1138

Specialia

EXPERIENTIA 32/9

A) HMT activity in the supernatant and particulate fractions of various regions of the rat brain during development, expressed in nmoles]g tissue/60 min 2 Days Brain regions

6 Days

S

P

Cortex 33.7nL27.9 Hypothalamus 86.8-c 15.0 Dieneephalon 75.0i18.0 Brain stem 105.3• Cerebellum 62.8~- 14.9

10 Days

S

13.7-E10.1 68.5~-18.1~ 62.3~ 15.3 109.1• ~ 22.9• 84.6~=14.0 22.1-~15.3 122.3• ~ 28.9~ 18.9 63.1•

Adult

P

S

P

S

P

19.7~=11.0b 86.64-19.2 ~ 27.1-t=15.1 38.7-E19.2 ~ 30.1•

71.1-E16.8 182.9• 19.2 ~ 107.1• ~ 125.6=E1 9 . 1 118.2~ 18.3 ~

22.7:t_12.5 131.0~=17.4 ~ 32.0~15.0 36.6i12.9 42.6~=15.3b

62.1-t=18.5 19.7~-12.2 163.2• 17.7 b 99.0• 89.1=L20.4 35.9• 110.2• 33.2• 98.5J 2 0 . 4 39.4i15.4

Each number is the mean value of the 10 animals -c SD.

B) PNMT activity in the supernatant and particulate fractions of the hemispheres and brain stem of the rat brain during development expressed in nmoles/g tissue/60 rain 9 Days

15 Days

21 Days

Brain regions

S

P

S

P

S

P

Hemispheres Brain stem

1.31-~0.72 2.10J 0.90

2.53~-0.96 4.83•

1.63~20.81 1.59-L0.63

3.88j=1.23 5.82=t_1.14

1.78~0.75 1.82-E0.66

4.33i1.17 5.32~-0.99

ap < 0.02; bp < 0.01; cp < 0.001 Each number is the mean value of 10 animals ~ SD.

Our results suggest t h a t t h e deciine of b r a i n h i s t a m i n e c o n t e n t a n d t h e a p p a r e n t increase of h i s t a m i n e t u r n o v e r a f t e r t h e 1st w e e k of life, m i g h t be t h e result of e n h a n c e d m e t h y l a t i o n b y t h e increased a c t i v i t y of H M T d u r i n g t h i s period. This is c o m p a t i b l e w i t h t h e o b s e r v a t i o n of SCI~WARTZ e t al. is t h a t e n d o g e n o u s h i s t a m i n e of r a t b r a i n 18 J.-C. SCHVe'ARTZ, H. POLLARD, S. BISCtIOFF a n d M. VERDIERE-

SAHQUE, Eur. J. Pharmae. )'6, 326 (1971). 19 j. T. COVLE and J. AXELROD, J. Neurochem. 19, 449 (1971). 2o j. T. COYLE and J. AXELROD, J. Neurochern. 19, 1117 (1972).

A l t e r a t i o n of C h o l e s t e r o l

j. c. DiAz-ZAGoYA,

Synthesis

M. E. HURTADO

rises s h a r p l y after i n h i b i t i o n of h i s t a m i n e m e t h y l a t i o n . F a c t o r s o t h e r t h a n m e t a b o l i s m rate, s u c h as m o d i f i c a t i o n s in c o m p a r t m e n t a t i o n 17 a n d release of h i s t a m i n e , m a y also c o n t r i b u t e to t h e a b o v e d e v e l o p m e n t a l changes. P N M T s h o w s a l m o s t a 2-fold increase in specific activit y b e t w e e n 17 d a y s of g e s t a t i o n a n d 14 d a y s a f t e r birth. No significant c h a n g e was o b s e r v e d a f t e r t h e end of t h e 2nd week, while, as s h o w n b y o t h e r i n v e s t i g a t o r s , t y r o s i n e h y d r o x y l a s e and d o p a m i n e - / % h y d r o x y l a s e , two o t h e r e n z y m e s of t h e same p a t h w a y , s h o w a f u r t h e r increase d u r i n g t h e following 2 weeks of life19, 20.

