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Circadian variations of plasma atrial natriuretic peptide and corticosterone in rats with continuous or restricted access to food
Life Sciences, Vol. 53, pp. 1795-1801 Printed in the USA
Pergamon Press
C I R C A D I A N V A R I A T I O N S OF P L A S M A A T R I A L N A T R I U R E T I C A N D C O R T I C O S T E R O N E IN R A T S W I T H C O N T I N U O U S O R R E S T R I C T E D A C C E S S TO F O O D M a n u e l H.A. O l i v e i r a , A n g e l a M.O. Leal, L u c i l a
PEPTIDE
Jos~ Antunes-Rodrigues (i), L.K. E l i a s a n d A y r t o n C. M o r e i r a
D i v i s i o n of E n d o c r i n o l o g y , D e p a r t m e n t of M e d i c i n e a n d D e p a r t m e n t of P h y s i o l o g y (I), F a c u l t y of M e d i c i n e 1 4 0 4 8 - 9 0 0 , R i b e i r a o Preto, SP, Brazil.
(Receivedinfinal~rm October6,1993) Summary In the p r e s e n t s t u d y p l a s m a A t r i a l N a t r i u r e t i c Peptide (ANP) a n d C o r t i c o s t e r o n e (B) l e v e l s w e r e d e t e r m i n e d in 2 g r o u p s of W i s t a r rats, a C o n t r o l g r o u p (C) w i t h free access to food, a n d a Food Shift group (FS) with a c c e s s to f o o d o n l y f r o m 0900 to ii00 h for 2 weeks. Blood samples were collected by d e c a p i t a t i o n at 4 h o u r intervals. ANOVA indicated that B v a r i e d o v e r t i m e in b o t h g r o u p s (F (6,46) = 10.14, p < 0.0001 for C a n d F (6,45) = 5.10, p = 0.0005 for FS) . T h e r e w a s a l s o time variation in plasma ANP levels in both g r o u p s (F (6,54) = 3.78, p = 0.003 for C a n d F (6,48) = 2.73, p = 0.02 for FS group). P l a s m a B presented circadian variations f r o m 78 + 17 n m o l / l (mean + SEM) at 0800 h to 339 + 79 n m o l / l at 2000 h. The day peak plasma ANP l e v e l (pmol/l) was a l s o a t t a i n e d at 2000 h (68 + 19). This value was higher than all others measured t h r o u g h o u t the day. The restricted feeding regimen r e s u l t e d in a 12 h o u r shift of peak B values (395 + 39 vs 125 + 24). The FS rats p r e s e n t e d the h i g h e s t A N P level at 5 8 0 0 h. The p r e s e n t study indicates similar circadian v a r i a t i o n s of p l a s m a A N P a n d B in rats o n an ad lib or restricted feeding regimen. ANP and B secretion may o c c u r in a n t i c i p a t i o n of the feeding p e r i o d or day activity. A t r i a l n a t r i u r e t i c peptide, A N P (99-126) is s y n t h e s i z e d and r e l e a s e d f r o m m a m m a l i a n c a r d i a c a t r i a a n d has s e v e r a l a c t i o n s on fluid and electrolyte homeostasis.Thus, systemic administration of ANP results in n a t r i u r e s i s and diuresis and a simultaneous decrease in p l a s m a renin, a n g i o t e n s i n a c t i v i t y and aldosterone secretion. Moreover ANP has been shown to be an effective i n h i b i t o r of v a s o p r e s s i n s e c r e t i o n a n d of salt a n d water intake in the rat (1,2). P e p t i d e s r e l a t e d to A N P are also synthesized l o c a l l y w i t h i n the C e n t r a l N e r v o u s S y s t e m ( C N S ) , w i t h p a r t i c u l a r l y high concentrations p r e s e n t in the hypothalamus, where atrial peptides m a y f u n c t i o n as n e u r o t r a n s m i t t e r s or neuromodulators. Recent studies have a l s o s u g g e s t e d that A N P m a y suppress the .
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A y r t o n C. M o r e i r a , Medicine 14048-900,
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MD, Division of Endocrinology, R i b e i r a o Preto, SP, Brazil. ~24-3~5/93 ~.00 + . ~
Copyri~t©1993PergamonPressLtd Allrightsrese~ed.
