Effect of prednisolone and hydrocortisone on distribution of mucopolysaccharides in the skin of rats

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135

M. D. MELAMED, Biochem. J., 92 (1964) i 2 P . M. JoTISZ, M. KAMINSXI AND J. LEGAULT-DEMARE, Biochim. Biophys. Acta, 23 (1957) 173M. B. RHODES, N. BENNETT AND R. E. FEENEX', J. Biol. Chem., 235 (196o) 1686. A. CttATTERJEE AND R. MONTGOMERY, A¥ch. Biochem. Biophys., 99 (1961) 426. E. FREDERICQ AND H. F. DEUTSCH, J. Biol. Chem., 181 (1949) 499. F. R. JEVONS, Biochim. Biophys, Acta, 45 (196o) 384 . D. AMINOFF, Biochem. J., 8I (1961) 384 . B. KETTERER, Life Sci., (1962) 163. F. K. HARTLEY AND F. R. JEVONS, Biochem. J., 84 (1962) I34. C. CESSI AND F. PILIEGO, Biochem. J., 77 (196o) 5o8. N. M. GREEN, J. Biol. Chem., 205 (1953) 535. G. W. SCHWERT AND Y. TAKENAKA, Biochim. Biophys. Acta, 16 (1955) 57 o.

Received November 25th, 1964 Biochim. Biophys. Acta, IOI (1965) 133-135

sc 83Ol 4

Effect of prednisolone and hydrocortisone on distribution of mucopolysaccharides in the skin of rats In an earlier publication SCHILLER AND DORFMAN 1 demonstrated decreased uptake of [I-14C~acetate by hyaluronic acid and chondroitin sulfate, as well as decreased uptake of 35S by the latter in skin of rats pretreated with cortisone acetate. They also reported that administration of hydrocortisone acetate caused a gradual decrease in the rate of turnover of the mucopolysaccharides in rat skin. Such studies on uptake or rates of disappearance of label suggest inhibition of synthesis. This interpretation, derived from the use of radioactive tracers, is reasonable only if a steady state is known to exist since similar results can be obtained from increases in pool sizes of mucopolysaccharides and/or their precursors. The present investigation was undertaken to study the levels of mucopolysaccharides in the skin of rats under the influence of hydrocortisone and prednisolone. Prednisolone was investigated because of its importance as an anti-inflammatory agent. Male rats of the Sprague-Dawley strain, weighing 15o-158 g were divided into four groups of 7 rats each. Cholesterol, hydrocortisone and prednisolone were suspended in a solution of 0.9% NaC1 containing lO°/0 ethanol and 2 drops of Tween 80 per ml. Cholesterol and hydrocortisone were diluted to a final concentration of 20 mg/ ml and prednisolone to 5 mg/ml. Each rat received subcutaneous injections of o.12 ml twice daily for IO days. The doses were selected on the basis of the fact that the anti-inflammatory activity of prednisolone is considered to be 3-5 times that of hydrocortisone. After 7 days of injections, the daily dose of hydrocortisone was reduced from 4.8 mg to 2.5 mg since the animals in this group were losing weight. Control rats were injected daily with o.12 ml of the diluent. The animals were killed 18 h after the last injection and the mucopolysaccharides were isolated from the skin b y quantitative techniques described in a previous publication 2. Uronic acid as determined b y the carbazole method of DlSCHE 3, was used as an indicator of mucopolysaccharide concentrations. By this method, the color equivalent of the uronic acid moiety of chondroitin sulfate from skin is depressed whereas that of heparin is enhanced. Biochim. Biophys. Acta, zoi (I965) 135-I37

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The results i n d i c a t e d in Table I show a m a r k e d decrease in c o n c e n t r a t i o n of b o t h c h o n d r o i t i n sulfate a n d h y a l u r o n i c acid in r a t s p r e t r e a t e d with hydrocortisone. I n c o n t r a s t , p r e d n i s o l o n e - t r e a t e d a n i m a l s d e m o n s t r a t e d a similar decrease in chond r o i t i n sulfate, b u t an increase in h y a l u r o n i c acid concentration. The m u c o p o l y saccharide c o n c e n t r a t i o n s of c h o l e s t e r o l - t r e a t e d animals were similar to those found in t h e skin of control animals, d e m o n s t r a t i n g t h a t the responses to h y d r o c o r t i s o n e a n d prednisolone were n o t due to a non-specific effect of the steroid nucleus. B o d y weights of t h e animals at the t i m e of sacrifice are p r e s e n t e d in Table I as the average of each group. A l t h o u g h weight loss was o b s e r v e d in h y d r o c o r t i s o n e i n i e c t e d rats, b o d y weights of p r e d n i s o l o n e - t r e a t e d animals r e m a i n e d u n c h a n g e d d u r i n g the entire e x p e r i m e n t a l period. Since previous studies 4 have shown t h a t weight loss per se a p p e a r s to e x e r t little, if any, descernible influence on the concent r a t i o n of h y a l u r o n i c acid or c h o n d r o i t i n sulfate in skin, it is inferred t h a t the loss of weight was not responsible for the results o b t a i n e d with hydrocortisone. TABI.E I T H E E F F E C T OF H Y D R O C O R T I S O N E IN S K I N OF RATS

Trea!~nenl

~on{2

AND PREDNISOLONE

Daily dose (rag)

Body weights (g)

O

224 222 1t 7 154

Cholesterol 4.8 Hydrocortisone 4.8" P~idniso]one 1.2

ON D I S T R I B U T I O N

OF M U C O P O L Y S A C C I I A R ] D E S

Distribution of mucopolysaccharides

. . . . . . . . . . . . . . . Hyaluronic Chondroilin Heparin acid sulfate (l*g uronic acid per g dry skin)

839 857 062 1049

361 337 244 264

I 5° 152 135 195

" For 7 days, then reduced to 2. 5 mg per day for the succeeding 3 days.