in Rat Liver as Induced by 4-Methyl-5-Hydroxy

Valeric Acid

and J. GONZ2{LEZ

Departamento Bioquimica, Facultad de ~fedicina, Universidad Nacional A utonoma de Mexico, A partado .Postal 70-759, Mexico 20, D. F., 8 March 1976. Summary. The r a t e of cholesterogenesis in r a t liver, m e a s u r e d b y t h e i n c o r p o r a t i o n of labelled a c e t a t e or m e v a l o n a t e i n t o cholesterol, was s i g n i f i c a n t l y s u p r e s s e d b y t h e use of 4 - m e t h y l - 5 - h y d r o x y valeric acid s o d i u m salt. This effect c a n n o t be e x p l a i n e d b y c h a n g e s in H M G CoA r e d u c t a s e a c t i v i t y . I t is generally agreed t h a t tile r a t e - E m i t t i n g s t e p in h e p a t i c cholesterogenesis is t h e f o r m a t i o n of m e v a l o n i c acid f r o m H M G CoA ( 3 - h y d r o x y - 3 - m e t h y l g l u t a r y l CoA) 1. This reaction, w h i c h is c a t a l y z e d b y tile e n z y m e H M G CoA r e d u c t a s e (EC 1.1.1.34), is altered b y d i e t a r y cholest e r o l c o n t e n t 2, h o r m o n a l c o n d i t i o n s a a n d drugs like c h o l e s t y r a m i n e 4. Several c o m p o u n d s , including c e r t a i n valeric acid d e r i v a t i v e s 5, h a v e b e e n t e s t e d as h y p o l i p e m i c agents, some of t h e m h a v i n g i m p o r t a n t i n h i b i t o r y effect on h e p a t i c cholesterogenesis b y m e c h a n i s m s n o t always i n v o l v i n g direct action of t h e d r u g u p o n t h e r e g u l a t o r y s t e p 6.

This r e p o r t deals w i t h t h e effect of 4 - m e t h y l - 5 - h y d r o x y valeric acid s o d i u m salt (MHVA) on h e p a t i c cholesterol b i o s y n t h e s i s in t h e rat. M H V A is o b t a i n e d as a side prod u c t in t h e c h e m i c a l s y n t h e s i s of p r e g n e n o l o n e f r o m diosgenin ~. Materials and methods. Male W i s t a r rats, 120 t o 150 g, m a i n t a i n e d on s t a n d a r d l a b o r a t o r y c h o w s u p p l e m e n t e d as i n d i c a t e d , were used in all e x p e r i m e n t s . 4 - m e t h y l - 5 h y d r o x y valeric acid was k i n d l y s u p p l i e d b y D r F. GIRAL (Nat. U n i v . lViexico, Sch. Chem.). 1-14C-sodium a c e t a t e (62 mCi/ mmole) a n d Dz-2-SH-mevalonic acid l a c t o n e (82 mCi/mmole) were o b t a i n e d f r o m T h e R a d i o c h e m i c a l