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hypothalamus-pituitary a d r e n a l axis b y i n t e r a c t i n g w i t h CRF 41 or by inhibiting pituitary corticotropin secretion (3 7). The role of ANP in a c u t e v o l u m e expansion has been extensively studied, but the i n f l u e n c e of A N P d u r i n g n o r m a l d a y - t o - d a y l i v i n g has b e e n not a d e q u a t e l y d e m o n s t r a t e d . It is w e l l e s t a b l i s h e d that the rhythmicity of the h y p o t h a l a m u s - p i t u i t a r y - a d r e n a l axis is r e l a t e d to p o s s i b l e n e u r a l mechanisms involved in sleep-wake, rest-activity, light-dark or feeding cycles. R a t s maintained under ad lib feeding or daytime food restriction manifest a circadian peak of p l a s m a B just p r i o r to the time of o n s e t of predominant food intake (8). Diurnal rhythm of plasma ANP c o n c e n t r a t i o n s w a s o b s e r v e d in normal volunteers or patients. However, detailed information on ANP periodicity in rats is scanty. Thus, we d e c i d e d to s t u d y the d a i l y v a r i a t i o n of plasma A N P in rats a n d the role of f o o d r e s t r i c t i o n on its s e c r e t i o n .
Methods Studies were p e r f o r m e d on 144 s i n g l y housed adult male Wistar rats weighing 200 g w h i c h had been acclimated to our l a b o r a t o r y for one week. L i g h t s on at 0700 h, off at 1900 h, r o o m t e m p e r a t u r e 24 C, l a b o r a t o r y c h o w a n d water ad lib, food and w a t e r c o n t a i n e r s r e p l e n i s h e d daily. Animals were handled daily and weighed by the same p e r s o n a n d the f o o d intake was also measured. A f t e r this first w e e k the a n i m a l s were divided into t w o groups, a C o n t r o l G r o u p (C) w i t h free a c c e s s to food, a n d a Food Shift G r o u p (FS) w i t h a c c e s s to food o n l y f r o m 0900 h to II00 h. O n the 21st day, a n i m a l s were sacrificed by decapitation a n d b l o o d w a s c o l l e c t e d at 0800, 1200, 1600, 2000, 2400, 0400 a n d 0800 h into tubes containing enzyme inhibitors, i0 ul Pepstatin A (500 uM), i0 ul p h e n y l m e t h y l s u l f o n i l f l u o r i d e (0,001 M), EDTA 1 mg/ml of blood for ANP determination, and into s e p a r a t e t u b e s for B d e t e r m i n a t i o n . P l a s m a B was d e t e r m i n e d b y a p r e v i o u s l y d e s c r i b e d R I A (9). The l o w e r l i m i t of s e n s i t i v i t y was 13 nmol/L, a n d the i n t r a - a n d i n t e r a s s a y c o e f f i c i e n t s of v a r i a t i o n (CVs) were 2% a n d 8%, r e s p e c t i v e l y . The i m m u n o r e a c t i v e A N P was e x t r a c t e d f r o m 1 ml of p l a s m a b y u s i n g S e p - P a k c a r t r i d g e s (Waters Associates, MilFord, MA) . P l a s m a A N P l e v e l s were measured by R I A as p r e v i o u s l y d e s c r i b e d (2). The mean recovery of s t a n d a r d A N P w a s 75 + 5% (mean + SD) . The intra- a n d interassay CVs were 8% and 12%, respectively. The s e n s i t i v i t y of the a s s a y w a s 2.5 p m o l / L . The antiserum used cross reacts with alpha-h ANP, 100%, rat A N P (Ile 12 a l p h a - h ANP), 100%, rat A t r i o p e p t i n III, 100%, ANP (8-33) (Ile 12 a l p h a - h A N P 3-28), 90%, ANP (18-28), 57%, rat ANP (13-28), 50%, rat A t r i o p e p t i n II, 27%, A u r i c u l i n A, 10%, and rat Atriopeptin I, 3%. The data were a n a l y z e d s t a t i s t i c a l l y b y one - w a y a n a l y s i s of v a r i a n c e (ANOVA), b y the W i l c o x o n M a n n - W h i t n e y Test and by the Spearman rank correlation coefficient. Weight and food intake data were a n a l y z e d b y the Student t-test. P v a l u e s less t h a n 0.05 were considered significant.