In a recent c o m m u n i c a t i o n , FEDIAY AND CLAY ~ r e p o r t e d t h a t the levels of h y a l u r o n i c acid a n d c h o n d r o i t i n sulfate were decreased in skin of r a t s following a 2 - d a y stress period, b u t were increased following a I 4 - d a y stress period. H o w e v e r , their values for e x p e r i m e n t a l a n d n o r m a l r a t s are v e r y m u c h lower t h a n those f o u n d c o n s i s t e n t l y b y t h e p r e s e n t authors. I n r a b b i t s t r e a t e d w i t h m e t h y l p r e d n i s o l o n e , KAPLAN AND F I S H E R 6 o b s e r v e d an increase in t h e uronic acid c o n t e n t of t h e v i t r e o u s h u m o r a n d an increase in t h e glueosamine c o n t e n t of t h e m u c o p o l y s a c c h a r i d e s of costal cartilage. They, therefore, p o s t u l a t e t h a t these effects are p r o d u c e d b y a d r e n a l corticosteroids. A l t h o u g h the increased level of h y a l u r o n i c acid f o u n d in skin of p r e d n i s o l o n e - t r e a t e d r a t s is in a g r e e m e n t w i t h the d a t a of KAPLAN AND F I S H E R 6, the p r e s e n t c o m m u n i c a t i o n d e m o n s t r a t e s , in addition, a difference in t h e response of t h e m u c o p o l y s a c c h a r i d e s of skin to h y d r o c o r t i s o n e a n d prednisolone. This difference in b e h a v i o r of t h e t w o steroids is of considerable i n t e r e s t since t h e y are q u a l i t a t i v e l y similar w i t h respect to t h e i r a n t i - i n f l a m m a t o r y a c t i v i t y . A dissociation in t h e b e h a v i o r of h y a l u r o n i c acid a n d c h o n d r o i t i n sulfate following h y p o p h y s e c t o m y or p r o p y l t h i o u r a c i l t r e a t m e n t has been r e p o r t e d preBiochim. Biophys. Acta, lOl (1965) 135-,I37

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137

viously 7. It is of interest to note t h a t the values for heparin fluctuate in the same direction as those for hyaluronic acid. This investigation was supported b y grants from The National F o u n d a t i o n and the National Institute of Arthritis and Metabolic Diseases of the United States Public Health Service (AM 05996). The authors are grateful to Dr. D. PETERSON of the Upjohn C o m p a n y for gifts of hydrocortisone and prednisolone.

La Rabida-University of Chicago Institute and the Departments of Biochemistry and Pediatrics, University of Chicago, Chicago, Ill. (U.S.A.) I 2 3 4 5 6 7

SARA SCHILLER* NELLIE BLUMENKRANTZ ALBERT DORFMAN

S. SCHILLERAND A. DORFMAN,Endocrinology, 60 (1957) 376. S. SCHILLER,G. A. SLOVERAND A. DORFMAN,J. Biol. Chem., 236 (I96I) 983. Z. DISCH~, J. Biol. Chem., I67 (1947) 189. S. SCHILLERAND A. DORFMAN,Biochim. Biophys. Acta, 78 (1963) 371. Z. FEDIAYAND M. M. CLAY, Nature, 202 (1964) 907. D. KAPLANAND B. FISHER, Biochim. Biophys. Acta, 83 (I964) lO2. S. SCHILLER,G. A. SLOVERAND A. DORFMAN,Biochim. Biophys. Acta, 58 (1962) 27.

Received October 2Ist, 1964 * Present address: Departments of Pathology and Biochemistry, New York Medical College, New York, U.S.A.

Biochim. Biophys. Acta, IOI (1965) 135-137

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On the metabolic role of~3-glucuronidase fl-Glucuronidase (fl-D-glucuronide glucuronohydrolase, EC 3.2.1.31) is an ubiquitous enzyme whose specific role in intermediary metabolism has not been ascertained, as yet. It has been implicated in the biosynthetic p a t h of L-ascorbic acid, since one of the products of the reaction is w-glucuronic acid, a clearly established precursor of vitamin C. However, a different p a t h of D-glucuronic acid formation has been suggested where fl-glucuronidase does not intervene:. Evidence for the participation of fl-glucuronidase stems from the fact t h a t the administration of drugs capable of stimulating L-ascorbic acid synthesis produces an increase of glucuronyl transferase, an enzyme responsible for the formation of fl-glucuronides, substrates of fl-glucuronidase:. To test the possibility t h a t fl-glucuronidase functions as postulated, the urinary excretion of L-ascorbic acid was measured 2 in two strains of mice, one (DBA/2j) with high and the other (C3H/Hej) with low enzyme activity3, 4 after the administration of chloretone, a c o m p o u n d causing augmentation of vitamin C excretion x. Fig. I illustrates the results of this experiment. It can be seen t h a t the two strains behaved in a similar manner. I t was further proved t h a t the corresponding levels of liver flglucuronidase in the two mice strains (4.3o and o.13/,moles of p r o d u c t per min per m g of protein) was not modified, as assayed b y the m e t h o d of LEVVY~, after extraction

Biochim. Biophys. Acta, ioi (1965) 137-14o