in conscious dogs by Brooks & Pickford (1958) and Chan & Sawyer (1961). OT at high doses (up to 12 ng kg-') did not cause natriuresis during water diuresis but only during low urine flows. (Note: conversion factors for AVP and OT: 2.5 pg pU-' and 2.0 pg pU-', respectively.) During water diuresis OT elicited natriuresis only at doses of 20 ng kg-' (Chan & Sawyer 1961). Assuming an apparent volume of distribution of 125 ml kg-' (Robinson 1980) and a rapid distribution the increment in POT in the latter experiment could have reached 160 pg ml-', i.e. markedly exceeding POT obtained in our experiment. T h e water diuresis in the two experiments was not sustained. Using an experimental set u p similar to ours Wesley & Gilmore (1985) investigated the effects of single injections of OT at a dose of 2 ng kg-' in anaesthetized macaque monkeys. Elevating POT by 10-20 pg ml-' they found no effects on creatinine clearance, free water clearance or the rate of excretion of sodium. T h e influence of anaesthetics or species differences may account for the difference between this study and ours. T h e distinct influence of OT on the natriuretic but not the antidiuretic effect of AVP in the present study is compatible with the proposal that the antidiuretic and natriuretic responses to OT are mediated by at least two different types of neurohypophysial hormone receptors (Chan & Hruby 1988). However, our results do not exclude the fact that other mechanisms are involved in natriuresis of neurohypophysial hormones. Further studies in conscious animals are needed to elucidate the osmotic regulation of both AVP and OT and the combined natriuretic action of the neurohypophysial hormones at the lower end of the physiological range of secretion. The authors thank Inge Pedersen, Karen Klausen and Sigurd Hansen for expert technical assistance. This work has been supported by grants from the Danish Medical Research Council and the Velux Foundation.
273
Preliminary results were presented at the International Symposium on Mechanisms of Sodium Homeostasis, Copenhagen, July 1989.
REFERENCES ALI, M.N. 1958. A comparison of some activities of arginine vasopressin and lysine vasopressin on kidney function in conscious dogs. B r i t 3 Pharmacol 13, 131-137. J.L. AMICO,J.A., CHALLINOR, S.M. & CAMERON, 1990. Pattern of oxytocin concentrations in the plasma and cerebrospinal fluid of lactating rhesus monkeys (Macaca mulatta) : Evidence for functionally independent oxytocinergic pathways in primates. 3 Clin Endocrinol Metab 71, 1531-1535. ANDERSEN,S.E. & BIE, P. 1990. Continuous servocontrolled replacement of urinary sodium loss in conscious dogs. A m 3 Physiol 259, R313-R316. BAERWOLF, M. & BIE,P. 1988. Effects of subpicomolar changes in vasopressin on urinary concentration. Am 3 Physiol 255, R94GR945. M.L. BALMENT, R.J., BRIMBLE, M.J. & FORSLING, 1980. Release of oxytocin induced by salt loading and its influence on renal excretion in the male rat. 3 Physiol 308, 439449. BALMENT, R.J., BRIMBLE, M.J., FORSLING, M.L. & MUSABAYANE, C.T. 1986. The influence of neurohypophysial hormones on renal function in the acutely hypophysectomized rat. 3 Physiol 381, 439-452. BALMENT, R.J., BRIMBLE, M.J., FORSLING, M.I.. & MUSABAYANE, C.T. 1986. A synergistic effect of oxytocin and vasopressin on sodium excretion in the neurohypophysectomized rat. 3 Physiol 381, 453464. BIE, P. 1976. Studies of cerebral osmoreceptors in anesthetized dogs. The effect of intravenous and intracarotid infusion of hyperosmolar sodium chloride during sustained water diuresis. Acta Physiol Scand 96, 306-318. BIE, P., MUNKSDORF, M. & WARBERG, J. 1984. Renal effects of overhydration during vasopressin infusion in conscious dogs. Am 3 Physiol 247, F103-F109. BRIMBLE, M.J., BALMENT, R.J., SMITH,C.P., WINDLE, M. 1990. Influence of oxytocin on R.J. & FORSLING, sodium excretion in the anaesthetized Brattleboro rat. 3 Endocrinol 129, 49-54. BRIMBLE,M.J., DYBALL,R.E.J. & FORSLING, M. 1977. Oxytocin release following osmotic activation of oxytocin neurones in the paraventricular and supraoptic nuclei. 3 Physiol 278, 69-78. BROOKS, F.P. & PICKFORD, M. 1958. The effect of posterior pituitary hormones on the excretion of electrolytes, in dogs. 3 Physiol 142, 468-493. CHAN,W.Y. & HRUBY, V.J. 1988. Natriuretic action of neurohypophysial peptides : Effects of agonists and antagonists an implication of natriuretic receptor. 3 Pharmacol Exp Ther 246, 597-602.