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Results There was no significant difference between the initial body weight (bw) g, m e a n + SE, of t h e t w o g r o u p s (192.6 + 1.5 x 1 9 4 . 7 + 1.23, C x FS) but the control animals were heavier than their FS counterparts (312.4 + 1.9 vs 246.4 + 2.0, p < 0 . 0 0 0 1 C x FS) at t h e e n d of t h e e x p e r i m e n t . T h e i n i t i a l f o o d i n t a k e (g/day) w a s t h e s a m e f o r the t w o g r o u p s (23.2 + 0.5 x 2 2 . 6 + 0.5, g / d a y , C x FS) . A l t h o u g h t h e FS group showed smaller food ~ntake than the C g r o u p on the 2 1 s t day (24.5 + 0.4 x 1 9 . 0 + 0.4, p < 0 . 0 0 0 1 , C x FS), w h e n the d a i l y i n t a k e w a s - e x p r e s s e d in g/day/100 g bw there was no significant difference in the initial food intake (9.8 + 1.4 x 9.5 + 1.8 g / d a y / 1 0 0 g bw, C x FS) o r in t h e f i n a l f o o d i n t a k e (7.8 ~ 1.0 x 7.7 + 1.3 g/ d a y / i00 g bw, C x FS). F i g u r e 1 s h o w s t h e r e s u l t s of B a n d A N P d e t e r m i n a t i o n s . ANOVA indicated that B varied over time in both groups (F (6,46) = 10.14, p < 0.0001 for C a n d F (6,45) = 5.10, p = 0 . 0 0 0 5 f o r FS) . There was also a time v a r i a t i o n in p l a s m a ANP l e v e l s in both groups (F (6,54) = 3.78, p = 0 . 0 0 3 f o r C a n d F (6,48) = 2.73, p = 0 . 0 2 f o r FS) . P l a s m a B p r e s e n t e d circadian v a r i a t i o n s f r o m 78 + 17 n m o l / l at 0 8 0 0 h to 339 + 79 n m o l / l (mean + SEM) at 2000 h? The restricted feeding r e g i m e n r e s u l t e d in 12 h o u r s h i f t of p e a k B values (395 + 39 vs 125 + 24). T h e d a y p e a k plasma ANP level (pmol/l) w a s a l s o attained at 2 0 0 0 h (68 + 19). T h i s v a l u e w a s higher than the remaining ones obtained throughout the day (2000 h x 0 8 0 0 h p = 0 . 0 0 0 4 , 2 0 0 0 h x 1 2 0 0 h p = 0.001, 2 0 0 0 h x 1 6 0 0 h p = 0.003, 2000 x 2400 h p = 0.04 and 2000 h x 0400 h p = 0.009). FS rats presented the highest ANP l e v e l at 0 8 0 0 h. However, this value was only significantly higher than the 1200 h value (p = 0.01) . In a d d i t i o n t h e r e w a s a p o s i t i v e c o r r e l a t i o n between ANP and B levels o b t a i n e d in t h e s a m e p l a s m a s a m p l e s f o r the C o n t r o l g r o u p (r = 0.39, p = 0.003) a n d f o r the FS group (r = 0.37, p = 0 . 0 0 7 1 ) .
Discussion The existence of a circadian rhythm of ANP in humans is s t i l l somewhat controversial. Nevertheless, the prevalent e v i d e n c e is t h a t in d i u r n a l l y a c t i v e s u b j e c t s , f o l l o w i n g a n o r m a l p a t t e r n of a c t i v i t y - r e s t , food and sleep at n i g h t , A N P has a circadian variation with a peak at 0 4 0 0 h, both in n o r m a l volunteers (10-14) a n d in h y p e r t e n s i v e (14) and congestive heart failure patients(15) . Contradictory results may reflect different ways of time sampling (16), p o s t u r e (17, 18), d i e t (16, 18), water intake, collection and h a n d l i n g of s a m p l e s (16, 18), a n d assays. Pulsatility has also been described for ANP and can both m i m i c a n d c o n c e a l u l t r a d i a n r h y t h m for A N P (19). N o studies of this type have been carried out on animals. Our results d e m o n s t r a t e t h e w e l l k n o w n c i r c a d i a n r h y t h m of p l a s m a B in r a t s w h i c h i n c r e a s e s b e f o r e the beginning of dark activity period, and peaks at 2 0 0 0 h. We also c o n f i r m e d as previously described (20, 21), t h a t B l e v e l s s h o w a 12 h o u r s h i f t when the f o o d is r e s t r i c t e d f r o m 0 9 0 0 to i i 0 0 h. T h e s e f i n d i n g s
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Fig. 1 periodicity of plasma B (IA) or ANP (IB) control rats under a d lib feeding ( ) or restricted feeding schedule (---) . V a l u e s + SEM. Solid bars indicate t i m e of d a r k n e s s .
