- Email: [email protected]
Cyclic 3′, 5′-adenosine monophosphate and cyclic 3′, 5′-guanosine monophosphate metabolism in malnutrition
NUTRITION RESEARCH, Vol. 6, pp. 589-599, 1986 0271-5317/86 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
CYCLIC 3',5'-ADENOSINE MONOPHOSPHATE AND C Y C L I C 3 ' , 5 ' - G U A N O S I N E M O N O P H O S P H A T E M E T A B O L I S H IN M A L N U T R I T I O N Uma S. S r i v a s t a v a , DSc, M a n o h a r Lal Thakur, MSc and C. Spach%, DSc D e p a r t m e n t of N u t r i t i o n , u n i v e r s i t y of M o n t r e a l , F a c u l t y of M e d i c i n e , M o n t r e a l , Quebec, C a n a d a H3C 3J7 and + C e n t r e Marcel Delepine, C.N.R.S. 45045 Orleans Cedex, France
ABSTRACT
When the concentration and turnover of t h e c y c l i c nucleotides cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate (cGHP) w e r e m e a s u r e d in the thymus, s p l e e n and c e r v i c a l l y m p h n o d e s of r a t s s u b j e c t e d to v a r i o u s m o d e l s of m a l n u t r i t i o n , and these same parameters of c A M P a l o n e a s s e s s e d in t h e i r liver, k i d n e y and brain, u n d e r n u t r i t i o n has b e e n found to m o d i f y c y c l i c n u c l e o t i d e m e t a b o l i s m . When the r e s u l t s on n u c l e i c a c i d and p r o t e i n m e t a b o l i s m are t a k e n into consideration w i t h the c h a n g e s in c y c l i c nucleotide metabolism, it was n o t e d t h a t the v a r i o u s p h a s e s of the c e l l c y c l e are p r o l o n g e d or b l o c k e d in the o r g a n s of malnourished rats. These data indicate a correlatzon b e t w e e n c y c l i c n u c l e o t i d e m e t a b o l z s m and impaired immune r e s p o n s i v e n e s s seen c o m m o n l y in u n d e r n o u r i s h e d rats. Key Words:
Cyclic nucleotides, metabolism, protein deficiency, lymphoid organs, rats
It is w e l l - k n o w n that protein deficient diets (PDDs) and malnutrition impair such p h y s i o l o g i c a l functions of l y m p h o c y t e s as immune and m i t o g e n i c r e s p o n s i v e n e s s , p r o l i f e r a t i o n , v i a b i l i t y , ionic exchange, ATPase activity, membrane integrity (1-6), and i m m u n o c o m p e t e n c e (7,8). Many i n v e s t i g a t o r s (5,6,9,10) h a v e reported that c A M P l e v e l s in l y m p h o c y t e s could act as h o r m o n a l m e d i a t o r s and p l a y an i m p o r t a n t r o l e , e s p e c l a l l y in c e l l u l a r proliferatzon and l y m p h o c y t e r e s p o n s e to a c t i v a t o r s or i n h i b i t o r s . Moreover, cAMP concentrations are f o u n d to i n c r e a s e in m i c r o b i a l cultures after s e r u m or a m i n o a c i d d e p r i v a t i o n b u t r e t u r n to n o r m a l a f t e r the a d d i t i o n of t h e s e n u t r i e n t s (ii). It h a s b e e n p o s t u l a t e d that the availability of n u t r i e n t s represents a major stimulus of c A M P m o d u l a t i o n , a h y p o t h e s i s w h i c h has been c o n f i r m e d by in v i v o and in v i t r o e x p e r i m e n t s (12-14). G o l d b e r g et al. (15) h a v e s u g g e s t e d that c G M P m i g h t also p l a y a s i g n i f i c a n t r o l e in p h y s i o l o g i c a l f u n c t i o n s either p a r a l l e l to or together with cA~4P ("dual hypothesis").
Correspondence: Uma S. Srivastava, Professor of F u n d a m e n t a l Nutrition, Department of N u t r i t i o n , University of M o n t r e a l , C.P. 6128, S u c c u r s a l e A, H o n t r e a l , Quebec, C a n a d a H3C 3J7.
589
U.S. SRIVASTAVA et al.
590
Effect
of PDDs on C y c l i c
Nucleotide
Metabolism
in Rat L y m p h o c y t e s
P D D s for 36 d a y s do not a l t e r c A M P l e v e l s in t h y m o c y t e s b u t e l e v a t e them in s p l e n i c and e s p e c i a l l y in l y m p h n o d e l y m p h o c y t e s (12). Such d i e t s e l i c i t an i n c r e a s e in the q u a n t i t y of 3 H - c A M P s y n t h e s i z e d during incubation with 2-3H-adenosine, i n d i c a t i n g an i n c r e m e n t in a d e n y l a t e c y c l a s e a c t i v i t y in thymocytes as w e l l as in splenic and lymphnode lymphocytes. T h i s is p r o b a b l y a l s o the c a s e w i t h c A M P phosphodiesterase activity, as s u g g e s t e d by an a u g m e n t a t i o n of r a d i o a c t i v e 5'-AMP and adenosine content. The enhanced a c t i v i t i e s of b o t h e n z y m e s are s i m i l a r in the t h y m u s w h i c h e x p l a i n s the m a i n t e n a n c e of normal c A M P l e v e l s after PDDs. The rise in a d e n y l a t e cyclase activities in s p l e e n and l y m p h n o d e lymphocytes is m o r e p r o n o u n c e d than the e l e v a t i o n of p h o s p h o d i e s t e r a s e activities. This i m b a l a n c e therefore leads to heightened c A M P c o n c e n t r a t i o n s induced by PDDs in the latter two organs (spleen and lymphnodes). It is n o w w e l l - e s t a b l i s h e d that an i n v e r s e r e l a t i o n s h i p exists between intracellular cAMP content and a d e n y l a t e cyclase and p h o s p h o d i e s t e r a s e a c t i v i t i e s on the one hand and the m i t o t i c rate of c o r r e s p o n d i n g c e l l s on the other. The r e s u l t s just described favor the h y p o t h e s i s that the t u r n o v e r of c A M P is i n v e r s e l y related to the proliferative potential of t h e s e t h r e e o r g a n s (thymus, spleen, lymphnodes) and, c o n s e q u e n t l y , to t h e i r c a p a c i t y to r e s i s t the stress of d i e t a r y protein d e f i c i e n c y (16). Indeed, the increase of newly-formed cAMP, d u e to p r o t e i n d e f i c i e n c y , is a l l the m o r e important s i n c e the w e i g h t and c e l l p o p u l a t i o n of the l y m p h o i d organs and the v i a b i l i t y of c o r r e s p o n d i n g l y m p h o c y t e s are decreased. A l l t h e s e d e f e c t s are p a r t i c u l a r l y m a r k e d in the t h y m u s w i t h l e s s pronounced manifestations in the l y m p h n o d e s (1,2). Effect of P D D s on c A M P and cGMP Levels in R a t U n f r a c t i o n a t e d S p l e e n L y m p h o c y t e s and T and B C e l l s
Thymoc~[tes__ L
The other p h y s i o l o g i c a l c y c l i c n u c l e o t i d e , cGMP, has a l s o been found to p l a y a p r o m i n e n t r o l e in c e l l u l a r function. For e x a m p l e , the i n h i b i t i o n of i m m u n e r e s p o n s i v e n e s s by thymectomy is r e v e r s e d in v i t r o by the a d d i t i o n of c G M P (17). It is e v e n s u g g e s t e d that the cAMP/cGMP r a t i o m a y be m o r e important t h a n e a c h of t h e t w o nucleotides in the r e g u l a t i o n of c e l l u l a r metabolism and in c e l l reactions to a c t i v a t o r s or i n h i b i t o r s (17). In a d d i t i o n , many reports d o c u m e n t the n e c e s s a r y c o o p e r a t i o n of two major categories of l y m p h o c y t e s in i m m u n e r e s p o n s i v e n e s s , the t h y m u s - d e r i v e d T l y m p h o c y t e s and the t h y m u s - i n d e p e n d e n t B lymphocytes. It has b e e n n o t e d t h a t P D D s do not i n f l u e n c e the c A M P c o n t e n t of thymus cells but largely increase it in u n f r a c t i o n a t e d spleen lymphocytes (13). Interestingly, the t h y m o c y t e c A M P l e v e l s are quantitatively l o w e r t h a n in s p l e n i c l y m p h o c y t e s . These changes could be i n v e r s e l y correlated to m i t o t i c activity which is relatively h i g h e r in the t h y m u s t h a n in the s p l e e n or l y m p h n o d e s . The a p p a r e n t a b s e n c e of an e f f e c t of P D D s on t h y m o c y t e s is a l s o demonstrated by the c G M P l e v e l s and c o n s e q u e n t l y by the c A M P / c G M P ratio, cGMP concentrations are h i g h e r in u n f r a c t i o n a t e d spleen lymphocytes t h a n in t h y m o c y t e s , the d i f f e r e n c e between the two organs being e v e n more pronounced than with cAMP. C o n s e q u e n t l y , the c A M P / c G M P ratio is lower in spleen c e l l s than in thymus cells. The c G M P l e v e l s are d i m i n i s h e d after p r o t e i n d e f i c i e n c y in unseparated s p l e n i c c e l l s and s p l e n i c T l y m p h o c y t e s , w h e r e a s the r i s e in c A M P content of u n s e p a r a t e d s p l e n i c c e l l s is found o n l y in B lymphocytes.
cAMP AND cGMP IN MALNUTRITION Thus, the c A M P / c G M P ratio is i n c r e a s e d l y m p h o c y t e extracts, e s p e c i a l l y in B c e l l s , in c A M P or a l o w e r i n g of c G M P l e v e l s . Effect of PDDs on C o r t i s o n e - i n d u c e d in R a t Thymoc_ytes~ U n f r a c t i o n a t e d Cells
591
in the t h r e e s p l e n i c either b e c a u s e of a rise
C h a n g e s in c A M P and Spleen Lymphocytes
cGMP Levels and T a n d B
Protein deprivation primarily destroys short-living, cortisones e n s i t i v e c o r t i c a l t h y m o c y t e s , w h i c h m a y be p a r t i c u l a r l y r i c h in c G M P in v i e w of t h e i r h i g h m i t o t i c a c t i v i t y (18). During protein d e f i c i e n c y , the few s u r v i v i n g , v i r t u a l l y m i t o t i c a l l y - i n a c t i v e cells s h o u l d c o n t a i n less c G M P and d i s p l a y an increased c A M P / c G M P ratio a c c o r d i n g to the "dual h y p o t h e s i s " of a r e l a t i o n s h i p b e t w e e n the two p a r a m e t e r s ( c A M P / c G M P ratio and c e l l u l a r p r o l i f e r a t i o n ) . W h i l e this is not the c a s e w i t h the t h y m u s (19), the t h e o r y is in a g r e e m e n t with the r e s u l t s obtained for s p l e e n lymphocytes. The cA~P/cGMP r a t i o is i n c r e a s e d after protein deprivation in unfractionated as w e l l as in T a n d e s p e c i a l l y in B s p l e e n lymphocytes. The g r e a t m a j o r i t y of m i t o t i c a l l y - a c t i v e splenic l y m p h o c y t e s are c o r t i s o n e - r e s i s t a n t in contrast to the t h y m o c y t e s (20). Thus, an attempt was made to d e t e r m i n e w h e t h e r a r e l a t i o n s h i p e x i s t s b e t w e e n the r e s p o n s i v e n e s s to c o r t i s o n e and the l e v e l s and p h y s i o l o g i c a l a c t i v i t y of the i n t r a c e l l u l a r c y c l i c n u c l e o t i d e s . The r e s u l t s showed that after a d a i l y injection of c o r t i s o n e acetate (3 m g / 1 0 0 g b o d y w e i g h t ) for 5 d a y s , the c A M P l e v e l s and c A M P / c G M P ratio were higher than in control thymocytes. S i m i l a r l y , the c A ~ P c o n t e n t and c A M P / c G M P ratio were m u c h higher than normal in s p l e n i c lymphocytes at 30 m i n u t e s , i, 4 a n d 24 h o u r s a f t e r the f i r s t c o r t i s o n e injection as w e l l as after 5 days of c o r t i s o n e treatment. E l e v a t e d v a l u e s were a l s o recorded at 30 m i n u t e s and 24 h o u r s in B cells and at 1 h o u r in T c e l l s a f t e r a s i n g l e i n j e c t i o n of the h o r m o n e as w e l l as a f t e r the f i f t h t r e a t m e n t d a y in b o t h T and B l ymphocytes. Thus, c y c l i c n u c l e o t i d e m e t a b o l i s m and the c A M P / c G M P ratio are a p p a r e n t l y m o r e s e n s i t i v e after c o r t i s o n e t r e a t m e n t in the s p l e e n t h a n in the t h y m u s . These results are s i m i l a r to t h o s e r e p o r t e d w i t h PDDs, w h i c h is n o t s u r p r i s i n g s i n c e the e f f e c t s of protein deprivation on i m m u n e or m i t o g e n i c e x p r e s s i o n r e s e m b l e in l a r g e m e a s u r e the c h a n g e s i n d u c e d b y c o r t i s o n e t r e a t m e n t in v i v o (6) . E f f e c t of PDDs on A n t i g e n - i n d u c e d Rat S p l e n i c L y m p h o c y t e s
Changes
in c A M P and c G M P L e v e l s
in
A t h y m u s - d e p e n d e n t antigen d e r i v e d from sheep red b l o o d c e l l s (SRBC) was injected once i n t r a p e r i t o n e a l l y in n o r m a l l y - f e d rats to study the e v o l u t i o n of cACIP and c G M P c o n t e n t in u n f r a c t i o n a t e d as w e l l as in s p l e n i c T and B l y m p h o c y t e s (14). A s i g n i f i c a n t fall in cA~4P l e v e l s occurred at 30 m i n u t e s and 1 hour after a d m i n i s t r a t i o n of the a n t i g e n . A t r a n s i e n t rise was noted at 2 h o u r s , b u t t h e s e v a l u e s r e t u r n e d to n o r m a l at 4 and 24 h o u r s . A second increase was o b s e r v e d on day 3 but again returned to normal after 5 days. The c G M P l e v e l s were b e l o w control v a l u e s until the f o u r t h hour, t h e n r o s e to the n o r m on d a y i, and p e a k e d on d a y 3. As w i t h c A M P , the c A M P / c G M P ratio was first depressed u n t i l the first hour, reached a peak at 2 hours, but then fell again a l t h o u g h it remained a b o v e initial v a l u e s until the end of the experiment.