15.9. 1976

1139

Speeialia

C e n t r e . 3 - 1 4 C - 3 - h y d r o x y - 3 - m e t h y i g l u t a r i c a c i d (2.19 m C i / mmole) was obtained from New England Nuclear Corp. All other chemicals were analytical reagents. L i v e r slices, a p p r o x i m a t e l y 1 m m t h i c k , w e r e p l a c e d in 2.5 m l of K r e b s R i n g e r p h o s p h a t e b u f f e r , p H 7.4, a n d i n c u b a t e d 2 h a t 37 ~ w i t h e i t h e r 5 tzCi of s o d i u m a c e t a t e o r 2 ~xCi of D L - m e v a l o n i c a c i d l a c t o n e , lVitlVA w a s d i s s o l v e d in 0.2 M p h o s p h a t e b u f f e r , p H 7.4. T h e r e a c t i o n w a s s t o p p e d b y t h e a d d i t i o n of 3 0 % a l c o h o l i c K O H a n d 5 m g of c h o l e s t e r o l a s c a r r i e r . F o r t h e in v i v o e x p e r i m e n t s , t h e animals received daily for 4 days, by stomach tube, the M H V A s o l u t i o n in 0.2 M p h o s p h a t e b u f f e r , p H 7.8, o r a n e q u i v a l e n t v o l u m e of b u f f e r s o l u t i o n . T h e a d m i n i s t r a t i o n w a s d o n e a t a p p r o x i m a t e l y t h e s a m e t i m e of t h e d a y . O n the 4th day, 1 h after the oral administration, each rat r e c e i v e d i.p. e i t h e r 5 ~zCi of s o d i u m a c e t a t e o r 2 ~zCi of D L - m e v a l o n i c a c i d l a c t o n e p e r 100 g of b o d y w e i g h t . T i l e r a t s w e r e k i l l e d 1 h l a t e r . T h e i s o l a t i o n of l i v e r c h o l e s t e r o l w a s c a r r i e d o u t a s i n d i c a t e d f o r t h e l i v e r slices, t h e c a r r i e r o m i t t e d . T o t a l s e r u m c h o l e s t e r o l w a s m e a s u r e d 8. C h o l e s terol from the liver was isolated with digitonin and radioactivity was measured in a liquid scintillation spectrom e t e r 9. T h e a c t i v i t y o f H M G C o A r e d u c t a s e i n t h e l i v e r m i c r o s o m e s w a s a s s a y e d ~~ p r e v i o u s l y p r e p a r i n g t h e a n h y d r y d e of 3 - x a C - 3 - h y d r o x y - 3 - m e t h y l g l u t a r i c a c i d 11. Microsomal protein was determined with Folin-phenol

R e s u l t s . T h e a d d i t i o n of d i f f e r e n t c o n c e n t r a t i o n s of M H V A p r o d u c e d a d e c r e a s e in a c e t a t e i n c o r p o r a t i o n i n t o c h o l e s t e r o l i n l i v e r s!ices. T h e e f f e c t w a s d o s e - d e p e n d e n t ( F i g u r e 1). I n t h e c a s e of m e v a l o n a t e i n c o r p o r a t i o n i n t o c h o l e s t e r o l , M H V A w a s also i n h i b i t o r y b u t a t h i g h e r c o n c e n t r a t i o n s ( F i g u r e 1). lV[HVA a d m i n i s t e r e d p a r e n t e r a l l y h a d n o e f f e c t o n acetate incorporation at doses that ranged from 0 to 1 m M p e r 100 g of b o d y w e i g h t . O n t h e o t h e r h a n d , o r a l administration had a critical influence on cholesterol s y n t h e s i s ( F i g u r e 2). T h e d o s e s - r e s p o n s e c u r v e s h o w s t h a t a c o n c e n t r a t i o n of 0.32 m M p e r 100 g of b o d y w e i g h t p r o duced 20% inhibition of acetate incorporation into cholesterol. The maximum inhibition was obtained with d o s e s f r o m 0.97 t o 1.95 m M p e r 100 g of b o d y w e i g h t . I n t h e r e s t of t h e e x p e r i m e n t 1.88 m M p e r 100 g of b o d y weight was employed. This dose inhibits 30% mevalonate i n c o r p o r a t i o n i n t o c h o l e s t e r o l (84 277 -E 5 280 d p m v s 57 253 :k 87 d p m , p e r g of f r e s h l i v e r , i n t h e c o n t r o l a n d M H V A - t r e a t e d r a t s , r e s p e c t i v e l y , p < 0.05, t - t e s t ) . S e r u m

100 %

r e a g e n t 12.

g_ 5C 100 9"~'x

-- - - ~ Mevalonate

%

0.;5

50

1.30

1.95

MHVA (mmotes per 100g of body weight)

o

~r

1'5 26 MHVA concentration

52 mM

Fig. 1. In vitro incorporation of Labelled acetate or mevalonate into cholesterol ill liver slices. The results represent the activity ill cholesterol from 200 mg liver slices, expressed as the percentage of the control value: 14061 i 2413 dpm per 200 mg of liver slices and 112121 • 16758 d p m per 200 rag of liver slices for acetate and mevalonate, respectively. Each point of the curve corresponds to the mean of 3 determinations.