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\\-.Y. S. SAVYER, 1V.H. 1961. Saliuretic actions of neuroh) poph!-sial peptides in conscious dogs. ,.in2 7 Ph.>'s/ol201, 799-803. C t f E S G , S.1V.T.& XoRm, \V.G. 1986. Responsiveness
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of os! tocin-producing neurons to acute salt loading in rats: Comparison with vasopressin-producing neurons. .IrurornJocrinoloR)i 42. 174-180. ENGSTRQL~. T. & VILH~RDT, H. 1989. Oxytocin concentrations in plasma, h! pothalamus and the pituitarv of rats following continuous infusion of osytocin. In X..k Thorn, H . i-ilhardt & 11.Freiner (eds). Proceedings of the Fourth International Con.firmre on the . ~ - r u r o h ~ ~ o p h ~pp. ~ s 98-1 i s , 00. Oxford Lni\-ersit)- Press, Oxford. K~s-rirc;,X.\\'. 1987. Simultaneous and independent release of vasopressin and oxytocin in the rat. Can 3 Ph,lljiol P h a r m r o l 66, 22-26. Rommsox, G.L. 1987. Ph!-siology of .IDH secretion. Kidnej, Int 32, S20-S26. ROBIYSOX, 1.C.A.F. 1980. The development and
el-aluation of a sensitive and specific radioimmunoassay for oxytocin in unextracted plasma. Journal of Irnrnunoassuy 1, 323-317. S4KLR.41, H., KANAI, A , , NOMURA, K., DEMURA, H. & SHIZCME, K. 1986. A simple and highly sensitive radioimmunoassay for 8-arginine vasopressin in human plasma using reversed-phase C,, silica column. 3 Tokyo Worn Med CoM 56, 39-03, STEELE, T.H. 1969. A modified semi-automated resorcinol method for determination of inulin. Clin Chem 15, 1072-1078. \VEITZMAN, R.E., GLATZ,T.H. & FISHER, D.A. 1978. The effect of hemorrhage and hypertonic saline upon plasma oxytocin and arginine vasopressin in conscious dogs. Endocrinology 103, 2154-2160. J.P. 1985. Effects of N ~ ~ S L EC.R. Y , & GILMORE, oxytocin, vasopressin and a-MSH on renal function in the nonhuman primate. Hormone Res 21, 55-59. \VISER, B.J. 1971. Statistical principles in experimental design (2nd ed.). McGraw-Hill, New York.