592
U.S. SRIVASTAVA et a l .
As s h o w n p r e v i o u s l y , P D D s r e d u c e d a n d d e l a y e d the a n t i g e n - i n d u c e d changes in c y c l i c nucleotide content in u n f r a c t i o n a t e d spleen lymphocytes (13). Thus, the f i r s t significant alteration (a s t r i k i n g d r o p in c A M P 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 o n l y 24 h o u r s after the a n t i g e n injection. These levels were subsequently e l e v a t e d but r e m a i n e d b e l o w initial v a l u e s until day i0. A moderate b u t l a t e i n c r e a s e in c G M P c o n t e n t w a s r e c o r d e d i0 d a y s a f t e r S R B C treatment. The c A ~ P / c G M P r a t i o was s o m e w h a t r e g u l a r l y d e c r e a s e d in c o m p a r i s o n to i n i t i a l v a l u e s , b e i n g a b o u t 50% l o w e r on d a y i0 in protein-deficient r a t s and 35% h i g h e r at the s a m e t i m e in c o n t r o l animals. There were similar significant differences in t h e evolution of c A M P and c G M P l e v e l s in T and B l y m p h o c y t e s from n o r m a l l y - f e d and p r o t e i n - d e p r i v e d rats. The e a r l y rise in c A M P c o n c e n t r a t i o n s in u n f r a c t i o n a t e d as w e l l as in T and B c e l l s c o u l d be due to a d e n y l a t e c y c l a s e a c t i v a t i o n which, in n o r m a l rats, m a y be the f i r s t e v e n t of the i m m u n e r e s p o n s e i n d u c e d b y the b i n d i n g of a n t i g e n to s e n s i t i v e c e l l s (21,22). The l a t e r i s e in c G ~ P l e v e l s in u n f r a c t i o n a t e d as w e l l as in T and B c e l l s c o u l d be e l i c i t e d b y t h e c o o p e r a t i v e s y s t e m (T c e l l s ) and c o u l d be the r e s u l t of g u a n y l a t e c y c l a s e a c t i v a t i o n which, in n o r m a l r a t s , m a y be the s e c o n d e v e n t in the i m m u n e r e s p o n s e t r i g g e r e d b y the p r o l i f e r a t i o n of -sensitive cells and the p r o d u c t i o n of antibodies. The i m m u n e r e s p o n s e , at l e a s t to t h y m u s - d e p e n d e n t antigens, would thus d e p e n d upon the c A M P / c G M P ratio. A c c o r d i n g to W a t s o n (23), an optimal ratio is n e c e s s a r y to g e n e r a t e the early, critical b i o c h e m i c a l e v e n t s in the r e s p o n s e of l y m p h o i d organs. This o p t i m a l r a t i o d e p e n d s u p o n the n u m b e r of s p e c i f i c s i t e s of c A ~ P and c G M P p r o t e i n k i n a s e s and of the s u b s t r a t e s a v a i l a b l e to t h e s e e n z y m e s (23) . It, therefore, seems r e a s o n a b l e to suggest that PDDs, like c o r t i s o n e treatment, impair immune responszveness v i a t h e i r e f f e c t s on t h e m e t a b o l i s m of c A M P and c G M P in l y m p h o c y t e s and via their i n f l u e n c e on t h e c A M P / c G M P ratio. Protein-restricted diets suppress two s i g n a l s : the e a r l y rise in cAL~P c o n t e n t and the later e l e v a t i o n of cGMP levels, especzally in s p l e n i c B l y m p h o c y t e s . On the o t h e r hand, after e x p o s u r e to PDDs or c o r t i s o n e treatment, the c A M P / c G M P r a t i o may be too high to a c c o m m o d a t e the i m m u n o l o g i c a l p r o c e s s (23). These data indicate that there may be a relationship b e t w e e n the d i s t u r b i n g impact of PDDs on the e v o l u t i o n of c A M P and c G M P l e v e l s as w e l l as the c A M P / c G M P ratzo in s p l e n i c l y m p h o c y t e s after a n t i g e n sensitization on the one hand, and the i m m u n o d e p r e s s l v e e f f e c t s of this m a l n u t r i t i o n on the other. E f f e c t of U n d e r n u t r i t i o n on the M e t a b o l i s m of c A M P in the T h y m u s and S p l e e n of F e m a l e Rats and Their N e o n a t a l and 2 1 - d a y - o l d P r o g e n y Our e a r l i e r d i s c u s s i o n was m a i n l y c e n t e r e d on the i n f l J e n c e of PDDs on c y c l i c n u c l e o t i d e m e t a b o l i s m in T and B l y m p h o c y t e s and on immune responsiveness in a d u l t rats. The f o c u s w i l l n o w be s h i f t e d to a s c e r t a i n how m a t e r n a l d i e t a r y r e s t r i c t i o n during g e s t a t i o n (G) and periods of g r o w t h , gestation and lactation (GGL) a f f e c t s the m e t a b o l i s m of c y c l i c n u c l e o t i d e s in the thymus and s p l e e n of a d u l t d a m s (D) and their neonatal (N) and 2 1 - d a y - o l d p r o g e n y (P).
cAMP AND cGMP IN MALNUTRITION
593
A T P - A D P and 5 ' - A M P in the t h y m u s and s p l e e n and a d e n o s i n e in the s p l e e n of Group G d a m s do not r e v e a l any m o d i f i c a t i o n s when c o m p a r e d to c o n t r o l a n i m a l s (C). T h e c A M P c o n t e n t of the t h y m u s and s p l e e n and a d e n o s i n e l e v e l s in the thymus increase c o n s i d e r a b l y in Group G. In a d d i t i o n , a l l n u c l e o t i d e s in the t h y m u s and s p l e e n of G G L d a m s are a u g m e n t e d in r e l a t i o n to either G r o u p C or G dams. The concentrations of v a r i o u s a d e n i n e n u c l e o t i d e s are s t r i k i n g l y enhanced in the t h y m u s a n d s p l e e n of the n e o n a t a l p r o g e n y of GGL dams (24). In contrast, the l e v e l s of ATP-ADP, c A M P and 5'-AMP are decreased, w h e r e a s a d e n o s i n e c o n t e n t is increased in the thymus of the n e o n a t a l p r o g e n y of Group G. A T P - A D P and a d e n o s i n e are e l e v a t e d w h i l e c A M P a n d 5 ' - A M P a r e r e d u c e d in the s p l e e n of the n e o n a t a l p r o g e n y of Group G. The concentrations of v a r i o u s nucleotides are augmented in the t h y m u s and s p l e e n of t h e 2 1 - d a y - o l d p r o g e n y of G r o u p s G and GGL. The o n l y e x c e p t i o n s are the A T P - A D P and 5'-AMP l e v e l s of the thymus and the a d e n o s i n e c o n t e n t of the s p l e e n of the 2 1 - d a y - o l d p r o g e n y of G r o u p G in which no c h a n g e s are noted. I n c o r p o r a t i o n d a t a r e v e a l an e n h a n c e d a c c u m u l a t i o n of r a d i o a c t i v i t y zn v a r i o u s n u c l e o t i d e s of the t h y m u s and s p l e e n of G and G G L dams. The s o l e e x c e p t i o n is t h e c A M P f r a c t i o n of the s p l e e n of G r o u p G w h i c h shows no c h a n g e in a c c u m u l a t i o n . S i m i l a r r e s u l t s are obtained with a d e n y l a t e cyclase, c A M P p h o s p h o d i e s t e r a s e and 5 ' - n u c l e o t i d a s e in the t h y m u s and s p l e e n of G and G G L d a m s . The a c t i v i t y of adenylate cyclase is much higher than that of cAMP phosphodiesterase, w h i l e 5 ' - n u c l e o t i d a s e i n c o r p o r a t l o n is zncreased preferentially in r e l a t i o n to t h e o t h e r two e n z y m e s in the t h y m u s and s p l e e n of u n d e r n o u r i s h e d dams. The data demonstrate t h a t the a c c u m u l a t i o n of r a d i o a c t i v i t y in v a r i o u s n u c l e o t i d e f r a c t i o n s is higher in the thymus and s p l e e n of the n e o n a t a l p r o g e n y of GGL d a m s but is l o w e r in G r o u p G (24). S i m i l a r data are obtained on the a c t i v i t y of a d e n y l a t e cyclase, c A M P phosphodiesterase and 5 ' - n u c l e o t i d a s e . As in G G L and G d a m s , adenylate cyclase levels are higher than those of cAMP phosphodiesterase, while 5'-nucleotidase content is i n c r e a s e d preferentially in c o m p a r i s o n to the o t h e r two e n z y m e s in the n e o n a t a l progeny. R a d i o a c t i v i t y studies show a greater a c c u m u l a t i o n of n u c l e o t i d e s in the t h y m u s and s p l e e n of 2 1 - d a y - o l d p r o g e n y of G r o u p s G and GGL. Similar results are noted for adenylate cyclase, cAMP phosphodiesterase and 5'-nucleotidase in the t h y m u s and s p l e e n of dams of b o t h t h e s e g r o u p s . Contrary to w h a t is o b s e r v e d in undernourished dams and thelr neonatal progeny, cAMP p h o s p h o d i e s t e r a s e a c t i v i t y is greater than that of a d e n y l a t e c y c l a s e in the thymus and s p l e e n of the 2 1 - d a y - o l d progeny. The l e v e l s of 5 ' - n u c l e o t i d a s e are higher than those of a d e n y l a t e c y c l a s e but lower than c A M P p h o s p h o d i e s t e r a s e in the thymus and s p l e e n of the progeny. F r o m the d a t a r e p o r t e d h e r e and e l s e w h e r e on n u c l e i c a c i d and protein metabolism in l y m p h o i d o r g a n s of u n d e r n o u r i s h e d d a m s (25) and t h e i r n e o n a t a l (24) and 2 1 - d a y - o l d p r o g e n y (26,27), it a p p e a r s that d i e t a r y r e s t r i c t i o n induces c h a n g e s in the c e l l u l a r g r o w t h of the t h y m u s and s p l e e n , z.e., c e l l diviszon c o u l d be a r r e s t e d p r e m a t u r e l y b y the p r o l o n g a t i o n or b l o c k a g e of v a r i o u s phases of the c e l l c y c l e (28,29), and t h e s e a l t e r a t i o n s c o u l d be r e l a t e d to an
594 impaired
U.S. SRIVASTAVA et al. immune
E f f e c t of Nu--~t~
responsiveness
(12).