1 M. D. SIPERSTEIN, Curr. Top cell. Reg. 2, 65 (1970). 2 D. SnAFIRO and V. W. RODWELL, Biochemistry Jl, 1042 (1972). a G. C. NESS, R. E. DUGAN, IV[. R. LAKSHMANAN, C. M. NEPOKROEFF and J. W. PORTER, Proc. natn. Acad. Sci., USA 70, 3839 (1973)i 4 S. GOLDFARB and H. C. PITO% J. Lipid Res. 13, 797 (1972). 5 D. KRITCHEVSKY, S. A. TELLER and J. A. STORY, Proc. Soc. exp. Biol. Med. 195, 12 (1974). s R. HESS and W. L. BANCZE. Experienda 24, 418 (1968). 7 F. GIRAL and J. GIRAL, her. Chem. her. 93, 2825 (1960). 8 L. L. ABELL, B. B. LEVY, B. B. BRODIE and F. E. KENDALL, J. biol. Chem. 195, 357 (1952). 9 j . C. DfAz-ZAGOYA, J. LAGUNA and J. GUZMXN-GARciA,Biochem. Pharm. 20, 3473 (1971). 10 S. SHEFER, S. HAUSER, V. LAPAR and E. H. MOSBACH, J. Lipid Res. 73, 402 (1972). 11 A. I. Louw, I. BEKERSKY and E. H. MOSBACH,Biochem. Prep. 13, 62 (1971). 13 E. F. HARTREE,Analyt. Biochem. dS, 422 (1972).

Fig. 2. In vivo incorporation of sodium acetate illto cholesterol in the liver of rats treated with MHVA. The results are expressed as the percentage of the control value: 810 ~ 121 dpm per mg of cholesterol. Each point corresponds to the mean of 4 rats, which received orally the indicated MHVA dose in phosphate buffer, pH 7.8 for 4 days. At the 4th day, 1 h after the MHVA administration, each rat received i.p. 5 vCi of (1-14C)-sodium acetate. The animals were sacrificed 1 h later. Cholesterol was isolated in the liver as the digitonide.

Table I. In vivo incorporation of radiolabeled acetate into cholesterol in rat liver Group

m M g-1 h-1

p

Control (4) ~ MHVA (4) 1% cholesterol (4) 1% choIesterol + MHVA (4) 1% diosgenin (4) 1% diosgenin + HMVA (4)

1600 :~ 511:~ 656 ~ 386 ~ 4624 ~ 2242 ~

0.10

50 b 87 241 130 338 432

The animals received the diets indicated for 6 days. Every day for 4 days before their sacrifice 1.88 m M per 100 g of body weight of MHVA were administered orally. 1 h before their killing each rat received i.p. 5 ~xCiper 100 g of body weight of (1-14C)-sodium acetate, Spec. act. 0.166 mCi/mM. The results are expressed as n m of acetate incorporated into cholesterol per gram of liver per h. ~Number of rats of each group is indicated in parenthesis, bStandard error of the mean. ~p-value when compared individually with control group, student t-test.

1140

Specialia

cholesterol in M H V A - t r e a t e d r a t s was 84.8 g: 4.1 m g / 100 m l v s 88.6 • 5.8 rag/100 m l in t h e c o n t r o l g r o u p (p > 0 . 1 0 ; t-test). The liver cholesterol c o n t e n t in M H V A t r e a t e d r a t s was also similar t o t h a t of c o n t r o l r a t s (265 • 17 rag/100 g vs 260 -k 4 m g / 1 0 0 g of fresh liver in t h e c o n t r o l group). On t h e c o n t r a r y , in r a t s w i t h a 1% cholesterol diet, t h e h e p a t i c cholesterol c o n t e n t s h o w e d an 8% increase. Table I s h o w s t h a t a diet 1% cholesterol plus 0.5% s o d i u m d e o x y c h o l a t e , offered for 48 h, prod u c e d 60% i n h i b i t i o n of a c e t a t e i n c o r p o r a t i o n into hep a t i c cholesterol. The a d m i n i s t r a t i o n of M H V A to cholesterol-fed r a t s p r o d u c e d 77% inhibition. M H V A - t r e a t e d

Table II. Activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the liver of MHVA-treated, diosgenin-fed or cholesterolfed rats Group

Activity of HMG CoA rectuctase

Control (9) 9 MHVA (4) 1% diosgenin (10) 1% cholesterol (4)

