Brain Research, 579 (1992) 113-118 (~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
113
BRES 17674
Central administration of atrial natriuretic peptide suppresses sodium and water intake of sheep R.S. Weisinger, J.R. Blair-West, D.A. Denton and E. Tarjan Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Vict. (Australia) (Accepted 17 December 1991)
Key words: Drinking behavior; Dehydration; Angiotensin II; Operant behavior
The effect of intracerebroventricular (i.c.v.) infusion (20/lg/h over 3 h) of human a-atrial natriuretic peptide (ANP) on Na and water intake of sheep was studied. I.c.v. infusion of ANP decreased (P < 0.01) Na and water intakes of water-deprived sheep but did not affect significantly Na or water intakes of Na and water-replete sheep. In addition, i,c.v, infusion of ANP decreased (P < 0.05) Na and water intakes of sheep infused i.c.v, with angiotensin II. The results suggest that ANP may act on brain mechanisms concerned with both Na appetite and thirst. These mechanisms may involve action on the angiotensin II component of sodium appetite but effects on other factors determinant of appetite cannot be excluded at present. INTRODUCTION
22,29,36 that the influence of A N P on regulation of b o d y
A t r i a l natriuretic p e p t i d e ( A N P ) 14'23 and its receptors 17'22 are distributed widely throughout the brain including m a n y of the areas thought to be involved in b o d y fluid balance, e.g. circumventricular organs (CVOs). A N P administered centrally has b e e n shown to inhibit angiotensin ( A N G ) II-induced changes in h o r m o n a l systems involved in b o d y fluid balance (e.g. A V P secretion 36, A C T H secretion13). In regard to the latter, A C T H
fluid and N a homeostasis m a y be due to its antagonism of the actions of A N G II. ANP, for example, has b e e n shown to block A N G II-induced neural excitation in the subfornical organ ( S F O ) 11. Given the evidence that the Na appetite of N a - d e p l e t e sheep is m e d i a t e d by circulating A N G II acting at brain areas without a b l o o d - b r a i n b a r r i e r 33, the evidence that there m a y be species differences in regard to the access to these brain areas by materials infused into the cerebral ventricles 5'6'8'33'35, and
has b e e n shown to have n u m e r o u s effects on b o d y fluid homeostasis in sheep, e.g. A C T H has b e e n shown to cause changes in plasma N a concentration and osmolality, plasma volume, N a and water intake and b l o o d aldosterone levels 24-26'31. In addition, central administration of A N P can influence both water and salt intakes of rats. F o r example, intracerebroventricular (i.c.v.) administration of A N P decreases the water intake caused by i.c.v, administration of A N G II 3'16A8 or by water deprivation3'16'18). H o w e v e r , A N P does not a p p e a r to influence the water intake of normally h y d r a t e d rats ~8. A l s o , the N a intake of spontaneously hypertensive rats 12 and of N a - d e p l e t e rats 4,t° is decreased by i.c.v, administration of ANP. In recent experiments, we d e m o n strated that while i.c.v, administration of A N P inhibited the increase in N a intake of w a t e r - d e p r i v e d o r Na-deplete rabbits 28, it did not alter the N a intake of Na-deplete sheep 19. Consistent with the o b s e r v e d overlap of cerebral A N G II and A N P receptors 17'22, it has b e e n suggested 13'16-1s'
the influence of A N P d e m o n s t r a t e d in the studies n o t e d above, we e x a m i n e d the influence of A N P on N a and water intake of sheep during infusion of A N G II and following water deprivation.
MATERIALS AND METHODS Fourteen cross-bred Merino ewes (35-40 kg body wt.) were used. The sheep were ovariectomized and had at least one carotid artery exteriorized in a skin loop. Prior to experimentation, all animals were surgically prepared with a guide tube (17-gauge stainless steel needle, 42 mm long) implanted 6-10 mm above each lateral brain ventricle. Animals were fed 0.8-0.9 kg oaten-lucerne chaff (Na content = 50-150 mmol/kg) at 16.30 h daily. The sheep were maintained in metabolism cages that allowed for the separate collection of urine and feces. The studies used operant conditioning because it has been shown to be a reliable method of measuring motivated behaviour in sheep 1. The cages contained two pedals. The animals were trained to press one pedal in order to obtain delivery of Na solution to a drinking cup (Na delivery = 25 ml of 0.5 M NaC1 = 12.5 mmol). A second pedal delivered 50 ml of water to a second drinking cup. The animals had 2 h daily (12.00-14.00 h) access to Na solution after the sounding of a tone and continuous access to water except in water deprivation exper-
Correspondence: R.S. Weisinger, Howard Florey Institute, Parkville, Vict. 3052, Australia. Fax: (61) (3) 348-1707.