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r e p r e s e n t e d the g o l d s t a n d a r d for the c o n t r o l of the e x p e r i m e n t . A N P l e v e l s in the c o n t r o l g r o u p also s h o w e d a p e a k at 2000 h, a v a l u e that was h i g h e r t h a n the r e m a i n i n g ones t h r o u g h o u t the day. Moreover, the circadian variation in ANP changed with the manipulation of r h y t h m by p h a s e - s h i f t of a s y n c h r o n i z e r as the food schedule ( r e s t r i c t i o n from 0900 to ii00 h) . Although there are c o n f l i c t i n g d a t a about the role of food a m o u n t for p r e f e e d i n g B p e a k s in rats (20, 22), in o u r e x p e r i m e n t there was no s i g n i f i c a n t d i f f e r e n c e in food intake (g/day/100 g bw) between restricted and ad lib fed animals. T h e r e f o r e we cannot e x p l a i n the v a r i a t i o n s in h o r m o n a l levels s o l e l y on this basis. S i n c e food r e s t r i c t i o n induces a d i s r u p t i o n of activityrest a n d s l e e p - w a k e p a t t e r n s w i t h c h a n g e s in h i p p o c a m p a l content of n o r e p i n e p h r i n e a n d serotonin, a n d in the c o r t i c a l c o n t e n t of serotonin (20), C R F - r e l a t e d n e u r o t r a n s m i t t e r s i n v o l v e d in f e e d i n g behavior (23), more studies are necessary to clarify the interactions among different neuropeptides (CRF,CCK, n e u r o t e n s i n , Pancreatic Polypeptide, galanin, ANP) and the c h a n g e s observed. The correlation between ANP and B under the experimental conditions used may have resulted from a common regulatory mechanism for both hormones as well as from a regulatory i n f l u e n c e of one hormone upon the other. Since in rats PRA, Aldosterone and B p e a k at the b e g i n n i n g of the dark activity p e r i o d (20,24,25), the s i m u l t a n e o u s i n c r e a s e of A N P m a y indicate a possible interaction between ANP and renin secretion as proposed by Donckier et al. (i0) . F u r t h e r support for the possibility that the circadian ANP concentration modulates plasma renin c o n c e n t r a t i o n is p r o v i d e d by the fact that when a p e r s o n is kept r e c u m b e n t for 24 hours and there is no n o c t u r n a l p e a k of A N P (17, 18), r e n i n c o n c e n t r a t i o n t h e n rises d u r i n g the n i g h t (26, 27). If not causal, this time relationship is very i m p o r t a n t due to the fact that ANP, just like r e n i n and cortisol, is a n t i c i p a t o r y in its p e r i o d i c c i r c a d i a n rise p r i o r to a w a k e n i n g in p r e p a r a t i o n for the day's a c t i v i t i e s (28). We additionally demonstrated a significant correlation between plasma ANP and B concentrations in both groups. Glucocorticoids s t i m u l a t e m y o c a r d i a l A N P gene a c t i v i t y (29), and secretion of ANP in vitro by cardiac myocytes (30), or hypothalamic neurones (31) in v i t r o and in v i v o in m a n (32, 33) or in rats (34). This s t i m u l a t i n g glucocorticoid e f f e c t on A N P s e c r e t i o n m a y be c o n s i s t e n t w i t h a p o s s i b l e hypothalamic-cardioa d r e n a l f e e d b a c k c o n t r o l mechanism, w h e r e b y A N P m a y be modulated by glucocorticoids and/or play a role in modulating the a c t i v a t i o n of the h y p o t h a l a m i c p i t u i t a r y a d r e n a l s y s t e m at one or m o r e levels. In c o n c l u s i o n , o u r r e s u l t s d e m o n s t r a t e that the circadian variations of ANP and B in rats on an ad lib or r e s t r i c t e d feeding regimen are parallel. Since the homeostasis of physiological functions usually relies on an interplay of antagonist factors, ANP may play a role in a putative hypothalamic-cardio-adrenal axis. The n a t u r e and m e a n i n g of these interactions deserve further investigation.