5.47 -~ 0.52 b 5.31 ~ 0.28 9.48 i 3.68 2.15 • 0.15

p > 0.10 p % 0.005 p < 0.005

The animals indicated received for 4 days before their sacrifice 1.88 nlM per 100 g of body weight of MHVA, dissolved in phosphate buffer pH 7.8, or the equivalent volume of phosphate buffer alone. The animals of groups 3 and 4 received the diets indicated 48 h before their sacrifice. Reductase activity is expressed as nm of mevalonate formed per h per mg of microsomal protein. Generally 4 trials were run from each animal. The assay system consisted of 0.8 ml containing 100 mM phosphate buffer pH 7.2, 3 mM MgCI2, 3 mM NADP, 10 mM glucose 6 phosphate, 2.5 units glucose 6 phosphate detlydrogenase, 50 mM reduced glutatbione, 0.2 mM (3-1aC)-HNiG CoA and 0.7-0.9 mg microsomal protein; ~Number of rats of each group is indicated in parenthesis, bStandard error of the mean.

EXPERIENTIA 32/9

r a t s s h o w e d 69% i n h i b i t i o n of a c e t a t e i n c o r p o r a t i o n into cholesterol. Diosgenin-fed r a t s showed, as i n d i c a t e previous r e p o r t s 9, increased i n c o r p o r a t i o n of a c e t a t e i n t o cholesterol. T h e a d m i n i s t r a t i o n of M H V A t o diosgeninfed r a t s p a r t l y r e v e r s e d t h e action of diosgenin. Finally, as M H V A i n h i b i t s b o t h a c e t a t e a n d m e v a l o n a t e incorp o r a t i o n i n t o cholesterol, t h i s effect does n o t correlate w i t h a d i r e c t action on H M G CoA r e d u c t a s e a c t i v i t y . This was c o n f i r m e d w h e n t h e e n z y m e was a s s a y e d in liver microsomes. M H V A did n o t i n h i b i t t h e a c t i v i t y of H M G CoA r e d u c t a s e (Table II). On t h e o t h e r h a n d , a l~ diosgenin diet p r o d u c e d a m o d e r a t e , s t a t i s t i c a l l y significant increase in t h e a c t i v i t y of t h e e n z y m e . Also, in a g r e e m e n t w i t h p r e v i o u s reportsla, a h i g h cholesterol d i e t i n h i b i t e d tile a c t i v i t y of H M G CoA r e d u c t a s e . Discussion. A l t h o u g h we c a n n o t a s c e r t a i n t h e m e c h a n i s m of a c t i o n of M H V A w i t h t h e d a t a p r e s e n t e d in t h i s p a p e r , tile r e s u l t s s h o w t h a t t h i s valeric acid d e r i v a t i v e i n h i b i t s h e p a t i c cholesterol s y n t h e s i s f r o m a c e t a t e or m e v a l o n a t e w i t h o u t i n h i b i t i n g H M G CoA r e d u c t a s e act i v i t y . On t h e o t h e r h a n d , diosgenin-fed r a t s showed a m o d e r a t e increase in H M G CoA r e d u c t a s e a c t i v i t y comp a r e d wittl t h e 3-fold increase in a c e t a t e i n c o r p o r a t i o n i n t o cholesterol, w i t h no c h a n g e in m e v a l o n a t e incorpor a t i o n into cholesterol 9. T h e r e f o r e diosgenin increases cholesterol b i o s y n t h e s i s in t h e liver a t least as a result of increasing t h e a c t i v i t y of t h e r a t e - l i m i t i n g s t e p in bios y n t h e t i c p a t h w a y . On t h e c o n t r a r y , M H V A i n h i b i t s h e p a t i c c h o l e s t e r o l s y n t h e s i s , b u t t h i s effect is n o t a d i r e c t a c t i o n on t h e a c t i v i t y of H M G CoA r e d u c t a s e . W e c a n n o t assure t h e M H V A action m e c h a n i s m a t t h e p r e s e n t s t a t e of our research, b u t t h e effect r e p o r t e d h e r e c o n t r i b u t e s to t h e d e v e l o p i n g of n e w orally-active a g e n t s for h u m a n t h e r a p y in cholesterol m e t a b o l i s m disorders. la D. J. SHAPIROand Y. W. RODWELL,J. biol. Chem. 246, 3210 (1971).