114 iments. All deliveries were consumed. The number of deliveries was counted and recorded continuously by computer, For i.c.v, infusion, on the day of the experiment, a blocker was removed from one of the guide tubes and a probe (20-gauge needie attached to a metal Lner-Lok cap) of appropriate length was inserted through the guide tube into the lateral brain ventricle. The probe was connected via polyethylene cannula to a 10-ml syringe held in an infusion pump (Perfusor, Braun). The cannula and sytinge were filled with a solution of the appropriate peptide dissolved in artificial cerebrospinal fluid (CSF; composition (in mM): [K] 2.8, [Ca] 1.1, [Na] 150, [C1] 157.5, [Mg] 0.9, [HPO4] 0.5. The i.c.v, infusion (i ml/h) was begun 1 h prior to Na access and continued for 3 h, i.e. until the end of the Na access period, CSF from the lateral ventricle was obtained by siphon prior to and after infusion. ANP (human a-atrial nattiuretic polypeptide 1-28, Peninsula) was infused at 20/,g/h. The dose of ANP used in the present experiments was based on the doses used in previous experiments in sheep 19. ANG II (Peninsula) was infused at 3.8/tg/h. This dose of ANG II has been shown to cause increased intake of Na and water in sheep 3°.
Calculation of the estimated Na balance The procedure used to calculate the estimated Na balance (see Table I) has been described previously34. The sheep were assumed to be in neutral sodium status at the start of the Na access period on the day prior to the experimental day. Neither Na intake in food nor Na loss in feces was measured and a small quantity of Na (e.g. 0-30 mmol) had to be corrected for in the daily balance calculation.
Analysis [Na] and [K] of saliva, urine and CSF were measured by Technicon Auto Analyser or Coming EEL-450 flame photometer. Usmolality of CSF was measured by Digimatic Osmometer (Advanced Instruments Inc.).
A N P h a d n o significant effect o n either N a or water intake. T h e r e were n o differences b e t w e e n baseline a n d infusion c o n d i t i o n s in terms of Na status or 22 h water intake prior to Na access at 12.00 h (Table I A ) . C S F composition was n o t significantly altered after the infusion of A N P (Table II).
Experiment 2. Effect o f i. c.v. infusion o f atrial natriureticpeptide on intake o f sodium and water o f sheep infused with A N G II Fig. 1B shows that relative to baseline, i n f u s i o n of A N G II at 3.8/~g/h caused a large increase in Na a n d water intake. I n f u s i o n of A N G II increased water i n t a k e d u r i n g the 1-h p e r i o d prior to N a access from 30 _+ 12 ml (baseline) to 1080 + 114 ml ( P < 0.001). Na i n t a k e was increased from 130 _+ 27 m m o l (baseline) to 293 _ 47 m m o l ( P < 0.001). T h e infusion of A N P significantly r e d u c e d the increase in water i n t a k e ( P < 0.001) a n d p r e v e n t e d the increase in N a i n t a k e caused by infusion of A N G II. T h e r e were n o significant differences b e t w e e n baseline a n d infusion c o n d i t i o n s in terms of N a status or 22 h water i n t a k e (Table IB). C S F [Na] a n d osmolality were decreased ( P < 0.01) after infusion of A N G II, a n d C S F [Na], [K] a n d osmolality were decreased (P's < 0.05) after infusion of A N G II plus A N P (Table II).
Experimental Design Experiment 1. Effect of i. c.v. infusion of atrial natriuretic peptide on intake of sodium solution and water. Sheep (n = 10) were infused with ANP.
Experiment 2. Effect of i.e.v, infusion of atrial natriuretic peptide on intake of sodium solution and water of sheep infused with angiotenSinplus ANp.II" Sheep (n = 10) were infused with ANG II or ANG II
INTAKE
Experiment 3. Effect of i.e.v, infusion of atrial natriuretic peptide on intake of sodium solution and water of water-deprived sheep.
0.5M
Sheep (n = 8) were deprived of water for 22 h or were deprived of water for 22 h and infused with ANP. The sheep were offered access to both Na and water at the end of the water-deprivation period, i.e. 12.00 h.