Effects on renal sodium and potassium excretion of vasopressin and oxytocin in conscious dogs S. E. A N D E R S E N , T. E N G S T R 0 M and P. B I E Department of Medical Physiology C, Panum Institute, University of Copenhagen, DK2200 Copenhagen N, Denmark ANDERSEN, s. E., ENGSTRBM, T. & BIE,P. 1992. Effects on renal sodium and potassium excretion of vasopressin and oxytocin in conscious dogs. Acta Physiol Scand 145, 267-273. Received 5 April 1991, accepted 7 November 1991. ISSN 0001-6772. Department of Medical Physiology C, University of Copenhagen, Denmark. Renal effects of arginine vasopressin and oxytocin were studied in conscious dogs, made water-diuretic by a waterload equivalent to 2% of body weight. Body water and content of sodium were maintained by separate servo-controlled infusions. Peptides were infused for 60 min at rates of 50 pg kg-' rnin-' (arginine vasopressin) or 1 ng kg-' min-' (oxytocin), either separately or combined. Infusions increased plasma arginine vasopressin to 1.9 f0.2 (arginine vasopressin alone) and 1.8f0.3 pg kg-' (arginine vasopressin plus oxytocin and plasma oxytocin to 7 2 f 5 (oxytocin alone) and 77 8 pg ml-' (oxytocin plus arginine vasopressin). Arginine vasopressin or arginine vasopressin plus oxytocin increased urine osmolality similarly by a factor of 13, decreased urine flow to between 5 and 7% of control and decreased free water clearance. Oxytocin reduced urine flow and free water clearance and increased urine osmolality by a factor of 2. Oxytocin and arginine vasopressin separately increased excretion of sodium from 4 f2 to 15 & 6 pmol min-' and from 7 f4 to 25 f 13 pmol min-', respectively. Arginine vasopressin plus oxytocin led to a pronounced natriuresis (13 f 4 to 101f27 pmol min-I). Arginine vasopressin and arginine vasopressin plus oxytocin increased the excretion of potassium by a factor of 2.5. Oxytocin and arginine vasopressin plus oxytocin increased urinary Na+/K+ ratio by a factor of 3.7. It is concluded, that oxytocin at plasma concentrations of 70-80 pg ml-' has modest antidiuretic and natriuretic effects and that the combined action of arginine vasopressin oxytocin may elicit supra-additive natriuretic effects. Key words : Neurohypophysial peptides, renal effects.
T h e role of vasopressin in the control of renal excretion of water and electrolytes in the conscious waterdiuretic dog is relatively well quantified (e.g. Baerwolf & Bie 1988, Bie et al. 1984). Oxytocin ( O T ) and arginine vasopressin (AVP) show extensive homologies (two amino acids [3-ILE] and [&LEU] distinguish OT from AVP), and both seem to be released in response to an increment in plasma osmolality (Weitzman et al. 1978). However, very few experiments on the renal effects of OT seem to have been Correspondence : Peter Bie, Department of Medical Physiology C, Panum Institutet, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark.
conducted in the dog (Ali 1958, Brooks 8~ Pickford 1958). These results indicated that OT may increase the rate of excretion of sodium at low urine flows and may act as an antidiuretic agent in water diuretic dogs. T h e observations were later confirmed by Chan & Sawyer (1961), who also found that OT in rather high doses could evoke natriuresis during water diuresis. Experiments in other species have yielded highly variable results, depending on species and the state of water and salt balance. Experiments in neurohypophysectomized and acutely hypophysectomized rats have demonstrated interactions between AVP and OT on renal functions (Balment et al. 1986). I t was
267
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observed that doses of OT, which by t h e m s e h e s had n o measurable renal effect, potentiate the narriuretic action of .-\I-P and that concurrent administration of both hormones largely reversed the renal sodium retention normally seen in neurohypophysectomized rats. T h c present stud!- was conducted in order to unveil a possible interaction of .41'P and OT on the renal excretion of sodium and potassium in the conscious dog under precisely defined experimental conditions with regard to body content of sodium and body w t .