Protein-calorie Malnutrition Earl~ C o n t e n t -i-n-Va---~us O r g a n s of Rats
in L i f e
on C~c~i__cc
Early protein-calorie malnutrition a l t e r s c A M P and c G M P l e v e l s d u r i n g the entire 3-week period of l a c t a t i o n in d i f f e r e n t organs of the r a t (30). c A M P c o n t e n t is i n c r e a s e d in the l i v e r , k i d n e y and b r a i n but d e c r e a s e d in the thymus and s p l e e n of pups s u b j e c t e d to 1 week of u n d e r n u t r i t i o n . The b r a i n and k i d n e y v a l u e s of c A M P return to c o n t r o l l e v e l s a f t e r 2 to 3 w e e k s of m a l n u t r i t i o n . The c G M P concentrations a r e a u g m e n t e d in the l i v e r , k i d n e y and t h y m u s , b u t are depressed in the s p l e e n of o f f s p r i n g exposed to 1 w e e k of malnutrition. V a r i a b l e c G M P r e s p o n s e s a r e n o t e d in the d i f f e r e n t o r g a n s after 2 to 3 weeks of u n d e r n u t r i t i o n . The c A M P / c G M P ratio is lower in the l i v e r and thymus but higher in t h e b r a i n a f t e r 1 a n d 2 w e e k s of m a l n u t r i t i o n . A f t e r 3 w e e k s , it r e t u r n s towards control v a l u e s in the l i v e r , but drops in the b r a i n and s t a y s l o w in the t h y m u s . T h e r a t i o r i s e s in the k i d n e y and s p l e e n after 1 w e e k of u n d e r n u t r i t i o n and e x h i b i t s an e l e v a t i o n in these oraans after 3 weeks. S p a c h and A s c h k e n a s y (13) n o t e d a d e c r e a s e in this ratio in T c e l l s of the thymus with an increase in b o t h T and B c e l l s of the s p l e e n of p r o t e i n - d e p r i v e d rats. In the p a s t , it h a s b e e n p o s t u l a t e d t h a t the c A M P / c G M P r a t i o is important in t h e r e g u l a t i o n of c e l l m e t a b o l i s m (16) and t h a t t h e o n s e t of i m m u n o l o g i c a l r e s p o n s i v e n e s s seems to be c o n d i t i o n e d by an o p t i m a l v a l u e of the c e l l u l a r c y c l i c n u c l e o t i d e ratio (13). Indeed, a r e d u c t i o n of c e l l u l a r g r o w t h (blocked m i t o t i c a c t i v i t y ) has been demonstrated in v a r i o u s o r g a n s , s u c h as t h e l i v e r , b r a i n , k i d n e y , t h y m u s a n d s p l e e n , in d i e t a r y - r e s t r i c t e d rats (12,24-29). On the b a s i s of t h e s e r e s u l t s , we c o u l d a s s u m e t h a t m i t o t i c inhibition i n d u c e d by d i e t a r y s t r e s s w o u l d be m u c h m o r e c o n d i t i o n e d by the cAMP/cGMP r a t i o t h a n by o n l y one of t h e s e c y c l i c nucleotides. F u r t h e r m o r e , a v a i l a b l e data on the c A M P / c G M P ratio c l e a r l y indicate that d i e t a r y d e p r i v a t i o n p r o v o k e s a f u n c t i o n a l i m p a i r m e n t of v a r i o u s organs, i n c l u d i n g the lymphoid organs, as attested by d e c r e a s e s or increases in the ratio. These o b s e r v a t i o n s on the l y m p h o i d organs c o n f i r m the r e s u l t s of o t h e r i n v e s t i g a t o r s (12,14), w h o h a v e a l s o r e p o r t e d s u p p r e s s e d immune r e s p o n s i v e n e s s in d i e t a r y - i n s u l t e d rats. E f f e c t of M a l n u t r i t i o n and Kidn-~y
on c A M P
Metabolism
in the R a t L i v e r ,
Brain
We will now mainly dlscuss the m a n n e r in w h i c h u n d e r n u t r i t i o n i n f l u e n c e s the m e t a b o l i c t u r n o v e r of v a r i o u s adenine n u c l e o t i d e s in the l i v e r , brain and k i d n e y of f e m a l e rats (28) and their 2 1 - d a y - o l d progeny. Malnutrition (50% d i e t a r y restriction) during the period of growth, g e s t a t i o n and l a c t a t i o n (19 weeks of life) causes a d e c r e a s e in the l e v e l s of v a r l o u s a d e n i n e n u c l e o t i d e s and n u c l e o s i d e s in the l i v e r with increases in the brain. The A T P - A D P and 5'-AMP c o n c e n t r a t i o n s in the k i d n e y s of u n d e r n o u r l s h e d f e m a l e r a t s do not r e v e a l a n y c h a n g e but c A M P and a d e n o s i n e are m a r k e d l y e l e v a t e d . The l e v e l s of v a r i o u s n u c l e o t i d e s are increased in the liver, brain and kidney of the 2 1 - d a y - o l d p r o g e n y of these dams. Incorporation
studies
demonstrate
that
the
turnover
of
various
cAMP AND cGMP IN MALNUTRITION
595
adenine n u c l e o t i d e s is heightened in the liver, brain and kidney of u n d e r n o u r i s h e d rats. A d e n y l a t e c y c l a s e is m o r e a c t i v e in c o m p a r i s o n to c A M P p h o s p h o d i e s t e r a s e , while 5'-nucleotidase is i n c r e a s e d p r e f e r e n t i a l l y in r e l a t i o n to both these enzymes in the liver, brain and kidney. The m e t a b o l i c t u r n o v e r of v a r i o u s n u c l e o t i d e s is e n h a n c e d in the l i v e r , b r a i n and k i d n e y of the 2 1 - d a y - o l d p r o g e n y of m a l n o u r i s h e d dams. Contrary to w h a t is o b s e r v e d in t h e i r m o t h e r s , cAMP phosphodiesterase is m u c h more a c t i v e than a d e n y l a t e c y c l a s e in all t h r e e o r g a n s of the o f f s p r i n g . The l e v e l s of 5 ' - n u c l e o t i d a s e are higher than those of a d e n y l a t e c y c l a s e and c A M P p h o s p h o d i e s t e r a s e in the l i v e r of the p r o g e n y . In the b r a i n , the a c t i v i t y of 5'n u c l e o t i d a s e is lower than that of both the other enzymes. However, 5'-nucleotidase is suppressed in comparison to cAMP phosphodiesterase b u t is g r e a t e r than adenylate cyclase in the k i d n e y of the p r o g e n y . T h e s e r e s u l t s and t h o s e d i s c u s s e d in the preceding section clearly indicate that dietary stress alters adenine n u c l e o t i d e m e t a b o l i s m in f e m a l e rats and their offspring and that this m o d u l a t i o n is organ-dependent. Furthermore, as in lymphoid organs, m a x i m a l d i e t a r y i n s u f f i c i e n c y p r o l o n g s or b l o c k s the G and S p h a s e s of the c e l l c y c l e in the l i v e r , b r a i n and k i d n e y of t h e s e dams and their progeny (28,29). E f f e c t of V i t a m i n E Deficiency Skele-tal M u s c l e o~-R~bbits
on
the
Turnover
of
cAMP
in
the
In a l l the f o r e g o i n g s e c t i o n s , we d i s c u s s e d the i m p a c t of d i e t a r y insufficiency (protein d e f i c i e n c y or p r o t e i n - c a l o r i e malnutrition) on the content and turnover of c y c l i c n u c l e o t i d e s in organs such as the l i v e r , brain, kidney, thymus and s p l e e n . In the p r e s e n t section, we w i l l examine the m e t a b o l i s m and t u r n o v e r of t h e s e enzymes in s k e l e t a l muscle. This parameter was chosen because v i t a m i n E d e f i c i e n c y causes profound d e g r a d a t i o n of m u s c l e protein and m u s c l e weakness, l e a d i n g to n u t r i t i o n a l l y - i n d u c e d muscular dystrophy. ATP-ADP is d e c r e a s e d in h o m o g e n a t e and n u c l e i - m y o f i b r i l s b u t is increased in m i t o c h o n d r i a l , m i c r o s o m a l and s o l u b l e fractions of the s k e l e t a l m u s c l e of t o c o p h e r o l - d e f i c i e n t (TD) rabbits (31,32). cAMP content is d e p r e s s e d in homogenate and all c e l l u l a r fractions with the e x c e p t i o n of the s o l u b l e fraction, in w h i c h no c h a n g e is manifest. A m a x i m a l d e c r e a s e is n o t e d in the n u c l e i - m y o f i b r i l l a r fraction. 5'-AMP activity is a u g m e n t e d in h o m o g e n a t e and v a r i o u s other fractions d e r i v e d from TD rabbits. Adenosine does not reveal any v a r i a t i o n in m u s c l e h o m o g e n a t e in these animals. However, it is d e c r e a s e d in n u c l e i - m y o f i b r i l s and in the m i c r o s o m a l fraction but is increased in the m i t o c h o n d r i a l and s o l u b l e fractions. The i n c o r p o r a t i o n of 8 - H 3 - a d e n i n e into A T P - A D P demonstrates a lowered accumulation of r a d i o a c t i v i t y in h o m o g e n a t e , nucleimyofibrils and the m i t o c h o n d r i a l fraction (32). The incorporation of r a d i o a c t i v i t y into c A M P is d i m i n i s h e d in the homogenate, n u c l e i myofibrils, microsomal and s o l u b l e fractions. Strangely enough, accumulation is enhanced in the mitochondrial fraction. Radioactivity in 5 ' - A M P is e l e v a t e d in t h e homogenate, mitochondrial, microsomal and s o l u b l e fractions but is lowered in the n u c l e i - m y o f i b r i l l a r fraction. The l a b e l l i n g of a d e n o s i n e is considerably decreased in the h o m o g e n a t e , nuclei-myofibrillar, microsomal and soluble fraction and is increased in the
596
U.S. SRIVASTAVAet al.
mitochondrial
fraction.