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Acknowledgements The authors thank Miss Adriana Rossi, Miss Marina Holanda a n d Mr. A d a l b e r t o Verceze for technical assistance and Miss Ana Cristina C. P e r e i r a for secretarial assistance. This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnol~gico, and HCFMRP-FAEPA.
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Pergamon Press
C I R C A D I A N V A R I A T I O N S OF P L A S M A A T R I A L N A T R I U R E T I C A N D C O R T I C O S T E R O N E IN R A T S W I T H C O N T I N U O U S O R R E S T R I C T E D A C C E S S TO F O O D M a n u e l H.A. O l i v e i r a , A n g e l a M.O. Leal, L u c i l a
PEPTIDE
Jos~ Antunes-Rodrigues (i), L.K. E l i a s a n d A y r t o n C. M o r e i r a
D i v i s i o n of E n d o c r i n o l o g y , D e p a r t m e n t of M e d i c i n e a n d D e p a r t m e n t of P h y s i o l o g y (I), F a c u l t y of M e d i c i n e 1 4 0 4 8 - 9 0 0 , R i b e i r a o Preto, SP, Brazil.
(Receivedinfinal~rm October6,1993) Summary In the p r e s e n t s t u d y p l a s m a A t r i a l N a t r i u r e t i c Peptide (ANP) a n d C o r t i c o s t e r o n e (B) l e v e l s w e r e d e t e r m i n e d in 2 g r o u p s of W i s t a r rats, a C o n t r o l g r o u p (C) w i t h free access to food, a n d a Food Shift group (FS) with a c c e s s to f o o d o n l y f r o m 0900 to ii00 h for 2 weeks. Blood samples were collected by d e c a p i t a t i o n at 4 h o u r intervals. ANOVA indicated that B v a r i e d o v e r t i m e in b o t h g r o u p s (F (6,46) = 10.14, p < 0.0001 for C a n d F (6,45) = 5.10, p = 0.0005 for FS) . T h e r e w a s a l s o time variation in plasma ANP levels in both g r o u p s (F (6,54) = 3.78, p = 0.003 for C a n d F (6,48) = 2.73, p = 0.02 for FS group). P l a s m a B presented circadian variations f r o m 78 + 17 n m o l / l (mean + SEM) at 0800 h to 339 + 79 n m o l / l at 2000 h. The day peak plasma ANP l e v e l (pmol/l) was a l s o a t t a i n e d at 2000 h (68 + 19). This value was higher than all others measured t h r o u g h o u t the day. The restricted feeding regimen r e s u l t e d in a 12 h o u r shift of peak B values (395 + 39 vs 125 + 24). The FS rats p r e s e n t e d the h i g h e s t A N P level at 5 8 0 0 h. The p r e s e n t study indicates similar circadian v a r i a t i o n s of p l a s m a A N P a n d B in rats o n an ad lib or restricted feeding regimen. ANP and B secretion may o c c u r in a n t i c i p a t i o n of the feeding p e r i o d or day activity. A t r i a l n a t r i u r e t i c peptide, A N P (99-126) is s y n t h e s i z e d and r e l e a s e d f r o m m a m m a l i a n c a r d i a c a t r i a a n d has s e v e r a l a c t i o n s on fluid and electrolyte homeostasis.Thus, systemic administration of ANP results in n a t r i u r e s i s and diuresis and a simultaneous decrease in p l a s m a renin, a n g i o t e n s i n a c t i v i t y and aldosterone secretion. Moreover ANP has been shown to be an effective i n h i b i t o r of v a s o p r e s s i n s e c r e t i o n a n d of salt a n d water intake in the rat (1,2). P e p t i d e s r e l a t e d to A N P are also synthesized l o c a l l y w i t h i n the C e n t r a l N e r v o u s S y s t e m ( C N S ) , w i t h p a r t i c u l a r l y high concentrations p r e s e n t in the hypothalamus, where atrial peptides m a y f u n c t i o n as n e u r o t r a n s m i t t e r s or neuromodulators. Recent studies have a l s o s u g g e s t e d that A N P m a y suppress the .
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A y r t o n C. M o r e i r a , Medicine 14048-900,
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MD, Division of Endocrinology, R i b e i r a o Preto, SP, Brazil. ~24-3~5/93 ~.00 + . ~
Copyri~t©1993PergamonPressLtd Allrightsrese~ed.