ZIATERI.ILS A N D M E T H O D S .4ninra/s. Experiments were performed on six trained, conscious female Beagle dogs weighing 10.5-13.5 kg. They \\ere fed once dail! with commercial food (Latz Komplett mised with Latz canned food) usually at 1400 hrs, and had free access to tap water. The intake of sodium and potassium was determined regularly and a\eraged 3.5k0.3 and 4.1 k 1.3mmol d q - ' kg bod! \ \ t I . respectively. The dogs were trained to accept percutaneous catheterization, catheterization of the hladder and to stand quietly for several hours supported by a canvas sling. The common carotid arteries had been placed in skin loops prior to the experiments. Prepuration. Each dog was used for all types of experiments with intervals of at least a week. .4 sterile catheter (Intracath'?) was placed in a saphenous vein and another in or near the right atrium via an external jugular yein. third catheter (Angiocath^) was placed in a carotid artery. .I modified silicone Foley catheter was inserted into the bladder. The venous catheters were used for blood sampling and infusion, while the arterial one was used to measure the arterial blood pressure (Sratham P50). Heart rate was measured hy registration of the electrocardiogram. Values of arterial blood pressure and heart rate were collected automatically every min using a Dialog 2000 monitor (Danica) interrogated bj- a DEC pVAX computer. Data were stored on disc and averaged over 15 min. Protocd. T h e dogs were hydrated via a gastric tube with a load of water at 37 "C equivalent to 2 O , of bodv wt. Thereafter, the body wt was kept within 102.0_+ 0.2O, of prehydration value by use of a seryomechanism (Bie 1976) infusing a hqpotonic solution of glucose and urea (G/U-solution), 40 and 25 mM, respectively. After hydration a 20 ml bolus injection of inulin, 40 g I-', was given, and a continuous infusion was started at a rate of 0.33 ml min-' in order to measure inulin clearance. During the experiment the quantity of sodium excreted per unit time was continuously replaced by a sodium servo-system
infusing a 400 rnhl solution of sodium chloride into the jugular catheter (Andersen & Bie 1990). Ninety min after h!-dration, when steady state water diuresis had been achieved, sampling of urine was started (time = 0). .-\fter a control period of 15 min, a continuous infusion of hormones lasting 60 min was carried out. ,4VP and OT were infused at rates of 50 pg kg-' min-' and 1 ng kg-' min-', respectively, either separately or in combination. In the control series only the vehicle was infused. Pure synthetic peptides (Ferring, Malmo, Sweden) were dissolved in a solution of acetic acid adjusted to p H = 4.5 and stored at - 18 "C. Immediately prior to infusion aliquats were thawed and diluted with G/U-solution and Haemaccels (Behringwerke, Marburg, Germany) in a ratio of 9 to 1. HaemacceF was added to avoid adsorption of peptide to tubing and glassware. Observations were continued in a 90min postinfusional phase. Urine was sampled at intervals of 15 min. Blood samples of 4.0ml (at 37.5, 97.5 and 157.5 min) and 16 ml (at 7.5,67.5 and 127.5 min) were obtained from the saphenous vein. After sampling the blood was centrifuged immediately at 4 "C. The erythrocytes were resuspended in isotonic sodium chloride and reinfused. Plasma samples were immediately frozen and stored at - 18 "C. Analyses. Na' and K' were measured on an IL 243 LED flame photometer. Osmolalities on an osmometer (Digimatic* osmometer model 3DI1, Advanced Instruments, MA, USA). Inulin was measured by the method described by Steele (1969) with minor modifications. Radioimmunoassay for AVP on extracted dog plasma was performed by the method described by Sakurai et al. (1986) with minor modifications. Extraction recovery averaged 7 2 yo.Specific and nonspecific binding was 39 and 2.2%, respectively. Intraassay coefficient of variation was about S.O"& All samples were run in one assay. Sensitivity of the assay was 0.15 pg tube-'. Half maximal binding (ED-50 value) occurred at 1.47 k 0.05 pg tube-'. Radioimmunoassay for oxytocin on infusates and unextracted dog plasma was performed by the method described by Engstrem et al. (1990). Specific and nonspecific binding was 35 and 2.0y0,respectively. Intraand interassay coefficients of variation were 7.894 and 7.700at 5 pg tube-'and 5.6%and 2.506at 20 pg tube. Sensitivity of the assay was 2 pg tube. Half maximal binding (ED-50 value) occurred at 14.5 Ifr 0.3 pg. Statistics. Results are given as mean+SE. Data were subjected to a one-way analysis of variance for repeated measures (Winer 1971). Because of variance inhomogeneity, as indicated by Bartlett's test, the data for sodium excretion and urine osmolality were transformed logarithmically prior to analysis of variance (Winer 1971). I n case of significantly large Fvalues all possible differences were evaluated systematically by Newman-Keuls' test (Winer 1971).