Effect of B e n z i m i d a z o l e on N i c o t i n a m i d e A d e n i n e D i n u c l e o t i d e P h o s p h a t e P h o s p h o m o n o e s t e r a s e Activity in Wheat L e a v e s 1 H. R. GODAVARI, C. K. CHIN 2 a n d E. R. WAYGOOD

Department o/ Botany, University o/ Manitoba, Winnipeg, Manitoba (Canada R 3 T 2N2), 22 March 7976. Summary. N i c o t i n a m i d e a d e n i n e dinucleotide p h o s p h a t e p h o s p h o m o n o e s t e r a s e was isolated a n d p a r t i a l l y purified f r o m w h e a t (Triticum aeslivum L. var. Selkirk) leaves. The e n z y m e h a d KNADP value of 1,4 • 10 -~ M a n d a p H o p t i m u m of 5.9. In vitro a c t i v i t y of t h i s e n z y m e was u n a f f e c t e d b y p r e c u r s o r s of N A D (nicotinamide a n d nicotinic acid) or c y t o kinins (kinetin a n d benzimidazole). H o w e v e r , w h e n d e t a c h e d w h e a t leaves were t r e a t e d w i t h solutions of t h e s e comp o u n d s , t h e p r e c u r s o r s lowered t h e specific a c t i v i t y while t h e c y t o k i n i n s e n h a n c e d t h e a c t i v i t y . I t is suggested t h a t s p a t i a l s e p a r a t i o n a n d c o m p a r t m e n t a t i o n of t h e e n z y m e a n d its s u b s t r a t e N A D P a c c o u n t for t h e similar effect of b e n z i m i d a z o l e on b o t h . E a r l i e r i n v e s t i g a t i o n s in t h i s l a b o r a t o r y i n t o t h e effects of b e n z i m i d a z o l e on t h e m e t a b o l i s m of excised leaves p r e s e n t e d a v a r i e d a n d c o m p l e x p a t t e r n of its influences a. A m o n g s t t h e s e are t h e effect of b e n z i m i d a z o l e on s t r u c t u r e a n d i n t e g r i t y of c h l o r o p l a s t s ~ a n d t h e m e t a b o l i s m of nic o t i n a m i d e n u c l e o t i d e s 5. A m a r k e d increase w a s r e p o r t e d in N A D P c o n t e n t a n d in t h e N A D P / N A D r a t i o in det a c h e d leaves of Wheat (Triticum aestivum vat. Selkirk) t r e a t e d w i t h solutions of b e n z i m i d a z o l e or k i n e t i n ~. B e n zimidazole t r e a t e d leaves fed w i t h r a d i o a c t i v e p r e c u r s o r s a c c u m u l a t e d r a d i o a c t i v i t y in N A D P > N A D , while leaves floated on w a t e r a c c u m u l a t e d t h e r a d i o a c t i v i t y in N A n > N A D P 5, L H o w e v e r N A D b u t n o t N A D P was r e p o r t e d

to accelarate t h e senescence of c h l o r o p l a s t s in plasmolyzed p r o t o p l a s t s of Elodea leaves 8. X~OSHIDA'S w o r k was e x t e n d e d in t h i s l a b o r a t o r y to d e t a c h e d u n p l a s m o l y z e d leaves of Elodea a n d w h e a t . I n b o t h cases N A n b u t n o t N A D P a c c e l e r a t e d t h e senescence (as m e a s u r e d b y chlorosis) of d e t a c h e d leaves a n d b e n z i m i d a z o l e o v e r c a m e t h i s effectL These studies suggest t h a t t h e senescence of d e t a c h e d w h e a t leaves a n d t h e effect of c y t o k i n i n s on senescence are d i r e c t l y or i n d i r e c t l y c o n n e c t e d to t h e ratio of t h e c o n c e n t r a t i o n s of N A D a n d N A D P in w h e a t leaves. N A D kinase (EC 2.7.1.23) w h i c h p h o s p h o r y l a t e s N A D to N A D P 9 a n d N A D P - p h o s p h a t a s e (EC 3.1.3.2) w h i c h h y d r o l y z e s N A D P t o N A D 10,11 a p p e a r t o p l a y i m p o r t a n t