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hypothalamus-pituitary a d r e n a l axis b y i n t e r a c t i n g w i t h CRF 41 or by inhibiting pituitary corticotropin secretion (3 7). The role of ANP in a c u t e v o l u m e expansion has been extensively studied, but the i n f l u e n c e of A N P d u r i n g n o r m a l d a y - t o - d a y l i v i n g has b e e n not a d e q u a t e l y d e m o n s t r a t e d . It is w e l l e s t a b l i s h e d that the rhythmicity of the h y p o t h a l a m u s - p i t u i t a r y - a d r e n a l axis is r e l a t e d to p o s s i b l e n e u r a l mechanisms involved in sleep-wake, rest-activity, light-dark or feeding cycles. R a t s maintained under ad lib feeding or daytime food restriction manifest a circadian peak of p l a s m a B just p r i o r to the time of o n s e t of predominant food intake (8). Diurnal rhythm of plasma ANP c o n c e n t r a t i o n s w a s o b s e r v e d in normal volunteers or patients. However, detailed information on ANP periodicity in rats is scanty. Thus, we d e c i d e d to s t u d y the d a i l y v a r i a t i o n of plasma A N P in rats a n d the role of f o o d r e s t r i c t i o n on its s e c r e t i o n .
Methods Studies were p e r f o r m e d on 144 s i n g l y housed adult male Wistar rats weighing 200 g w h i c h had been acclimated to our l a b o r a t o r y for one week. L i g h t s on at 0700 h, off at 1900 h, r o o m t e m p e r a t u r e 24 C, l a b o r a t o r y c h o w a n d water ad lib, food and w a t e r c o n t a i n e r s r e p l e n i s h e d daily. Animals were handled daily and weighed by the same p e r s o n a n d the f o o d intake was also measured. A f t e r this first w e e k the a n i m a l s were divided into t w o groups, a C o n t r o l G r o u p (C) w i t h free a c c e s s to food, a n d a Food Shift G r o u p (FS) w i t h a c c e s s to food o n l y f r o m 0900 h to II00 h. O n the 21st day, a n i m a l s were sacrificed by decapitation a n d b l o o d w a s c o l l e c t e d at 0800, 1200, 1600, 2000, 2400, 0400 a n d 0800 h into tubes containing enzyme inhibitors, i0 ul Pepstatin A (500 uM), i0 ul p h e n y l m e t h y l s u l f o n i l f l u o r i d e (0,001 M), EDTA 1 mg/ml of blood for ANP determination, and into s e p a r a t e t u b e s for B d e t e r m i n a t i o n . P l a s m a B was d e t e r m i n e d b y a p r e v i o u s l y d e s c r i b e d R I A (9). The l o w e r l i m i t of s e n s i t i v i t y was 13 nmol/L, a n d the i n t r a - a n d i n t e r a s s a y c o e f f i c i e n t s of v a r i a t i o n (CVs) were 2% a n d 8%, r e s p e c t i v e l y . The i m m u n o r e a c t i v e A N P was e x t r a c t e d f r o m 1 ml of p l a s m a b y u s i n g S e p - P a k c a r t r i d g e s (Waters Associates, MilFord, MA) . P l a s m a A N P l e v e l s were measured by R I A as p r e v i o u s l y d e s c r i b e d (2). The mean recovery of s t a n d a r d A N P w a s 75 + 5% (mean + SD) . The intra- a n d interassay CVs were 8% and 12%, respectively. The s e n s i t i v i t y of the a s s a y w a s 2.5 p m o l / L . The antiserum used cross reacts with alpha-h ANP, 100%, rat A N P (Ile 12 a l p h a - h ANP), 100%, rat A t r i o p e p t i n III, 100%, ANP (8-33) (Ile 12 a l p h a - h A N P 3-28), 90%, ANP (18-28), 57%, rat ANP (13-28), 50%, rat A t r i o p e p t i n II, 27%, A u r i c u l i n A, 10%, and rat Atriopeptin I, 3%. The data were a n a l y z e d s t a t i s t i c a l l y b y one - w a y a n a l y s i s of v a r i a n c e (ANOVA), b y the W i l c o x o n M a n n - W h i t n e y Test and by the Spearman rank correlation coefficient. Weight and food intake data were a n a l y z e d b y the Student t-test. P v a l u e s less t h a n 0.05 were considered significant.