Effects of AVP andlor O T on renal Na' and K+
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'The l e d of significance was 0.0.5 and u = 6 unless othern ise indicated.
KF:SLI.'"S Infirsion /!/' . I I .P
Infusion of -4i-P at -iO pg lip-' m i n - ' increased plasma concentration of -41P (p.11.P) from 0.9 f0 . 1 to I .9 f0 . 2 pg mI '. After cessation of infusion p.\VP had returned to 0.9 0.1 pg ml-'. Urine flov decreased from 3.8k0.4 to 0 . 2 i 0.1 ml min I ( P < O.01, Table I ) . J.ihenise free \$att'r clearance (CH,,,) decreased fimm 3.0 i0.3 to -0..7&(J.2ml min-' (P < 0.01, Fig. I ) . This \$as accompanied b!- a 13-fold increase in urine osmolalit! (uOsm), from 0 2 i 3 to 821 1X4 mOsm kg ( P < 0.01, 'Table 1). T h e rate of excrction of sodium showed an increase of ( 7 k 4 to 25 f 13 pmol min-.'; 18 pmoI min Fig. 2) as did the rate of excretion of potassium ( 1 3 iL t o 31 6 pmol min~-'). \lean arterial blood pressure (MABP) (mean 1 I3 & 1 mmHg), heart rate (FIR), plasma osmolality (pOsm), plasma concentrations of sodium (pNa') and potassium (PIC') remained constant throughout the experiment. Infusion ( f 0 T
Infusion of 07' at 1 ng kg-' min-' increased plasm;i concentration of OT ( p O T ) from below
1
*
limit of detecting to 72 f5 pg ml-'. After cessation of infusion p O T returned to undetectable IeL-els. Urine flow (Table 1) and CH,O (Fig. 1) decreased from 4.2 i0.5 to 3.0 f 0 . 7 mi min-' and from 3.3f0.4 to 2.2+0.6ml min-', respectively, and Uosm doubled (Table 1). After the termination of hormone infusion both urine flow and CH,O increased to levels significantly higher than values observed in control and AVP experiments. T h e rate of excretion of sodium showed a persistent increment averaging 11 pmol min-' and did not return to control level (Fig. 2). T h e rate of excretion of potassium tended to increase, but the rate was not significantl!- higher than that during the preinfusional period. At the end of the experiment, though, the rate of excretion was significantly higher than in control and AVP experiments. Plasma K' decreased from 4.1 kO.1 to 3.7+ 0.1 mmol I-' during the infusion. Administration of OT did not affect MARP (mean 108f 1 mmHg), HR, pOsm, pAVP, or pNA+.
Conihinivi infirsion o f .4 VP und 0 T Combined infusion of AVP and OT 5 0 pg kg-' rnin and 1 ng kg-' min-', respectively, produced plasma concentrations of hormones similar to those found after single infusions (pAVP = 1.8i0.3 pg ml-' ( n = 4) and POT = 77 f 8 pg ml.'). T h e effects on urine flow (Table l), uOsm (Table 1) and C,.,,, (Fig. 1) h~eresimilar to the changes seen in dogs during single infusion of -1L-P. How-ever, during recovery urine flow increased to a level significantly higher than seen in control and .4\)-P experiments. T h e rate of excretion of sodium, however, increasing by 89 pmol min-' from 13 f4 to 101 +27 pmol min-.' (P< 0.01), greatly exceeded the sum of the excretion rates (18f 11 pmol min-') measured in the single hormone series (Fig. 2). T h e masimum was reached shortly after termination of infusion at a time when urine flow was still reduced. T h e pronounced natriuresis was accompanied by an increase in the rate of excretion of potassium (13 f3 to 35 f7 pmol min-') similar to that seen with AVP alone. T h e effect of combined infusion on the rate of excretion of potassium was prolonged, and at the end of the experiment the rate of excretion was signilicantl!- higher compared to control and -4L-Pesperiments. A trend towards a decrease in
Efects o f AVP andlor OT on renal Na' and Ki
271
II INFUSION
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TIME (MIN)
-
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Time (rnin) Fig. 2. Rate of excretion of sodium (Pmol min-'). Hormone or vehicle were infused from min 15 to min 75. = controls, 0 = AVP 50 pg kg-' min-',
A = OT 1 ng kg-' min-', 4 = AVP SO pg kg-' min-' and OT 1 ng kg-' min-'. Dotted line indicates sum of single hormone effects. Values significantly different from preinfusion values (" = 0.05 and * = 0.01) of the same series are indicated. Values are means SEM ; n = 6 dogs.