Statistical analysis The mean of the values obtained on the day prior to each infusion for each animal was used in determining the baseline value. A t-test matched pairs design (Fig. 1A, Table I A ) or a one-way anal-
ysis of variance, repeated measures design (Figs. 1B and 2; Table IB and IC) or independent measures design (Table II) and subsequent t-tests (using the error mean square from the ANOVA as the
300
_Lmean +SEM (n = 10) .... --" =0
113
BRES 17674
Central administration of atrial natriuretic peptide suppresses sodium and water intake of sheep R.S. Weisinger, J.R. Blair-West, D.A. Denton and E. Tarjan Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Vict. (Australia) (Accepted 17 December 1991)
Key words: Drinking behavior; Dehydration; Angiotensin II; Operant behavior
The effect of intracerebroventricular (i.c.v.) infusion (20/lg/h over 3 h) of human a-atrial natriuretic peptide (ANP) on Na and water intake of sheep was studied. I.c.v. infusion of ANP decreased (P < 0.01) Na and water intakes of water-deprived sheep but did not affect significantly Na or water intakes of Na and water-replete sheep. In addition, i,c.v, infusion of ANP decreased (P < 0.05) Na and water intakes of sheep infused i.c.v, with angiotensin II. The results suggest that ANP may act on brain mechanisms concerned with both Na appetite and thirst. These mechanisms may involve action on the angiotensin II component of sodium appetite but effects on other factors determinant of appetite cannot be excluded at present. INTRODUCTION
22,29,36 that the influence of A N P on regulation of b o d y
A t r i a l natriuretic p e p t i d e ( A N P ) 14'23 and its receptors 17'22 are distributed widely throughout the brain including m a n y of the areas thought to be involved in b o d y fluid balance, e.g. circumventricular organs (CVOs). A N P administered centrally has b e e n shown to inhibit angiotensin ( A N G ) II-induced changes in h o r m o n a l systems involved in b o d y fluid balance (e.g. A V P secretion 36, A C T H secretion13). In regard to the latter, A C T H
fluid and N a homeostasis m a y be due to its antagonism of the actions of A N G II. ANP, for example, has b e e n shown to block A N G II-induced neural excitation in the subfornical organ ( S F O ) 11. Given the evidence that the Na appetite of N a - d e p l e t e sheep is m e d i a t e d by circulating A N G II acting at brain areas without a b l o o d - b r a i n b a r r i e r 33, the evidence that there m a y be species differences in regard to the access to these brain areas by materials infused into the cerebral ventricles 5'6'8'33'35, and
has b e e n shown to have n u m e r o u s effects on b o d y fluid homeostasis in sheep, e.g. A C T H has b e e n shown to cause changes in plasma N a concentration and osmolality, plasma volume, N a and water intake and b l o o d aldosterone levels 24-26'31. In addition, central administration of A N P can influence both water and salt intakes of rats. F o r example, intracerebroventricular (i.c.v.) administration of A N P decreases the water intake caused by i.c.v, administration of A N G II 3'16A8 or by water deprivation3'16'18). H o w e v e r , A N P does not a p p e a r to influence the water intake of normally h y d r a t e d rats ~8. A l s o , the N a intake of spontaneously hypertensive rats 12 and of N a - d e p l e t e rats 4,t° is decreased by i.c.v, administration of ANP. In recent experiments, we d e m o n strated that while i.c.v, administration of A N P inhibited the increase in N a intake of w a t e r - d e p r i v e d o r Na-deplete rabbits 28, it did not alter the N a intake of Na-deplete sheep 19. Consistent with the o b s e r v e d overlap of cerebral A N G II and A N P receptors 17'22, it has b e e n suggested 13'16-1s'
the influence of A N P d e m o n s t r a t e d in the studies n o t e d above, we e x a m i n e d the influence of A N P on N a and water intake of sheep during infusion of A N G II and following water deprivation.
MATERIALS AND METHODS Fourteen cross-bred Merino ewes (35-40 kg body wt.) were used. The sheep were ovariectomized and had at least one carotid artery exteriorized in a skin loop. Prior to experimentation, all animals were surgically prepared with a guide tube (17-gauge stainless steel needle, 42 mm long) implanted 6-10 mm above each lateral brain ventricle. Animals were fed 0.8-0.9 kg oaten-lucerne chaff (Na content = 50-150 mmol/kg) at 16.30 h daily. The sheep were maintained in metabolism cages that allowed for the separate collection of urine and feces. The studies used operant conditioning because it has been shown to be a reliable method of measuring motivated behaviour in sheep 1. The cages contained two pedals. The animals were trained to press one pedal in order to obtain delivery of Na solution to a drinking cup (Na delivery = 25 ml of 0.5 M NaC1 = 12.5 mmol). A second pedal delivered 50 ml of water to a second drinking cup. The animals had 2 h daily (12.00-14.00 h) access to Na solution after the sounding of a tone and continuous access to water except in water deprivation exper-
Correspondence: R.S. Weisinger, Howard Florey Institute, Parkville, Vict. 3052, Australia. Fax: (61) (3) 348-1707.