From these results, it can b e s e e n t h a t the a c t i v i t i e s of a d e n y l c y c l a s e and c A M P p h o s p h o d i e s t e r a s e are h e i g h t e n e d to v a r y i n g d e g r e e s in the n u c l e i - m y o f i b r i l l a r , m i c r o s o m a l and s o l u b l e f r a c t i o n s of the s k e l e t a l m u s c l e of TD rabbits, cAMP phosphodiesterase l e v e l s are always higher than adenyl cyclase concentrations in t h e s e t h r e e cellular fractions. Maximal increases in the a c t i v i t y of c A M P phosphodiesterase a r e e v i d e n t in the m i c r o s o m a l fraction, but are abated to i n t e r m e d i a t e l e v e l s in the s o l u b l e fraction and are l o w e s t in the n u c l e i - m y o f i b r i l l a r fraction. In contrast, the a c t i v i t y of adenyl cyclase is m a r k e d l y augmented whereas that of c A M P phosphodiesterase is o n l y s l i g h t l y e l e v a t e d in the m i t o c h o n d r i a of the s k e l e t a l m u s c l e of TD rabbits. These o b s e r v a t i o n s are further c o n f i r m e d by the u t i l i z a t i o n of r a d i o a c t i v e p r e c u r s o r s specific to adenyl c y c l a s e (ATP-814C), c A M P p h o s p h o d i e s t e r a s e ( c A M P - 8 1 4 C ) and 5'-nucleotidase (5'-AMP-814C). A d m i n i s t r a t i o n of t o c o p h e r o l to rabbits after 2 weeks of v i t a m i n E deficiency partially restores the m e t a b o l i c chain of e n z y m e s involved in t h e m e t a b o l i s m of c A M P , These results clearly d e m o n s t r a t e that v i t a m i n E p l a y s an i m p o r t a n t role in the m e t a b o l i c turnover of c y c l i c nucleotides, b u t its m e c h a n i s m of a c t i o n is obscure at the p r e s e n t time. F r o m the d a t a r e p o r t e d in t h i s r e v i e w , it c o u l d be c o n c l u d e d that p r o t e i n d e f i c i e n c y and d i e t a r y r e s t r i c t i o n in a d u l t rats and their progeny modulate cyclic nucleotide metabolism and t u r n o v e r w h i c h could, in turn, cause i m m u n o - d e p r e s s i o n and exert a p r o f o u n d a d v e r s e i n f l u e n c e on i m m u n o - c o m p e t e n c e .
REFERENCES
i.
Aschkenasy A. 1971; 376-378.
Nutrition
et
hematopolese.
Paris:
C.N.R.S.,
2.
A s c h k e n a s y A. Etude q u a n t i t a t i v e de la p o p u l a t i o n c e l l u l a i r e du t h y m u s , d e s g a n g l i o n s et d e la r a t e c h e z d e s r a t s n o r m a u x et d e s rats c a r e n c e s en proteines. Role joue par l'atrophie thymique dans l ' h y p o p l a s i e s p l e n o g a n g l i o n n a i r e p r o v o q u e e par l'inanition proteique. Arch. Sci. Physiol. 1972; 26: 359-366.
3.
A s c h k e n a s y A. Differing e f f e c t s of d i e t a r y protein d e p r i v a t i o n on the p r o d u c t i o n of r o s e t t e - f o r m i n g c e l l s in the l y m p h n o d e s and the s p l e e n and on the l e v e l s of s e r u m h a e m a g g l u t i n i n s in r a t s i m m u n i z e d to s h e e p red c e l l s . I m m u n o l o g y 1973; 24: 6 1 7 633.
4.
A s c h k e n a s y A. H y p e r p l a s i e des g a n g l i o n s l y m p h a t i q u e s et de la rate apres i m m u n i s a t i o n c o n t r e des g l o b u l e s rouges de m o u t o n et p a r t i c i p a t i o n c o m p a r e e des l y m p h o c y t e s t h y m o - d e p e n d a n t s a cette hyperplasle chez des rats carences en p r o t e l n e s et d e s non carences. Ann. Nutr. Aliment. 1973; 27: 147-160.
5.
S p a c h C, A s c h k e n a s y A. Effets d'une carence alimentaire en proteines sur l e s t e n e u r s en Na et K et les a c t i v i t e s ATPa s i q u e s des l y m p h o c y t e s thymiques, s p l e n i q u e s , g a n g l i o n n a i r e s et s a n g u i n s c h e z le rat. C.R. Soc. B i o l . (Paris) 1973; 167: 1 5 4 0 1544.
cAMP AND cGMP IN MALNUTRITION
597
6.
Aschkenasy A. E f f e c t s of a p r o t e i n - f r e e d i e t on the D N A s y n t h e s i z z n g and d i v i d i n g c e l l s in the l y m p h o i d o r g a n s of rats. C h a n g e s i n d u c e d by p h y t o h e m a g g l u t i n i n and c o r t i s o n e in v i v o . Life Sci. 1977; 21: 253-260.
7.
C h a n d r a RK. I m m u n o c o m p e t e n c e in l o w b i r t h w e i g h t infants after intrauterlne malnutrition. Lancet 1974; II: 1 3 9 3 - 1 3 9 4 .
8.
F e r g u s o n AC, N e w m a n CG, Oh W, S t e i n ER. Decreased rosetteforming lymphocytes in m a l n u t r i t i o n and i n t r a u t e r i n e g r o w t h retardation. J. Pediat. 1974; 85: 717-723.
9.
Sutherland EW. S t u d i e s on S c l e n c e 1972; 177: 401-408.
i0.
R o b i n s o n GS, B u t c h e r A c a d e m i c Press, 1971;
ii.
S h e p p a r d JR. The r o l e of c y c l i c A M P in the c o n t r o l of c e l l division. In: Braun W, L i c h t e n s t e i n LM, Parker CW, ed. Cyclic AMP, C e l l G r o w t h and the Immune Response, B e r l i n - H e i d e l b e r g - N e w York: S p r i n g e r - V e r l a g , 1974; 290-301.
12.
S r i v a s t a v a U, Spach C, A s c h k e n a s y A. C y c l i c A M P m e t a b o l l s m and n u c l e i c a c i d c o n t e n t in the l y m p h o c y t e s of the t h y m u s , s p l e e n and l y m p h n o d e s of p r o t e i n - d e f i c i e n t rats. J. Nutr. 1975; 105: 924-938.
13.
Spach C, A s c h k e n a s y A. C y c l i c A M P and G M P l e v e l s in t h y m o c y t e s and s p l e n i c T and B l y m p h o c y t e s in p r o t e i n d e p r i v e d rats. J. Nutr. 1977; 107: 1729-1736.
14.
S p a c h C, A s c h k e n a s y A. E f f e c t s of a p r o t e i n - f r e e d i e t on the changes in c y c l i c AMP and cyclic GMP levels induced by immunization in s p l e n i c T and B l y m p h o c y t e s in the rat. J. Nutr. 1979; 109: 1 2 6 5 - 1 2 7 3 .
15.
G o l d b e r g ND, H a d d o x MK, E s t e n s e n R, W h i t e JC, L o p e z C, H a d d e n JW. E v i d e n c e of a d u a l i s m b e t w e e n c y c l i c G M P and c y c l i c A M P in the regulation of c e l l proliferation and other cellular processes. In: Braun W, L i c h t e n s t e i n LM, Parker CW, ed. Cyclic AMP, C e l l G r o w t h and the Immune Response, B e r l i n - H e i d e l b e r g - N e w York: S p r i n g e r - V e r l a g , 1974; 246-262.
16.
Abell CW, Monahan TM. The r o l e of a d e n o s i n e m o n o p h o s p h a t e in the r e g u l a t i o n of m a m m a l i a n c e l l Cell. Biol. 1973; 59: 549-558.
the
mechanism
RW, S u t h e r l a n d 427.
W.
of
hormone
Cyclic
17. W a t s o n J, Epstein R, Cohn M. C y c l ic i n t r a c e l l u l a r m e d i a t o r s of the e x p r e s s l o n of cells. Nature (London) 1973; 246: 405-409.
AMP.
action.
New York:
3',5'-cyclic divlsion. J.
nucleotides as antigen-sensitive
18.
Spach C, A s c h k e n a s y A. Effet de l ' i m m u n l s a t i o n sur les teneurs en A~4P c y c l i q u e des l y m p h o c y t e s s p l e n l q u e s chez des rats n o r m a u x ou c a r e n c e s en p r o t e i n e s . J. P h y s i o l . (Paris) 1975; 71: A b s t r a c t 314A.
19.
Aschkenasy regulation
A. Some data and of the l y m p h o p o i e s z s
hypotheses and i m m u n e
On t h e h o r m o n a l responsiveness in
598
U.S. SRIVASTAVA et al. protein deficient rats. Proc. 10th Nutrition, Kyoto, Japan, 1975; 132-133.
Inter.
Congress
of
20.
A s c h k e n a s y A. C o m p a r a t i v e effects of a protein d e p r i v e d diet on the l y m p h o c y t e c o n t e n t s and the number of DNA synthesizing and d i v i d i n g c e l l s in the lymphoid organs of the rat. I n f l u e n c e of the d i e t on the s t i m u l a t i n g action of p h y t o h e m a g g l u t i n i n and the i n h i b i t o r y e f f e c t of c o r t i s o n e . Ann. Nutr. A l i m e n t . 1978; 32: 15-40.
21.
Shearer WT, Wedner HJ, P h i l p o t t GW. E a r l y changes in c y c l i c A M P and c y c l i c G M P in a n t i b o d y s t i m u l a t e d tumor cells. Fed. Proc. 1974; 33: A b s t r a c t 3382.
22.
H e n n e y C. Relationships between the c y t o l y t i c activity of thymus-derived lymphocytes and c e l l u l a r cyclic nucleotide concentrations. In: B r a u n W, L i c h t e n s t e i n LM, P a r k e r CW, ed. Cyclic AMP, Cell G r o w t h and the I m m u n e R e s p o n s e , BerlinH e i d e l b e r g - N e w York: S p r i n g e r - V e r l a g , 1974; 195-208.
23. W a t s o n J. C y c l i c cell activation.
nucleotides as i n t r a c e l l u l a r mediators Transplant Rev. 1975; 23: 223-249.
of B
24.
Srivastava, U, R a k s h i t AK, S e e b a g M, O m o l o k o C, T h a k u r ML. M e t a b o l i s m of adenine n u c l e o s i d e s and n u c l e o t i d e s , n u c l e i c acids and p r o t e i n s in the t h y m u s and s p l e e n of n e o n a t a l p r o g e n y of d i e t a r y restricted rats. Nutr. Rep. Inter. 1981; 23: 1035-1042.
25.
Srivastava US, B h a t n a g a r G, B h a r g a v a S, M i t c h e l l J. Nucleic a c i d and p r o t e i n m e t a b o l i s m in the p a n c r e a s , spleen, thymus, l u n g , c a r d i a c and s k e l e t a l m u s c l e of u n d e r n o u r i s h e d rats. J. P h y s i o l . (Paris) 1979; 75: 545-554.
26.
S r i v a s t a v a U, Goswami T, Vu ML. M e t a b o l i s m of protein and RNA in the organs of the young of u n d e r n o u r i s h e d rats. II. Changes in the pancreas, s p l e e n and thymus. Nutr. Rep. Inter. 1978; 17: 257-265.
27.
G o s w a m i T, Vu ML, S r i v a s t a v a U. Quantitative c h a n g e s in DNA, R N A and p r o t e i n c o n t e n t of t h e v a r i o u s o r g a n s of the y o u n g of undernourished female rats. J. Nutr. 1974; 104: 1257-1264.
28.
Srivastava U, O m o l o k o C. Metabolism of a d e n i n e n u c l e o t i d e s , nucleic a c i d s and p r o t e i n s in the l i v e r , b r a i n and k i d n e y of undernourished f e m a l e rats. Nutr. Rep. Inter. 1981; 23: 1 0 2 1 1033.
29.
Srivastava US, O m o l o k o C. M e t a b o l i s m of a d e n i n e n u c l e o t i d e s , n u c l e i c acids and proteins in the liver, brain and kidney of 21day-old progeny of u n d e r n o u r i s h e d female rats. Ann. Nutr. Metab. 1983; 27: 261-267.
30.
S r i v a s t a v a US, Thakur ML. C y c l i c n u c l e o t i d e content in organs of rats m a l n o u r i s h e d at v a r i o u s ages e a r l y in life. Nutr. Rep. Inter. 1985; 31: 1033-1046.
31.
S r i v a s t a v a U, Robin L. Effects of a tocopherol d e f i c i e n t diet on the t u r n o v e r of c y c l i c AMP in the s k e l e t a l m u s c l e of rabbits. Proc. 10th Inter. C o n g r e s s of N u t r i t i o n , K y o t o , J a p a n , 1975; 168.
cAMP AND cGMP IN MALNUTRITION 32.
599
Srivastava U, R o b i n L. Turnover of c y c l i c - A M P in v a r i o u s cellular fractions of s k e l e t a l m u s c l e of t o c o p h e r o l deficient rabbits. Proc. Inter. C o n g r e s s of B i o c h e m i s t r y , Hamburg, West Germany, 1976; 389.