Fig. 3. Urinary Na'/K+ ratio. Hormone or vehicle were infused from min 15 to min 75. =controls, = AVP 50 pg kg-' min-', A = OT 1 ng kg-' min-', 4 = AVP 50 pg kg-' min-' and OT 1 ng kg-' min-'. Values significantly different from preinfusion values (" = 0.05 and * = 0.01) of the Same series are indicated. Values are means+SEM; n = 6 dogs.
T
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er
ttx pK+, statistically insignificant but of similar magnitude to the decrease found in OT single hormone series, was observed. pOsm showed a decrease from 292+ 1 to 289+ 1 mosm kg-l ( P < 0.01). T h e concurrent administration of hormones did not affect HR, MABP (mean 117 2 1 mmHg) or pNa+ measurably.
a~
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45
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105
135
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Fig. 4. Fractional excretion of sodium (0J. Hormone During the control series pOsm, pNa+ and p K + = or vehicle were infused from min 15 to min 75. did not change significantly. Likewise, detectable controls, = AVP 50 pg kg-' min-', A= changes did not occur in HR, urine flow, CHZO OT 1 ng kg-' min-', 4 = AVP 50 pg kg-' min-' and (Fig. l), uOsm (Table I), rates of excretion of OT 1 ng kg-' min '. Values significantly different sodium (Fig. 2) and potassium or pAVP. p O T from preinfusion values (" = 0.05 and * = 0.01) ofthe was below the detection limit of the assay same series are indicated. Values are means SEM ; throughout the experiment. Towards the end of n = 6 dogs. the post-infusion phase MABP increased slightly maximum obtained during combined hormone from look5 to 1 0 9 + 3 mmHg. During infusion of OT and combined infusion infusion was significantly higher than in control of AVP and OT the urinary sodium/potassium and AVP experiments. T h e urinary sodium/ ratio increased from 0 . 4 5 0 . 2 to 1.7 F0.8 and potassium ratio did not change during control 1.1k0.3 to 4.lf 1.3, respectively (Fig. 3 ) . T h e and AVP experiments. 13-2
272
S . E. Anderseii et al.
I h t a f’or inuiin clearance are g i w n in ‘Table 1 For certain periods in time it wa5 mident that the urine flou- data did not represent a steady state. T h e values for these periods h a w been omitted. I l o w e x r , assuming only minor changes in GFK, calculations of the fractional escretions of electrol!-tes remain d i d . During the control, .%W,and (IT series fractional escretion of sodium did not change significantly (Fig. 4). T h e combined infusion of A1.P and OT, hon e\-er, increased fractional sodium escretion significanti!- from 0 . 2 6 ~ 0 . 1 0 t o1.21 i O . 3 7 , i . e . almost h! a fhctor of 5.