114 iments. All deliveries were consumed. The number of deliveries was counted and recorded continuously by computer, For i.c.v, infusion, on the day of the experiment, a blocker was removed from one of the guide tubes and a probe (20-gauge needie attached to a metal Lner-Lok cap) of appropriate length was inserted through the guide tube into the lateral brain ventricle. The probe was connected via polyethylene cannula to a 10-ml syringe held in an infusion pump (Perfusor, Braun). The cannula and sytinge were filled with a solution of the appropriate peptide dissolved in artificial cerebrospinal fluid (CSF; composition (in mM): [K] 2.8, [Ca] 1.1, [Na] 150, [C1] 157.5, [Mg] 0.9, [HPO4] 0.5. The i.c.v, infusion (i ml/h) was begun 1 h prior to Na access and continued for 3 h, i.e. until the end of the Na access period, CSF from the lateral ventricle was obtained by siphon prior to and after infusion. ANP (human a-atrial nattiuretic polypeptide 1-28, Peninsula) was infused at 20/,g/h. The dose of ANP used in the present experiments was based on the doses used in previous experiments in sheep 19. ANG II (Peninsula) was infused at 3.8/tg/h. This dose of ANG II has been shown to cause increased intake of Na and water in sheep 3°.
Calculation of the estimated Na balance The procedure used to calculate the estimated Na balance (see Table I) has been described previously34. The sheep were assumed to be in neutral sodium status at the start of the Na access period on the day prior to the experimental day. Neither Na intake in food nor Na loss in feces was measured and a small quantity of Na (e.g. 0-30 mmol) had to be corrected for in the daily balance calculation.
Analysis [Na] and [K] of saliva, urine and CSF were measured by Technicon Auto Analyser or Coming EEL-450 flame photometer. Usmolality of CSF was measured by Digimatic Osmometer (Advanced Instruments Inc.).
A N P h a d n o significant effect o n either N a or water intake. T h e r e were n o differences b e t w e e n baseline a n d infusion c o n d i t i o n s in terms of Na status or 22 h water intake prior to Na access at 12.00 h (Table I A ) . C S F composition was n o t significantly altered after the infusion of A N P (Table II).
Experiment 2. Effect o f i. c.v. infusion o f atrial natriureticpeptide on intake o f sodium and water o f sheep infused with A N G II Fig. 1B shows that relative to baseline, i n f u s i o n of A N G II at 3.8/~g/h caused a large increase in Na a n d water intake. I n f u s i o n of A N G II increased water i n t a k e d u r i n g the 1-h p e r i o d prior to N a access from 30 _+ 12 ml (baseline) to 1080 + 114 ml ( P < 0.001). Na i n t a k e was increased from 130 _+ 27 m m o l (baseline) to 293 _ 47 m m o l ( P < 0.001). T h e infusion of A N P significantly r e d u c e d the increase in water i n t a k e ( P < 0.001) a n d p r e v e n t e d the increase in N a i n t a k e caused by infusion of A N G II. T h e r e were n o significant differences b e t w e e n baseline a n d infusion c o n d i t i o n s in terms of N a status or 22 h water i n t a k e (Table IB). C S F [Na] a n d osmolality were decreased ( P < 0.01) after infusion of A N G II, a n d C S F [Na], [K] a n d osmolality were decreased (P's < 0.05) after infusion of A N G II plus A N P (Table II).
Experimental Design Experiment 1. Effect of i. c.v. infusion of atrial natriuretic peptide on intake of sodium solution and water. Sheep (n = 10) were infused with ANP.
Experiment 2. Effect of i.e.v, infusion of atrial natriuretic peptide on intake of sodium solution and water of sheep infused with angiotenSinplus ANp.II" Sheep (n = 10) were infused with ANG II or ANG II
INTAKE
Experiment 3. Effect of i.e.v, infusion of atrial natriuretic peptide on intake of sodium solution and water of water-deprived sheep.
0.5M
Sheep (n = 8) were deprived of water for 22 h or were deprived of water for 22 h and infused with ANP. The sheep were offered access to both Na and water at the end of the water-deprivation period, i.e. 12.00 h.
Statistical analysis The mean of the values obtained on the day prior to each infusion for each animal was used in determining the baseline value. A t-test matched pairs design (Fig. 1A, Table I A ) or a one-way anal-
ysis of variance, repeated measures design (Figs. 1B and 2; Table IB and IC) or independent measures design (Table II) and subsequent t-tests (using the error mean square from the ANOVA as the
300
_Lmean +SEM (n = 10) .... --" =0