Accepted for publication April 18, 1986
CYCLIC 3',5'-ADENOSINE MONOPHOSPHATE AND C Y C L I C 3 ' , 5 ' - G U A N O S I N E M O N O P H O S P H A T E M E T A B O L I S H IN M A L N U T R I T I O N Uma S. S r i v a s t a v a , DSc, M a n o h a r Lal Thakur, MSc and C. Spach%, DSc D e p a r t m e n t of N u t r i t i o n , u n i v e r s i t y of M o n t r e a l , F a c u l t y of M e d i c i n e , M o n t r e a l , Quebec, C a n a d a H3C 3J7 and + C e n t r e Marcel Delepine, C.N.R.S. 45045 Orleans Cedex, France
ABSTRACT
When the concentration and turnover of t h e c y c l i c nucleotides cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate (cGHP) w e r e m e a s u r e d in the thymus, s p l e e n and c e r v i c a l l y m p h n o d e s of r a t s s u b j e c t e d to v a r i o u s m o d e l s of m a l n u t r i t i o n , and these same parameters of c A M P a l o n e a s s e s s e d in t h e i r liver, k i d n e y and brain, u n d e r n u t r i t i o n has b e e n found to m o d i f y c y c l i c n u c l e o t i d e m e t a b o l i s m . When the r e s u l t s on n u c l e i c a c i d and p r o t e i n m e t a b o l i s m are t a k e n into consideration w i t h the c h a n g e s in c y c l i c nucleotide metabolism, it was n o t e d t h a t the v a r i o u s p h a s e s of the c e l l c y c l e are p r o l o n g e d or b l o c k e d in the o r g a n s of malnourished rats. These data indicate a correlatzon b e t w e e n c y c l i c n u c l e o t i d e m e t a b o l z s m and impaired immune r e s p o n s i v e n e s s seen c o m m o n l y in u n d e r n o u r i s h e d rats. Key Words:
Cyclic nucleotides, metabolism, protein deficiency, lymphoid organs, rats
It is w e l l - k n o w n that protein deficient diets (PDDs) and malnutrition impair such p h y s i o l o g i c a l functions of l y m p h o c y t e s as immune and m i t o g e n i c r e s p o n s i v e n e s s , p r o l i f e r a t i o n , v i a b i l i t y , ionic exchange, ATPase activity, membrane integrity (1-6), and i m m u n o c o m p e t e n c e (7,8). Many i n v e s t i g a t o r s (5,6,9,10) h a v e reported that c A M P l e v e l s in l y m p h o c y t e s could act as h o r m o n a l m e d i a t o r s and p l a y an i m p o r t a n t r o l e , e s p e c l a l l y in c e l l u l a r proliferatzon and l y m p h o c y t e r e s p o n s e to a c t i v a t o r s or i n h i b i t o r s . Moreover, cAMP concentrations are f o u n d to i n c r e a s e in m i c r o b i a l cultures after s e r u m or a m i n o a c i d d e p r i v a t i o n b u t r e t u r n to n o r m a l a f t e r the a d d i t i o n of t h e s e n u t r i e n t s (ii). It h a s b e e n p o s t u l a t e d that the availability of n u t r i e n t s represents a major stimulus of c A M P m o d u l a t i o n , a h y p o t h e s i s w h i c h has been c o n f i r m e d by in v i v o and in v i t r o e x p e r i m e n t s (12-14). G o l d b e r g et al. (15) h a v e s u g g e s t e d that c G M P m i g h t also p l a y a s i g n i f i c a n t r o l e in p h y s i o l o g i c a l f u n c t i o n s either p a r a l l e l to or together with cA~4P ("dual hypothesis").
Correspondence: Uma S. Srivastava, Professor of F u n d a m e n t a l Nutrition, Department of N u t r i t i o n , University of M o n t r e a l , C.P. 6128, S u c c u r s a l e A, H o n t r e a l , Quebec, C a n a d a H3C 3J7.
589
U.S. SRIVASTAVA et al.
590
Effect
of PDDs on C y c l i c
Nucleotide
Metabolism
in Rat L y m p h o c y t e s
P D D s for 36 d a y s do not a l t e r c A M P l e v e l s in t h y m o c y t e s b u t e l e v a t e them in s p l e n i c and e s p e c i a l l y in l y m p h n o d e l y m p h o c y t e s (12). Such d i e t s e l i c i t an i n c r e a s e in the q u a n t i t y of 3 H - c A M P s y n t h e s i z e d during incubation with 2-3H-adenosine, i n d i c a t i n g an i n c r e m e n t in a d e n y l a t e c y c l a s e a c t i v i t y in thymocytes as w e l l as in splenic and lymphnode lymphocytes. T h i s is p r o b a b l y a l s o the c a s e w i t h c A M P phosphodiesterase activity, as s u g g e s t e d by an a u g m e n t a t i o n of r a d i o a c t i v e 5'-AMP and adenosine content. The enhanced a c t i v i t i e s of b o t h e n z y m e s are s i m i l a r in the t h y m u s w h i c h e x p l a i n s the m a i n t e n a n c e of normal c A M P l e v e l s after PDDs. The rise in a d e n y l a t e cyclase activities in s p l e e n and l y m p h n o d e lymphocytes is m o r e p r o n o u n c e d than the e l e v a t i o n of p h o s p h o d i e s t e r a s e activities. This i m b a l a n c e therefore leads to heightened c A M P c o n c e n t r a t i o n s induced by PDDs in the latter two organs (spleen and lymphnodes). It is n o w w e l l - e s t a b l i s h e d that an i n v e r s e r e l a t i o n s h i p exists between intracellular cAMP content and a d e n y l a t e cyclase and p h o s p h o d i e s t e r a s e a c t i v i t i e s on the one hand and the m i t o t i c rate of c o r r e s p o n d i n g c e l l s on the other. The r e s u l t s just described favor the h y p o t h e s i s that the t u r n o v e r of c A M P is i n v e r s e l y related to the proliferative potential of t h e s e t h r e e o r g a n s (thymus, spleen, lymphnodes) and, c o n s e q u e n t l y , to t h e i r c a p a c i t y to r e s i s t the stress of d i e t a r y protein d e f i c i e n c y (16). Indeed, the increase of newly-formed cAMP, d u e to p r o t e i n d e f i c i e n c y , is a l l the m o r e important s i n c e the w e i g h t and c e l l p o p u l a t i o n of the l y m p h o i d organs and the v i a b i l i t y of c o r r e s p o n d i n g l y m p h o c y t e s are decreased. A l l t h e s e d e f e c t s are p a r t i c u l a r l y m a r k e d in the t h y m u s w i t h l e s s pronounced manifestations in the l y m p h n o d e s (1,2). Effect of P D D s on c A M P and cGMP Levels in R a t U n f r a c t i o n a t e d S p l e e n L y m p h o c y t e s and T and B C e l l s
Thymoc~[tes__ L
The other p h y s i o l o g i c a l c y c l i c n u c l e o t i d e , cGMP, has a l s o been found to p l a y a p r o m i n e n t r o l e in c e l l u l a r function. For e x a m p l e , the i n h i b i t i o n of i m m u n e r e s p o n s i v e n e s s by thymectomy is r e v e r s e d in v i t r o by the a d d i t i o n of c G M P (17). It is e v e n s u g g e s t e d that the cAMP/cGMP r a t i o m a y be m o r e important t h a n e a c h of t h e t w o nucleotides in the r e g u l a t i o n of c e l l u l a r metabolism and in c e l l reactions to a c t i v a t o r s or i n h i b i t o r s (17). In a d d i t i o n , many reports d o c u m e n t the n e c e s s a r y c o o p e r a t i o n of two major categories of l y m p h o c y t e s in i m m u n e r e s p o n s i v e n e s s , the t h y m u s - d e r i v e d T l y m p h o c y t e s and the t h y m u s - i n d e p e n d e n t B lymphocytes. It has b e e n n o t e d t h a t P D D s do not i n f l u e n c e the c A M P c o n t e n t of thymus cells but largely increase it in u n f r a c t i o n a t e d spleen lymphocytes (13). Interestingly, the t h y m o c y t e c A M P l e v e l s are quantitatively l o w e r t h a n in s p l e n i c l y m p h o c y t e s . These changes could be i n v e r s e l y correlated to m i t o t i c activity which is relatively h i g h e r in the t h y m u s t h a n in the s p l e e n or l y m p h n o d e s . The a p p a r e n t a b s e n c e of an e f f e c t of P D D s on t h y m o c y t e s is a l s o demonstrated by the c G M P l e v e l s and c o n s e q u e n t l y by the c A M P / c G M P ratio, cGMP concentrations are h i g h e r in u n f r a c t i o n a t e d spleen lymphocytes t h a n in t h y m o c y t e s , the d i f f e r e n c e between the two organs being e v e n more pronounced than with cAMP. C o n s e q u e n t l y , the c A M P / c G M P ratio is lower in spleen c e l l s than in thymus cells. The c G M P l e v e l s are d i m i n i s h e d after p r o t e i n d e f i c i e n c y in unseparated s p l e n i c c e l l s and s p l e n i c T l y m p h o c y t e s , w h e r e a s the r i s e in c A M P content of u n s e p a r a t e d s p l e n i c c e l l s is found o n l y in B lymphocytes.
cAMP AND cGMP IN MALNUTRITION Thus, the c A M P / c G M P ratio is i n c r e a s e d l y m p h o c y t e extracts, e s p e c i a l l y in B c e l l s , in c A M P or a l o w e r i n g of c G M P l e v e l s . Effect of PDDs on C o r t i s o n e - i n d u c e d in R a t Thymoc_ytes~ U n f r a c t i o n a t e d Cells
591
in the t h r e e s p l e n i c either b e c a u s e of a rise
C h a n g e s in c A M P and Spleen Lymphocytes
cGMP Levels and T a n d B
Protein deprivation primarily destroys short-living, cortisones e n s i t i v e c o r t i c a l t h y m o c y t e s , w h i c h m a y be p a r t i c u l a r l y r i c h in c G M P in v i e w of t h e i r h i g h m i t o t i c a c t i v i t y (18). During protein d e f i c i e n c y , the few s u r v i v i n g , v i r t u a l l y m i t o t i c a l l y - i n a c t i v e cells s h o u l d c o n t a i n less c G M P and d i s p l a y an increased c A M P / c G M P ratio a c c o r d i n g to the "dual h y p o t h e s i s " of a r e l a t i o n s h i p b e t w e e n the two p a r a m e t e r s ( c A M P / c G M P ratio and c e l l u l a r p r o l i f e r a t i o n ) . W h i l e this is not the c a s e w i t h the t h y m u s (19), the t h e o r y is in a g r e e m e n t with the r e s u l t s obtained for s p l e e n lymphocytes. The cA~P/cGMP r a t i o is i n c r e a s e d after protein deprivation in unfractionated as w e l l as in T a n d e s p e c i a l l y in B s p l e e n lymphocytes. The g r e a t m a j o r i t y of m i t o t i c a l l y - a c t i v e splenic l y m p h o c y t e s are c o r t i s o n e - r e s i s t a n t in contrast to the t h y m o c y t e s (20). Thus, an attempt was made to d e t e r m i n e w h e t h e r a r e l a t i o n s h i p e x i s t s b e t w e e n the r e s p o n s i v e n e s s to c o r t i s o n e and the l e v e l s and p h y s i o l o g i c a l a c t i v i t y of the i n t r a c e l l u l a r c y c l i c n u c l e o t i d e s . The r e s u l t s showed that after a d a i l y injection of c o r t i s o n e acetate (3 m g / 1 0 0 g b o d y w e i g h t ) for 5 d a y s , the c A M P l e v e l s and c A M P / c G M P ratio were higher than in control thymocytes. S i m i l a r l y , the c A ~ P c o n t e n t and c A M P / c G M P ratio were m u c h higher than normal in s p l e n i c lymphocytes at 30 m i n u t e s , i, 4 a n d 24 h o u r s a f t e r the f i r s t c o r t i s o n e injection as w e l l as after 5 days of c o r t i s o n e treatment. E l e v a t e d v a l u e s were a l s o recorded at 30 m i n u t e s and 24 h o u r s in B cells and at 1 h o u r in T c e l l s a f t e r a s i n g l e i n j e c t i o n of the h o r m o n e as w e l l as a f t e r the f i f t h t r e a t m e n t d a y in b o t h T and B l ymphocytes. Thus, c y c l i c n u c l e o t i d e m e t a b o l i s m and the c A M P / c G M P ratio are a p p a r e n t l y m o r e s e n s i t i v e after c o r t i s o n e t r e a t m e n t in the s p l e e n t h a n in the t h y m u s . These results are s i m i l a r to t h o s e r e p o r t e d w i t h PDDs, w h i c h is n o t s u r p r i s i n g s i n c e the e f f e c t s of protein deprivation on i m m u n e or m i t o g e n i c e x p r e s s i o n r e s e m b l e in l a r g e m e a s u r e the c h a n g e s i n d u c e d b y c o r t i s o n e t r e a t m e n t in v i v o (6) . E f f e c t of PDDs on A n t i g e n - i n d u c e d Rat S p l e n i c L y m p h o c y t e s
Changes
in c A M P and c G M P L e v e l s
in
A t h y m u s - d e p e n d e n t antigen d e r i v e d from sheep red b l o o d c e l l s (SRBC) was injected once i n t r a p e r i t o n e a l l y in n o r m a l l y - f e d rats to study the e v o l u t i o n of cACIP and c G M P c o n t e n t in u n f r a c t i o n a t e d as w e l l as in s p l e n i c T and B l y m p h o c y t e s (14). A s i g n i f i c a n t fall in cA~4P l e v e l s occurred at 30 m i n u t e s and 1 hour after a d m i n i s t r a t i o n of the a n t i g e n . A t r a n s i e n t rise was noted at 2 h o u r s , b u t t h e s e v a l u e s r e t u r n e d to n o r m a l at 4 and 24 h o u r s . A second increase was o b s e r v e d on day 3 but again returned to normal after 5 days. The c G M P l e v e l s were b e l o w control v a l u e s until the f o u r t h hour, t h e n r o s e to the n o r m on d a y i, and p e a k e d on d a y 3. As w i t h c A M P , the c A M P / c G M P ratio was first depressed u n t i l the first hour, reached a peak at 2 hours, but then fell again a l t h o u g h it remained a b o v e initial v a l u e s until the end of the experiment.