I ) I s c,
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T h e main findings of the stud!- were a modest antiduretic and natriuretic effect of OT infused alone, and supra-additive effects of -41-Pand OT on renal escretion of sodium. In the pattern of the response it should be noted that the changes in the rate of’ercretion of sodium are reflected in urinarl- X a - / I C ratio and in the fractional excretion rates and that the peak sodium excretion occurred a t the nadir of urine flo\\. ‘I’ahcn together this demonstrates that washout of‘ the dead space of the urinar!- drainage s!-stem can not account for our main findings. T o the contrar!, the dead space (1-olume of the lower urinary tract and external drainage tubing) is liliel! to hake caused deminuation and dela! of the ohserxeetl events. Calculation of inulin clearance requires stead!state conditions. ..lt times of rapid changes in urinc flon inulin clearances do not express GFR. In the present study the d u e s obtained prior to hormone infusions and at the end of the infusion and recover!- periods were all obtained a t stead!state urine flov and are, therefore, most likel!- to reflect GFR. Other clearance data should be interpreted with great caution. T h e rate of excretion of sodium in the control period of the ..lI-P+OT series was slightly greater than in the -41-Pand OT series (Fig. 2 ) . I i o a e ~ e rat , the beginning of the infusion period prior to the appearance of other effects of the hormones no significant differences between rates of excretion of sodium of the three series could be detected. I t seems thus 1-ery unliliel!-. that the obserwd supraadditive natriuretic effect was due to small initial differences in sodium excretion betlieen the series.
U-hen AVP $+as infused separately, the expected reduction in urine flow and C H L 0was accompanied by a minor increase in the rate of escretion of sodium. This result is in agreement with earlier experiments on the effect of’ overh!-dration during different AVP infusions (Bie t t a / . 1984). I n that study larger doses caused further elevation in sodium excretion. I n a subsequent study it w-as shown that infusions of -41.P a t the very low rates of 2 and 5 pg kg-’ min-’ reduced urine flow by 39 and 61 O o , respectivel!-, without measurable alterations in sodium excretion (Baerwolf & Bie 1988). These results together demonstrate a threshold for the natriuretic action of AVP which is rather low. None of these studies included measurements of hormone concentrations in plasma. Physiological rele1-ance of the supra-additive effect of AVP and OT on renal excretion of sodium requires simultaneous release of both hormones in response to physiological stimuli, e.g. increasing plasma osmolality. While the osmotic regulation of AVP secretion is well established (e.g. Robertson 1987), the role of plasma osmolalitl- in regulation of OT secretion is poorly elucidated also in dogs. I n conscious dogs plasma concentrations of both hormones increase in response to increments in plasma osmolalit>- from 3 0 4 5 1 mOsm kg-’ to 316f 1 mOsm kg-’ (Weitzman et 01. 1978). I n rats a strong positive correlation between plasma osmolality and plasma oxytocin concentration (Balment et ul. 1980) and osmotic sensitivity of OTand ri1-P-producing neurons has been observed (Rrimble 1977, Cheng & North 1986). Simultaneous release of both hormones in response to a varier>-of stimuli e.g. intravenous injection of hypertonic saline and isotonic hypovolemia has been demonstrated (Kasting 1988). T h e plasma concentrations of AVP measured during the present study are well u-ithin the normal physiological range. Increments in piasma OT, though, may be considered substantial or supraphysiological. However, POT of the same magnitude has been observed in lactating rhesus monkeys during nursing (Amico et t i / . 1990) and in Brattleboro rats in response to an increment in pOsm from 3 0 7 i 3 to 3 1 9 i 2 mosm kg-’ (Brimble et a / . 1990). I t might be questionable whether the demonstrated supra-additive effect of AVP and OT is part of physiological sodium homeostasis, but the phen-