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Results There was no significant difference between the initial body weight (bw) g, m e a n + SE, of t h e t w o g r o u p s (192.6 + 1.5 x 1 9 4 . 7 + 1.23, C x FS) but the control animals were heavier than their FS counterparts (312.4 + 1.9 vs 246.4 + 2.0, p < 0 . 0 0 0 1 C x FS) at t h e e n d of t h e e x p e r i m e n t . T h e i n i t i a l f o o d i n t a k e (g/day) w a s t h e s a m e f o r the t w o g r o u p s (23.2 + 0.5 x 2 2 . 6 + 0.5, g / d a y , C x FS) . A l t h o u g h t h e FS group showed smaller food ~ntake than the C g r o u p on the 2 1 s t day (24.5 + 0.4 x 1 9 . 0 + 0.4, p < 0 . 0 0 0 1 , C x FS), w h e n the d a i l y i n t a k e w a s - e x p r e s s e d in g/day/100 g bw there was no significant difference in the initial food intake (9.8 + 1.4 x 9.5 + 1.8 g / d a y / 1 0 0 g bw, C x FS) o r in t h e f i n a l f o o d i n t a k e (7.8 ~ 1.0 x 7.7 + 1.3 g/ d a y / i00 g bw, C x FS). F i g u r e 1 s h o w s t h e r e s u l t s of B a n d A N P d e t e r m i n a t i o n s . ANOVA indicated that B varied over time in both groups (F (6,46) = 10.14, p < 0.0001 for C a n d F (6,45) = 5.10, p = 0 . 0 0 0 5 f o r FS) . There was also a time v a r i a t i o n in p l a s m a ANP l e v e l s in both groups (F (6,54) = 3.78, p = 0 . 0 0 3 f o r C a n d F (6,48) = 2.73, p = 0 . 0 2 f o r FS) . P l a s m a B p r e s e n t e d circadian v a r i a t i o n s f r o m 78 + 17 n m o l / l at 0 8 0 0 h to 339 + 79 n m o l / l (mean + SEM) at 2000 h? The restricted feeding r e g i m e n r e s u l t e d in 12 h o u r s h i f t of p e a k B values (395 + 39 vs 125 + 24). T h e d a y p e a k plasma ANP level (pmol/l) w a s a l s o attained at 2 0 0 0 h (68 + 19). T h i s v a l u e w a s higher than the remaining ones obtained throughout the day (2000 h x 0 8 0 0 h p = 0 . 0 0 0 4 , 2 0 0 0 h x 1 2 0 0 h p = 0.001, 2 0 0 0 h x 1 6 0 0 h p = 0.003, 2000 x 2400 h p = 0.04 and 2000 h x 0400 h p = 0.009). FS rats presented the highest ANP l e v e l at 0 8 0 0 h. However, this value was only significantly higher than the 1200 h value (p = 0.01) . In a d d i t i o n t h e r e w a s a p o s i t i v e c o r r e l a t i o n between ANP and B levels o b t a i n e d in t h e s a m e p l a s m a s a m p l e s f o r the C o n t r o l g r o u p (r = 0.39, p = 0.003) a n d f o r the FS group (r = 0.37, p = 0 . 0 0 7 1 ) .
Discussion The existence of a circadian rhythm of ANP in humans is s t i l l somewhat controversial. Nevertheless, the prevalent e v i d e n c e is t h a t in d i u r n a l l y a c t i v e s u b j e c t s , f o l l o w i n g a n o r m a l p a t t e r n of a c t i v i t y - r e s t , food and sleep at n i g h t , A N P has a circadian variation with a peak at 0 4 0 0 h, both in n o r m a l volunteers (10-14) a n d in h y p e r t e n s i v e (14) and congestive heart failure patients(15) . Contradictory results may reflect different ways of time sampling (16), p o s t u r e (17, 18), d i e t (16, 18), water intake, collection and h a n d l i n g of s a m p l e s (16, 18), a n d assays. Pulsatility has also been described for ANP and can both m i m i c a n d c o n c e a l u l t r a d i a n r h y t h m for A N P (19). N o studies of this type have been carried out on animals. Our results d e m o n s t r a t e t h e w e l l k n o w n c i r c a d i a n r h y t h m of p l a s m a B in r a t s w h i c h i n c r e a s e s b e f o r e the beginning of dark activity period, and peaks at 2 0 0 0 h. We also c o n f i r m e d as previously described (20, 21), t h a t B l e v e l s s h o w a 12 h o u r s h i f t when the f o o d is r e s t r i c t e d f r o m 0 9 0 0 to i i 0 0 h. T h e s e f i n d i n g s