592
U.S. SRIVASTAVA et a l .
As s h o w n p r e v i o u s l y , P D D s r e d u c e d a n d d e l a y e d the a n t i g e n - i n d u c e d changes in c y c l i c nucleotide content in u n f r a c t i o n a t e d spleen lymphocytes (13). Thus, the f i r s t significant alteration (a s t r i k i n g d r o p in c A M P 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 o n l y 24 h o u r s after the a n t i g e n injection. These levels were subsequently e l e v a t e d but r e m a i n e d b e l o w initial v a l u e s until day i0. A moderate b u t l a t e i n c r e a s e in c G M P c o n t e n t w a s r e c o r d e d i0 d a y s a f t e r S R B C treatment. The c A ~ P / c G M P r a t i o was s o m e w h a t r e g u l a r l y d e c r e a s e d in c o m p a r i s o n to i n i t i a l v a l u e s , b e i n g a b o u t 50% l o w e r on d a y i0 in protein-deficient r a t s and 35% h i g h e r at the s a m e t i m e in c o n t r o l animals. There were similar significant differences in t h e evolution of c A M P and c G M P l e v e l s in T and B l y m p h o c y t e s from n o r m a l l y - f e d and p r o t e i n - d e p r i v e d rats. The e a r l y rise in c A M P c o n c e n t r a t i o n s in u n f r a c t i o n a t e d as w e l l as in T and B c e l l s c o u l d be due to a d e n y l a t e c y c l a s e a c t i v a t i o n which, in n o r m a l rats, m a y be the f i r s t e v e n t of the i m m u n e r e s p o n s e i n d u c e d b y the b i n d i n g of a n t i g e n to s e n s i t i v e c e l l s (21,22). The l a t e r i s e in c G ~ P l e v e l s in u n f r a c t i o n a t e d as w e l l as in T and B c e l l s c o u l d be e l i c i t e d b y t h e c o o p e r a t i v e s y s t e m (T c e l l s ) and c o u l d be the r e s u l t of g u a n y l a t e c y c l a s e a c t i v a t i o n which, in n o r m a l r a t s , m a y be the s e c o n d e v e n t in the i m m u n e r e s p o n s e t r i g g e r e d b y the p r o l i f e r a t i o n of -sensitive cells and the p r o d u c t i o n of antibodies. The i m m u n e r e s p o n s e , at l e a s t to t h y m u s - d e p e n d e n t antigens, would thus d e p e n d upon the c A M P / c G M P ratio. A c c o r d i n g to W a t s o n (23), an optimal ratio is n e c e s s a r y to g e n e r a t e the early, critical b i o c h e m i c a l e v e n t s in the r e s p o n s e of l y m p h o i d organs. This o p t i m a l r a t i o d e p e n d s u p o n the n u m b e r of s p e c i f i c s i t e s of c A ~ P and c G M P p r o t e i n k i n a s e s and of the s u b s t r a t e s a v a i l a b l e to t h e s e e n z y m e s (23) . It, therefore, seems r e a s o n a b l e to suggest that PDDs, like c o r t i s o n e treatment, impair immune responszveness v i a t h e i r e f f e c t s on t h e m e t a b o l i s m of c A M P and c G M P in l y m p h o c y t e s and via their i n f l u e n c e on t h e c A M P / c G M P ratio. Protein-restricted diets suppress two s i g n a l s : the e a r l y rise in cAL~P c o n t e n t and the later e l e v a t i o n of cGMP levels, especzally in s p l e n i c B l y m p h o c y t e s . On the o t h e r hand, after e x p o s u r e to PDDs or c o r t i s o n e treatment, the c A M P / c G M P r a t i o may be too high to a c c o m m o d a t e the i m m u n o l o g i c a l p r o c e s s (23). These data indicate that there may be a relationship b e t w e e n the d i s t u r b i n g impact of PDDs on the e v o l u t i o n of c A M P and c G M P l e v e l s as w e l l as the c A M P / c G M P ratzo in s p l e n i c l y m p h o c y t e s after a n t i g e n sensitization on the one hand, and the i m m u n o d e p r e s s l v e e f f e c t s of this m a l n u t r i t i o n on the other. E f f e c t of U n d e r n u t r i t i o n on the M e t a b o l i s m of c A M P in the T h y m u s and S p l e e n of F e m a l e Rats and Their N e o n a t a l and 2 1 - d a y - o l d P r o g e n y Our e a r l i e r d i s c u s s i o n was m a i n l y c e n t e r e d on the i n f l J e n c e of PDDs on c y c l i c n u c l e o t i d e m e t a b o l i s m in T and B l y m p h o c y t e s and on immune responsiveness in a d u l t rats. The f o c u s w i l l n o w be s h i f t e d to a s c e r t a i n how m a t e r n a l d i e t a r y r e s t r i c t i o n during g e s t a t i o n (G) and periods of g r o w t h , gestation and lactation (GGL) a f f e c t s the m e t a b o l i s m of c y c l i c n u c l e o t i d e s in the thymus and s p l e e n of a d u l t d a m s (D) and their neonatal (N) and 2 1 - d a y - o l d p r o g e n y (P).
cAMP AND cGMP IN MALNUTRITION
593
A T P - A D P and 5 ' - A M P in the t h y m u s and s p l e e n and a d e n o s i n e in the s p l e e n of Group G d a m s do not r e v e a l any m o d i f i c a t i o n s when c o m p a r e d to c o n t r o l a n i m a l s (C). T h e c A M P c o n t e n t of the t h y m u s and s p l e e n and a d e n o s i n e l e v e l s in the thymus increase c o n s i d e r a b l y in Group G. In a d d i t i o n , a l l n u c l e o t i d e s in the t h y m u s and s p l e e n of G G L d a m s are a u g m e n t e d in r e l a t i o n to either G r o u p C or G dams. The concentrations of v a r i o u s a d e n i n e n u c l e o t i d e s are s t r i k i n g l y enhanced in the t h y m u s a n d s p l e e n of the n e o n a t a l p r o g e n y of GGL dams (24). In contrast, the l e v e l s of ATP-ADP, c A M P and 5'-AMP are decreased, w h e r e a s a d e n o s i n e c o n t e n t is increased in the thymus of the n e o n a t a l p r o g e n y of Group G. A T P - A D P and a d e n o s i n e are e l e v a t e d w h i l e c A M P a n d 5 ' - A M P a r e r e d u c e d in the s p l e e n of the n e o n a t a l p r o g e n y of Group G. The concentrations of v a r i o u s nucleotides are augmented in the t h y m u s and s p l e e n of t h e 2 1 - d a y - o l d p r o g e n y of G r o u p s G and GGL. The o n l y e x c e p t i o n s are the A T P - A D P and 5'-AMP l e v e l s of the thymus and the a d e n o s i n e c o n t e n t of the s p l e e n of the 2 1 - d a y - o l d p r o g e n y of G r o u p G in which no c h a n g e s are noted. I n c o r p o r a t i o n d a t a r e v e a l an e n h a n c e d a c c u m u l a t i o n of r a d i o a c t i v i t y zn v a r i o u s n u c l e o t i d e s of the t h y m u s and s p l e e n of G and G G L dams. The s o l e e x c e p t i o n is t h e c A M P f r a c t i o n of the s p l e e n of G r o u p G w h i c h shows no c h a n g e in a c c u m u l a t i o n . S i m i l a r r e s u l t s are obtained with a d e n y l a t e cyclase, c A M P p h o s p h o d i e s t e r a s e and 5 ' - n u c l e o t i d a s e in the t h y m u s and s p l e e n of G and G G L d a m s . The a c t i v i t y of adenylate cyclase is much higher than that of cAMP phosphodiesterase, w h i l e 5 ' - n u c l e o t i d a s e i n c o r p o r a t l o n is zncreased preferentially in r e l a t i o n to t h e o t h e r two e n z y m e s in the t h y m u s and s p l e e n of u n d e r n o u r i s h e d dams. The data demonstrate t h a t the a c c u m u l a t i o n of r a d i o a c t i v i t y in v a r i o u s n u c l e o t i d e f r a c t i o n s is higher in the thymus and s p l e e n of the n e o n a t a l p r o g e n y of GGL d a m s but is l o w e r in G r o u p G (24). S i m i l a r data are obtained on the a c t i v i t y of a d e n y l a t e cyclase, c A M P phosphodiesterase and 5 ' - n u c l e o t i d a s e . As in G G L and G d a m s , adenylate cyclase levels are higher than those of cAMP phosphodiesterase, while 5'-nucleotidase content is i n c r e a s e d preferentially in c o m p a r i s o n to the o t h e r two e n z y m e s in the n e o n a t a l progeny. R a d i o a c t i v i t y studies show a greater a c c u m u l a t i o n of n u c l e o t i d e s in the t h y m u s and s p l e e n of 2 1 - d a y - o l d p r o g e n y of G r o u p s G and GGL. Similar results are noted for adenylate cyclase, cAMP phosphodiesterase and 5'-nucleotidase in the t h y m u s and s p l e e n of dams of b o t h t h e s e g r o u p s . Contrary to w h a t is o b s e r v e d in undernourished dams and thelr neonatal progeny, cAMP p h o s p h o d i e s t e r a s e a c t i v i t y is greater than that of a d e n y l a t e c y c l a s e in the thymus and s p l e e n of the 2 1 - d a y - o l d progeny. The l e v e l s of 5 ' - n u c l e o t i d a s e are higher than those of a d e n y l a t e c y c l a s e but lower than c A M P p h o s p h o d i e s t e r a s e in the thymus and s p l e e n of the progeny. F r o m the d a t a r e p o r t e d h e r e and e l s e w h e r e on n u c l e i c a c i d and protein metabolism in l y m p h o i d o r g a n s of u n d e r n o u r i s h e d d a m s (25) and t h e i r n e o n a t a l (24) and 2 1 - d a y - o l d p r o g e n y (26,27), it a p p e a r s that d i e t a r y r e s t r i c t i o n induces c h a n g e s in the c e l l u l a r g r o w t h of the t h y m u s and s p l e e n , z.e., c e l l diviszon c o u l d be a r r e s t e d p r e m a t u r e l y b y the p r o l o n g a t i o n or b l o c k a g e of v a r i o u s phases of the c e l l c y c l e (28,29), and t h e s e a l t e r a t i o n s c o u l d be r e l a t e d to an
594 impaired
U.S. SRIVASTAVA et al. immune
E f f e c t of Nu--~t~
responsiveness
(12).