Efects of AVP andlor OT on renal Na' and Kf omenon may obviously be of relevance to body fluid control during labour and nursing. T h e renal effect of normal as well as supranormal concentrations of OT is a topic of some controversy. I n the present study we observed a modest natriuretic and antidiuretic effect of OT (1 ng kg-' min-' giving 70 pg ml-' in plasma). This is discordant with results obtained in conscious dogs by Brooks & Pickford (1958) and Chan & Sawyer (1961). OT at high doses (up to 12 ng kg-') did not cause natriuresis during water diuresis but only during low urine flows. (Note: conversion factors for AVP and OT: 2.5 pg pU-' and 2.0 pg pU-', respectively.) During water diuresis OT elicited natriuresis only at doses of 20 ng kg-' (Chan & Sawyer 1961). Assuming an apparent volume of distribution of 125 ml kg-' (Robinson 1980) and a rapid distribution the increment in POT in the latter experiment could have reached 160 pg ml-', i.e. markedly exceeding POT obtained in our experiment. T h e water diuresis in the two experiments was not sustained. Using an experimental set u p similar to ours Wesley & Gilmore (1985) investigated the effects of single injections of OT at a dose of 2 ng kg-' in anaesthetized macaque monkeys. Elevating POT by 10-20 pg ml-' they found no effects on creatinine clearance, free water clearance or the rate of excretion of sodium. T h e influence of anaesthetics or species differences may account for the difference between this study and ours. T h e distinct influence of OT on the natriuretic but not the antidiuretic effect of AVP in the present study is compatible with the proposal that the antidiuretic and natriuretic responses to OT are mediated by at least two different types of neurohypophysial hormone receptors (Chan & Hruby 1988). However, our results do not exclude the fact that other mechanisms are involved in natriuresis of neurohypophysial hormones. Further studies in conscious animals are needed to elucidate the osmotic regulation of both AVP and OT and the combined natriuretic action of the neurohypophysial hormones at the lower end of the physiological range of secretion. The authors thank Inge Pedersen, Karen Klausen and Sigurd Hansen for expert technical assistance. This work has been supported by grants from the Danish Medical Research Council and the Velux Foundation.
273
Preliminary results were presented at the International Symposium on Mechanisms of Sodium Homeostasis, Copenhagen, July 1989.
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\\-.Y. S. SAVYER, 1V.H. 1961. Saliuretic actions of neuroh) poph!-sial peptides in conscious dogs. ,.in2 7 Ph.>'s/ol201, 799-803. C t f E S G , S.1V.T.& XoRm, \V.G. 1986. Responsiveness
Cti.A\,
of os! tocin-producing neurons to acute salt loading in rats: Comparison with vasopressin-producing neurons. .IrurornJocrinoloR)i 42. 174-180. ENGSTRQL~. T. & VILH~RDT, H. 1989. Oxytocin concentrations in plasma, h! pothalamus and the pituitarv of rats following continuous infusion of osytocin. In X..k Thorn, H . i-ilhardt & 11.Freiner (eds). Proceedings of the Fourth International Con.firmre on the . ~ - r u r o h ~ ~ o p h ~pp. ~ s 98-1 i s , 00. Oxford Lni\-ersit)- Press, Oxford. K~s-rirc;,X.\\'. 1987. Simultaneous and independent release of vasopressin and oxytocin in the rat. Can 3 Ph,lljiol P h a r m r o l 66, 22-26. Rommsox, G.L. 1987. Ph!-siology of .IDH secretion. Kidnej, Int 32, S20-S26. ROBIYSOX, 1.C.A.F. 1980. The development and
el-aluation of a sensitive and specific radioimmunoassay for oxytocin in unextracted plasma. Journal of Irnrnunoassuy 1, 323-317. S4KLR.41, H., KANAI, A , , NOMURA, K., DEMURA, H. & SHIZCME, K. 1986. A simple and highly sensitive radioimmunoassay for 8-arginine vasopressin in human plasma using reversed-phase C,, silica column. 3 Tokyo Worn Med CoM 56, 39-03, STEELE, T.H. 1969. A modified semi-automated resorcinol method for determination of inulin. Clin Chem 15, 1072-1078. \VEITZMAN, R.E., GLATZ,T.H. & FISHER, D.A. 1978. The effect of hemorrhage and hypertonic saline upon plasma oxytocin and arginine vasopressin in conscious dogs. Endocrinology 103, 2154-2160. J.P. 1985. Effects of N ~ ~ S L EC.R. Y , & GILMORE, oxytocin, vasopressin and a-MSH on renal function in the nonhuman primate. Hormone Res 21, 55-59. \VISER, B.J. 1971. Statistical principles in experimental design (2nd ed.). McGraw-Hill, New York.