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r e p r e s e n t e d the g o l d s t a n d a r d for the c o n t r o l of the e x p e r i m e n t . A N P l e v e l s in the c o n t r o l g r o u p also s h o w e d a p e a k at 2000 h, a v a l u e that was h i g h e r t h a n the r e m a i n i n g ones t h r o u g h o u t the day. Moreover, the circadian variation in ANP changed with the manipulation of r h y t h m by p h a s e - s h i f t of a s y n c h r o n i z e r as the food schedule ( r e s t r i c t i o n from 0900 to ii00 h) . Although there are c o n f l i c t i n g d a t a about the role of food a m o u n t for p r e f e e d i n g B p e a k s in rats (20, 22), in o u r e x p e r i m e n t there was no s i g n i f i c a n t d i f f e r e n c e in food intake (g/day/100 g bw) between restricted and ad lib fed animals. T h e r e f o r e we cannot e x p l a i n the v a r i a t i o n s in h o r m o n a l levels s o l e l y on this basis. S i n c e food r e s t r i c t i o n induces a d i s r u p t i o n of activityrest a n d s l e e p - w a k e p a t t e r n s w i t h c h a n g e s in h i p p o c a m p a l content of n o r e p i n e p h r i n e a n d serotonin, a n d in the c o r t i c a l c o n t e n t of serotonin (20), C R F - r e l a t e d n e u r o t r a n s m i t t e r s i n v o l v e d in f e e d i n g behavior (23), more studies are necessary to clarify the interactions among different neuropeptides (CRF,CCK, n e u r o t e n s i n , Pancreatic Polypeptide, galanin, ANP) and the c h a n g e s observed. The correlation between ANP and B under the experimental conditions used may have resulted from a common regulatory mechanism for both hormones as well as from a regulatory i n f l u e n c e of one hormone upon the other. Since in rats PRA, Aldosterone and B p e a k at the b e g i n n i n g of the dark activity p e r i o d (20,24,25), the s i m u l t a n e o u s i n c r e a s e of A N P m a y indicate a possible interaction between ANP and renin secretion as proposed by Donckier et al. (i0) . F u r t h e r support for the possibility that the circadian ANP concentration modulates plasma renin c o n c e n t r a t i o n is p r o v i d e d by the fact that when a p e r s o n is kept r e c u m b e n t for 24 hours and there is no n o c t u r n a l p e a k of A N P (17, 18), r e n i n c o n c e n t r a t i o n t h e n rises d u r i n g the n i g h t (26, 27). If not causal, this time relationship is very i m p o r t a n t due to the fact that ANP, just like r e n i n and cortisol, is a n t i c i p a t o r y in its p e r i o d i c c i r c a d i a n rise p r i o r to a w a k e n i n g in p r e p a r a t i o n for the day's a c t i v i t i e s (28). We additionally demonstrated a significant correlation between plasma ANP and B concentrations in both groups. Glucocorticoids s t i m u l a t e m y o c a r d i a l A N P gene a c t i v i t y (29), and secretion of ANP in vitro by cardiac myocytes (30), or hypothalamic neurones (31) in v i t r o and in v i v o in m a n (32, 33) or in rats (34). This s t i m u l a t i n g glucocorticoid e f f e c t on A N P s e c r e t i o n m a y be c o n s i s t e n t w i t h a p o s s i b l e hypothalamic-cardioa d r e n a l f e e d b a c k c o n t r o l mechanism, w h e r e b y A N P m a y be modulated by glucocorticoids and/or play a role in modulating the a c t i v a t i o n of the h y p o t h a l a m i c p i t u i t a r y a d r e n a l s y s t e m at one or m o r e levels. In c o n c l u s i o n , o u r r e s u l t s d e m o n s t r a t e that the circadian variations of ANP and B in rats on an ad lib or r e s t r i c t e d feeding regimen are parallel. Since the homeostasis of physiological functions usually relies on an interplay of antagonist factors, ANP may play a role in a putative hypothalamic-cardio-adrenal axis. The n a t u r e and m e a n i n g of these interactions deserve further investigation.
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Acknowledgements The authors thank Miss Adriana Rossi, Miss Marina Holanda a n d Mr. A d a l b e r t o Verceze for technical assistance and Miss Ana Cristina C. P e r e i r a for secretarial assistance. This work was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnol~gico, and HCFMRP-FAEPA.
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