Protein-calorie Malnutrition Earl~ C o n t e n t -i-n-Va---~us O r g a n s of Rats
in L i f e
on C~c~i__cc
Early protein-calorie malnutrition a l t e r s c A M P and c G M P l e v e l s d u r i n g the entire 3-week period of l a c t a t i o n in d i f f e r e n t organs of the r a t (30). c A M P c o n t e n t is i n c r e a s e d in the l i v e r , k i d n e y and b r a i n but d e c r e a s e d in the thymus and s p l e e n of pups s u b j e c t e d to 1 week of u n d e r n u t r i t i o n . The b r a i n and k i d n e y v a l u e s of c A M P return to c o n t r o l l e v e l s a f t e r 2 to 3 w e e k s of m a l n u t r i t i o n . The c G M P concentrations a r e a u g m e n t e d in the l i v e r , k i d n e y and t h y m u s , b u t are depressed in the s p l e e n of o f f s p r i n g exposed to 1 w e e k of malnutrition. V a r i a b l e c G M P r e s p o n s e s a r e n o t e d in the d i f f e r e n t o r g a n s after 2 to 3 weeks of u n d e r n u t r i t i o n . The c A M P / c G M P ratio is lower in the l i v e r and thymus but higher in t h e b r a i n a f t e r 1 a n d 2 w e e k s of m a l n u t r i t i o n . A f t e r 3 w e e k s , it r e t u r n s towards control v a l u e s in the l i v e r , but drops in the b r a i n and s t a y s l o w in the t h y m u s . T h e r a t i o r i s e s in the k i d n e y and s p l e e n after 1 w e e k of u n d e r n u t r i t i o n and e x h i b i t s an e l e v a t i o n in these oraans after 3 weeks. S p a c h and A s c h k e n a s y (13) n o t e d a d e c r e a s e in this ratio in T c e l l s of the thymus with an increase in b o t h T and B c e l l s of the s p l e e n of p r o t e i n - d e p r i v e d rats. In the p a s t , it h a s b e e n p o s t u l a t e d t h a t the c A M P / c G M P r a t i o is important in t h e r e g u l a t i o n of c e l l m e t a b o l i s m (16) and t h a t t h e o n s e t of i m m u n o l o g i c a l r e s p o n s i v e n e s s seems to be c o n d i t i o n e d by an o p t i m a l v a l u e of the c e l l u l a r c y c l i c n u c l e o t i d e ratio (13). Indeed, a r e d u c t i o n of c e l l u l a r g r o w t h (blocked m i t o t i c a c t i v i t y ) has been demonstrated in v a r i o u s o r g a n s , s u c h as t h e l i v e r , b r a i n , k i d n e y , t h y m u s a n d s p l e e n , in d i e t a r y - r e s t r i c t e d rats (12,24-29). On the b a s i s of t h e s e r e s u l t s , we c o u l d a s s u m e t h a t m i t o t i c inhibition i n d u c e d by d i e t a r y s t r e s s w o u l d be m u c h m o r e c o n d i t i o n e d by the cAMP/cGMP r a t i o t h a n by o n l y one of t h e s e c y c l i c nucleotides. F u r t h e r m o r e , a v a i l a b l e data on the c A M P / c G M P ratio c l e a r l y indicate that d i e t a r y d e p r i v a t i o n p r o v o k e s a f u n c t i o n a l i m p a i r m e n t of v a r i o u s organs, i n c l u d i n g the lymphoid organs, as attested by d e c r e a s e s or increases in the ratio. These o b s e r v a t i o n s on the l y m p h o i d organs c o n f i r m the r e s u l t s of o t h e r i n v e s t i g a t o r s (12,14), w h o h a v e a l s o r e p o r t e d s u p p r e s s e d immune r e s p o n s i v e n e s s in d i e t a r y - i n s u l t e d rats. E f f e c t of M a l n u t r i t i o n and Kidn-~y
on c A M P
Metabolism
in the R a t L i v e r ,
Brain
We will now mainly dlscuss the m a n n e r in w h i c h u n d e r n u t r i t i o n i n f l u e n c e s the m e t a b o l i c t u r n o v e r of v a r i o u s adenine n u c l e o t i d e s in the l i v e r , brain and k i d n e y of f e m a l e rats (28) and their 2 1 - d a y - o l d progeny. Malnutrition (50% d i e t a r y restriction) during the period of growth, g e s t a t i o n and l a c t a t i o n (19 weeks of life) causes a d e c r e a s e in the l e v e l s of v a r l o u s a d e n i n e n u c l e o t i d e s and n u c l e o s i d e s in the l i v e r with increases in the brain. The A T P - A D P and 5'-AMP c o n c e n t r a t i o n s in the k i d n e y s of u n d e r n o u r l s h e d f e m a l e r a t s do not r e v e a l a n y c h a n g e but c A M P and a d e n o s i n e are m a r k e d l y e l e v a t e d . The l e v e l s of v a r i o u s n u c l e o t i d e s are increased in the liver, brain and kidney of the 2 1 - d a y - o l d p r o g e n y of these dams. Incorporation
studies
demonstrate
that
the
turnover
of
various
cAMP AND cGMP IN MALNUTRITION
595
adenine n u c l e o t i d e s is heightened in the liver, brain and kidney of u n d e r n o u r i s h e d rats. A d e n y l a t e c y c l a s e is m o r e a c t i v e in c o m p a r i s o n to c A M P p h o s p h o d i e s t e r a s e , while 5'-nucleotidase is i n c r e a s e d p r e f e r e n t i a l l y in r e l a t i o n to both these enzymes in the liver, brain and kidney. The m e t a b o l i c t u r n o v e r of v a r i o u s n u c l e o t i d e s is e n h a n c e d in the l i v e r , b r a i n and k i d n e y of the 2 1 - d a y - o l d p r o g e n y of m a l n o u r i s h e d dams. Contrary to w h a t is o b s e r v e d in t h e i r m o t h e r s , cAMP phosphodiesterase is m u c h more a c t i v e than a d e n y l a t e c y c l a s e in all t h r e e o r g a n s of the o f f s p r i n g . The l e v e l s of 5 ' - n u c l e o t i d a s e are higher than those of a d e n y l a t e c y c l a s e and c A M P p h o s p h o d i e s t e r a s e in the l i v e r of the p r o g e n y . In the b r a i n , the a c t i v i t y of 5'n u c l e o t i d a s e is lower than that of both the other enzymes. However, 5'-nucleotidase is suppressed in comparison to cAMP phosphodiesterase b u t is g r e a t e r than adenylate cyclase in the k i d n e y of the p r o g e n y . T h e s e r e s u l t s and t h o s e d i s c u s s e d in the preceding section clearly indicate that dietary stress alters adenine n u c l e o t i d e m e t a b o l i s m in f e m a l e rats and their offspring and that this m o d u l a t i o n is organ-dependent. Furthermore, as in lymphoid organs, m a x i m a l d i e t a r y i n s u f f i c i e n c y p r o l o n g s or b l o c k s the G and S p h a s e s of the c e l l c y c l e in the l i v e r , b r a i n and k i d n e y of t h e s e dams and their progeny (28,29). E f f e c t of V i t a m i n E Deficiency Skele-tal M u s c l e o~-R~bbits
on
the
Turnover
of
cAMP
in
the
In a l l the f o r e g o i n g s e c t i o n s , we d i s c u s s e d the i m p a c t of d i e t a r y insufficiency (protein d e f i c i e n c y or p r o t e i n - c a l o r i e malnutrition) on the content and turnover of c y c l i c n u c l e o t i d e s in organs such as the l i v e r , brain, kidney, thymus and s p l e e n . In the p r e s e n t section, we w i l l examine the m e t a b o l i s m and t u r n o v e r of t h e s e enzymes in s k e l e t a l muscle. This parameter was chosen because v i t a m i n E d e f i c i e n c y causes profound d e g r a d a t i o n of m u s c l e protein and m u s c l e weakness, l e a d i n g to n u t r i t i o n a l l y - i n d u c e d muscular dystrophy. ATP-ADP is d e c r e a s e d in h o m o g e n a t e and n u c l e i - m y o f i b r i l s b u t is increased in m i t o c h o n d r i a l , m i c r o s o m a l and s o l u b l e fractions of the s k e l e t a l m u s c l e of t o c o p h e r o l - d e f i c i e n t (TD) rabbits (31,32). cAMP content is d e p r e s s e d in homogenate and all c e l l u l a r fractions with the e x c e p t i o n of the s o l u b l e fraction, in w h i c h no c h a n g e is manifest. A m a x i m a l d e c r e a s e is n o t e d in the n u c l e i - m y o f i b r i l l a r fraction. 5'-AMP activity is a u g m e n t e d in h o m o g e n a t e and v a r i o u s other fractions d e r i v e d from TD rabbits. Adenosine does not reveal any v a r i a t i o n in m u s c l e h o m o g e n a t e in these animals. However, it is d e c r e a s e d in n u c l e i - m y o f i b r i l s and in the m i c r o s o m a l fraction but is increased in the m i t o c h o n d r i a l and s o l u b l e fractions. The i n c o r p o r a t i o n of 8 - H 3 - a d e n i n e into A T P - A D P demonstrates a lowered accumulation of r a d i o a c t i v i t y in h o m o g e n a t e , nucleimyofibrils and the m i t o c h o n d r i a l fraction (32). The incorporation of r a d i o a c t i v i t y into c A M P is d i m i n i s h e d in the homogenate, n u c l e i myofibrils, microsomal and s o l u b l e fractions. Strangely enough, accumulation is enhanced in the mitochondrial fraction. Radioactivity in 5 ' - A M P is e l e v a t e d in t h e homogenate, mitochondrial, microsomal and s o l u b l e fractions but is lowered in the n u c l e i - m y o f i b r i l l a r fraction. The l a b e l l i n g of a d e n o s i n e is considerably decreased in the h o m o g e n a t e , nuclei-myofibrillar, microsomal and soluble fraction and is increased in the
596
U.S. SRIVASTAVAet al.
mitochondrial
fraction.
From these results, it can b e s e e n t h a t the a c t i v i t i e s of a d e n y l c y c l a s e and c A M P p h o s p h o d i e s t e r a s e are h e i g h t e n e d to v a r y i n g d e g r e e s in the n u c l e i - m y o f i b r i l l a r , m i c r o s o m a l and s o l u b l e f r a c t i o n s of the s k e l e t a l m u s c l e of TD rabbits, cAMP phosphodiesterase l e v e l s are always higher than adenyl cyclase concentrations in t h e s e t h r e e cellular fractions. Maximal increases in the a c t i v i t y of c A M P phosphodiesterase a r e e v i d e n t in the m i c r o s o m a l fraction, but are abated to i n t e r m e d i a t e l e v e l s in the s o l u b l e fraction and are l o w e s t in the n u c l e i - m y o f i b r i l l a r fraction. In contrast, the a c t i v i t y of adenyl cyclase is m a r k e d l y augmented whereas that of c A M P phosphodiesterase is o n l y s l i g h t l y e l e v a t e d in the m i t o c h o n d r i a of the s k e l e t a l m u s c l e of TD rabbits. These o b s e r v a t i o n s are further c o n f i r m e d by the u t i l i z a t i o n of r a d i o a c t i v e p r e c u r s o r s specific to adenyl c y c l a s e (ATP-814C), c A M P p h o s p h o d i e s t e r a s e ( c A M P - 8 1 4 C ) and 5'-nucleotidase (5'-AMP-814C). A d m i n i s t r a t i o n of t o c o p h e r o l to rabbits after 2 weeks of v i t a m i n E deficiency partially restores the m e t a b o l i c chain of e n z y m e s involved in t h e m e t a b o l i s m of c A M P , These results clearly d e m o n s t r a t e that v i t a m i n E p l a y s an i m p o r t a n t role in the m e t a b o l i c turnover of c y c l i c nucleotides, b u t its m e c h a n i s m of a c t i o n is obscure at the p r e s e n t time. F r o m the d a t a r e p o r t e d in t h i s r e v i e w , it c o u l d be c o n c l u d e d that p r o t e i n d e f i c i e n c y and d i e t a r y r e s t r i c t i o n in a d u l t rats and their progeny modulate cyclic nucleotide metabolism and t u r n o v e r w h i c h could, in turn, cause i m m u n o - d e p r e s s i o n and exert a p r o f o u n d a d v e r s e i n f l u e n c e on i m m u n o - c o m p e t e n c e .
REFERENCES
i.
Aschkenasy A. 1971; 376-378.
Nutrition
et
hematopolese.
Paris:
C.N.R.S.,
2.
A s c h k e n a s y A. Etude q u a n t i t a t i v e de la p o p u l a t i o n c e l l u l a i r e du t h y m u s , d e s g a n g l i o n s et d e la r a t e c h e z d e s r a t s n o r m a u x et d e s rats c a r e n c e s en proteines. Role joue par l'atrophie thymique dans l ' h y p o p l a s i e s p l e n o g a n g l i o n n a i r e p r o v o q u e e par l'inanition proteique. Arch. Sci. Physiol. 1972; 26: 359-366.
3.
A s c h k e n a s y A. Differing e f f e c t s of d i e t a r y protein d e p r i v a t i o n on the p r o d u c t i o n of r o s e t t e - f o r m i n g c e l l s in the l y m p h n o d e s and the s p l e e n and on the l e v e l s of s e r u m h a e m a g g l u t i n i n s in r a t s i m m u n i z e d to s h e e p red c e l l s . I m m u n o l o g y 1973; 24: 6 1 7 633.
4.
A s c h k e n a s y A. H y p e r p l a s i e des g a n g l i o n s l y m p h a t i q u e s et de la rate apres i m m u n i s a t i o n c o n t r e des g l o b u l e s rouges de m o u t o n et p a r t i c i p a t i o n c o m p a r e e des l y m p h o c y t e s t h y m o - d e p e n d a n t s a cette hyperplasle chez des rats carences en p r o t e l n e s et d e s non carences. Ann. Nutr. Aliment. 1973; 27: 147-160.
5.
S p a c h C, A s c h k e n a s y A. Effets d'une carence alimentaire en proteines sur l e s t e n e u r s en Na et K et les a c t i v i t e s ATPa s i q u e s des l y m p h o c y t e s thymiques, s p l e n i q u e s , g a n g l i o n n a i r e s et s a n g u i n s c h e z le rat. C.R. Soc. B i o l . (Paris) 1973; 167: 1 5 4 0 1544.
cAMP AND cGMP IN MALNUTRITION
597
6.
Aschkenasy A. E f f e c t s of a p r o t e i n - f r e e d i e t on the D N A s y n t h e s i z z n g and d i v i d i n g c e l l s in the l y m p h o i d o r g a n s of rats. C h a n g e s i n d u c e d by p h y t o h e m a g g l u t i n i n and c o r t i s o n e in v i v o . Life Sci. 1977; 21: 253-260.
7.
C h a n d r a RK. I m m u n o c o m p e t e n c e in l o w b i r t h w e i g h t infants after intrauterlne malnutrition. Lancet 1974; II: 1 3 9 3 - 1 3 9 4 .
8.
F e r g u s o n AC, N e w m a n CG, Oh W, S t e i n ER. Decreased rosetteforming lymphocytes in m a l n u t r i t i o n and i n t r a u t e r i n e g r o w t h retardation. J. Pediat. 1974; 85: 717-723.
9.
Sutherland EW. S t u d i e s on S c l e n c e 1972; 177: 401-408.
i0.
R o b i n s o n GS, B u t c h e r A c a d e m i c Press, 1971;
ii.
S h e p p a r d JR. The r o l e of c y c l i c A M P in the c o n t r o l of c e l l division. In: Braun W, L i c h t e n s t e i n LM, Parker CW, ed. Cyclic AMP, C e l l G r o w t h and the Immune Response, B e r l i n - H e i d e l b e r g - N e w York: S p r i n g e r - V e r l a g , 1974; 290-301.
12.
S r i v a s t a v a U, Spach C, A s c h k e n a s y A. C y c l i c A M P m e t a b o l l s m and n u c l e i c a c i d c o n t e n t in the l y m p h o c y t e s of the t h y m u s , s p l e e n and l y m p h n o d e s of p r o t e i n - d e f i c i e n t rats. J. Nutr. 1975; 105: 924-938.
13.
Spach C, A s c h k e n a s y A. C y c l i c A M P and G M P l e v e l s in t h y m o c y t e s and s p l e n i c T and B l y m p h o c y t e s in p r o t e i n d e p r i v e d rats. J. Nutr. 1977; 107: 1729-1736.
14.
S p a c h C, A s c h k e n a s y A. E f f e c t s of a p r o t e i n - f r e e d i e t on the changes in c y c l i c AMP and cyclic GMP levels induced by immunization in s p l e n i c T and B l y m p h o c y t e s in the rat. J. Nutr. 1979; 109: 1 2 6 5 - 1 2 7 3 .
15.
G o l d b e r g ND, H a d d o x MK, E s t e n s e n R, W h i t e JC, L o p e z C, H a d d e n JW. E v i d e n c e of a d u a l i s m b e t w e e n c y c l i c G M P and c y c l i c A M P in the regulation of c e l l proliferation and other cellular processes. In: Braun W, L i c h t e n s t e i n LM, Parker CW, ed. Cyclic AMP, C e l l G r o w t h and the Immune Response, B e r l i n - H e i d e l b e r g - N e w York: S p r i n g e r - V e r l a g , 1974; 246-262.
16.
Abell CW, Monahan TM. The r o l e of a d e n o s i n e m o n o p h o s p h a t e in the r e g u l a t i o n of m a m m a l i a n c e l l Cell. Biol. 1973; 59: 549-558.
the
mechanism
RW, S u t h e r l a n d 427.
W.
of
hormone
Cyclic
17. W a t s o n J, Epstein R, Cohn M. C y c l ic i n t r a c e l l u l a r m e d i a t o r s of the e x p r e s s l o n of cells. Nature (London) 1973; 246: 405-409.
AMP.
action.
New York:
3',5'-cyclic divlsion. J.
nucleotides as antigen-sensitive
18.
Spach C, A s c h k e n a s y A. Effet de l ' i m m u n l s a t i o n sur les teneurs en A~4P c y c l i q u e des l y m p h o c y t e s s p l e n l q u e s chez des rats n o r m a u x ou c a r e n c e s en p r o t e i n e s . J. P h y s i o l . (Paris) 1975; 71: A b s t r a c t 314A.
19.
Aschkenasy regulation
A. Some data and of the l y m p h o p o i e s z s
hypotheses and i m m u n e
On t h e h o r m o n a l responsiveness in
598
U.S. SRIVASTAVA et al. protein deficient rats. Proc. 10th Nutrition, Kyoto, Japan, 1975; 132-133.
Inter.
Congress
of
20.
A s c h k e n a s y A. C o m p a r a t i v e effects of a protein d e p r i v e d diet on the l y m p h o c y t e c o n t e n t s and the number of DNA synthesizing and d i v i d i n g c e l l s in the lymphoid organs of the rat. I n f l u e n c e of the d i e t on the s t i m u l a t i n g action of p h y t o h e m a g g l u t i n i n and the i n h i b i t o r y e f f e c t of c o r t i s o n e . Ann. Nutr. A l i m e n t . 1978; 32: 15-40.
21.
Shearer WT, Wedner HJ, P h i l p o t t GW. E a r l y changes in c y c l i c A M P and c y c l i c G M P in a n t i b o d y s t i m u l a t e d tumor cells. Fed. Proc. 1974; 33: A b s t r a c t 3382.
22.
H e n n e y C. Relationships between the c y t o l y t i c activity of thymus-derived lymphocytes and c e l l u l a r cyclic nucleotide concentrations. In: B r a u n W, L i c h t e n s t e i n LM, P a r k e r CW, ed. Cyclic AMP, Cell G r o w t h and the I m m u n e R e s p o n s e , BerlinH e i d e l b e r g - N e w York: S p r i n g e r - V e r l a g , 1974; 195-208.
23. W a t s o n J. C y c l i c cell activation.
nucleotides as i n t r a c e l l u l a r mediators Transplant Rev. 1975; 23: 223-249.
of B
24.
Srivastava, U, R a k s h i t AK, S e e b a g M, O m o l o k o C, T h a k u r ML. M e t a b o l i s m of adenine n u c l e o s i d e s and n u c l e o t i d e s , n u c l e i c acids and p r o t e i n s in the t h y m u s and s p l e e n of n e o n a t a l p r o g e n y of d i e t a r y restricted rats. Nutr. Rep. Inter. 1981; 23: 1035-1042.
25.
Srivastava US, B h a t n a g a r G, B h a r g a v a S, M i t c h e l l J. Nucleic a c i d and p r o t e i n m e t a b o l i s m in the p a n c r e a s , spleen, thymus, l u n g , c a r d i a c and s k e l e t a l m u s c l e of u n d e r n o u r i s h e d rats. J. P h y s i o l . (Paris) 1979; 75: 545-554.
26.
S r i v a s t a v a U, Goswami T, Vu ML. M e t a b o l i s m of protein and RNA in the organs of the young of u n d e r n o u r i s h e d rats. II. Changes in the pancreas, s p l e e n and thymus. Nutr. Rep. Inter. 1978; 17: 257-265.
27.
G o s w a m i T, Vu ML, S r i v a s t a v a U. Quantitative c h a n g e s in DNA, R N A and p r o t e i n c o n t e n t of t h e v a r i o u s o r g a n s of the y o u n g of undernourished female rats. J. Nutr. 1974; 104: 1257-1264.
28.
Srivastava U, O m o l o k o C. Metabolism of a d e n i n e n u c l e o t i d e s , nucleic a c i d s and p r o t e i n s in the l i v e r , b r a i n and k i d n e y of undernourished f e m a l e rats. Nutr. Rep. Inter. 1981; 23: 1 0 2 1 1033.
29.
Srivastava US, O m o l o k o C. M e t a b o l i s m of a d e n i n e n u c l e o t i d e s , n u c l e i c acids and proteins in the liver, brain and kidney of 21day-old progeny of u n d e r n o u r i s h e d female rats. Ann. Nutr. Metab. 1983; 27: 261-267.
30.
S r i v a s t a v a US, Thakur ML. C y c l i c n u c l e o t i d e content in organs of rats m a l n o u r i s h e d at v a r i o u s ages e a r l y in life. Nutr. Rep. Inter. 1985; 31: 1033-1046.
31.
S r i v a s t a v a U, Robin L. Effects of a tocopherol d e f i c i e n t diet on the t u r n o v e r of c y c l i c AMP in the s k e l e t a l m u s c l e of rabbits. Proc. 10th Inter. C o n g r e s s of N u t r i t i o n , K y o t o , J a p a n , 1975; 168.
cAMP AND cGMP IN MALNUTRITION 32.
599
Srivastava U, R o b i n L. Turnover of c y c l i c - A M P in v a r i o u s cellular fractions of s k e l e t a l m u s c l e of t o c o p h e r o l deficient rabbits. Proc. Inter. C o n g r e s s of B i o c h e m i s t r y , Hamburg, West Germany, 1976; 389.
Accepted for publication April 18, 1986