15.2. 1975

Specialia

was 400 m g / k g ; 42 c o n t r o l r a t s were i n j e c t e d w i t h s i m i l a r q u a n t i t i e s of p r o p a n d i o l M E R C K only. A t d a y s 1, 3, 7, 10, 14, 17 a n d 21, a f t e r t h e f i r s t dose 3 a n i m a l s of e a c h g r o u p were r e m o v e d a n d a s a m p l e of b l o o d t a k e n f r o m t h e r e t r o o r b i t a l plexus. Besides h a e m a t o c r i t e r y t h r o c y t e s , r e t i c u l o c y t e s a n d H e i n z - b o d i e s were c o u n t e d b y m e a n s of C o u l t e r c o u n t e r , m e t h y l e n e blue s t a i n i n g a n d nile b l u e s t a i n i n g respectively. H a e m o g l o b i n was e s t i m a t e d b y t h e haemoglobin-cyanide method. The remaining animals were a l l o w e d to r e c o v e r for 21 d a y s d u r i n g w h i c h t i m e b l o o d was t a k e n in a s i m i l a r m a n n e r as a b o v e , a n d t h e same parameters measured at similar times after stopping dosing. A n o t h e r g r o u p of 5 a n i m a l s were t r e a t e d in t h e s a m e w a y w i t h A I A U in a d a i l y o v e r a l l dose of 500 m g / k g for 42 days, a n d o n t h e 43rd d a y t h e t h r o m b o c y t e s were c o u n t e d b y t h e cocaine m e t h o d a n d c o m p a r e d to 5 c o n t r o l animals. Results and discussion. If r a t s are t r e a t e d for a r e l a t i v e l y l o n g p e r i o d w i t h A I A U , t h e y show m a r k e d d i s t u r b a n c e s in l i v e r h a e m m e t a b o l i s m , w h i c h is p a r a l l e l e d b y a E M a n d h i s t o l o g i c a l l y visible p i g m e n t a t i o n of t h i s tissue 9, a n d i n c r e a s e d u r i n a r y e x c r e t i o n of p o r p h y r i n s a n d t h e i r precursors. V a r i a t i o n s were also n o t e d in s e r u m e n z y m e s a n d o t h e r s e r u m p a r a m e t e r s (RENTSCH a n d JOHNSTON 10). I n o u r e x p e r i m e n t s we were u n a b l e t o f i n d m a j o r c h a n g e s in t h e h a e m a t o p o e t i c s y s t e m . E r y t h r o c y t e s , h a e m a t o c r i t a n d h a e m o g l o b i n v a r i a t i o n s were w i t h i n t h e m a r g i n of e r r o r of o u r t e c h n i q u e s . D u r i n g t h e e x p e r i m e n t t h e r e t i c u l o c y t e s were in t h e r a n g e of 21-145 cells/1000, w h i c h is w i t h i n t h e p h y s i o l o g i c a l n o r m for t h e r a t s used. H e i n z - b o d i e s , m a r k e r s of i n t r a - e r y t h r o c y t e h a e m o g l o b i n d e s t r u c t i o n (RENTSCH a n d WITTEKIND 11), were n o t found. H e n c e i t c a n b e seen t h a t A I A U p r o d u c e s n o s i g n i f i c a n t a l t e r a t i o n s in t h e p a r a m e t e r s m e a s u r e d , e x c e p t for a s l i g h t e l e v a t i o n of t h e t h r o m b o c y t e s in t h e h i g h - d o s e

Thromboeytes of rats dosed with AIAU 500 mg/kg (T) compared with those of controls (K)

209

a n i m a l s , a r e s u l t c o n t r a r y to t h a t f o u n d in h u m a n s h y p e r s e n s i t i v e t o A I A U (see Table). LEVlN et al. 12 s u g g e s t t h a t A I A U e x e r t s its p a t h o p h y s i o l o g i c a l effects for 2 r e a s o n s : 1. t h e r e is a n allyl g r o u p in t h e molecule, a n d 2. t h e c o m p o u n d is c o n v e r t e d i n t o a n a c t i v e m e t a b o l i t e . I t is also s u g g e s t e d t h a t t h e a c t i v e f o r m is a n e p o x i d e on t h e allyl group. T h i s e p o x i d e is t h e n t h o u g h t t o r e a c t w i t h h a e m c a u s i n g its d e s t r u c t i o n in liver. W h y t h e n does A I A U n o t cause h a e m o g l o b i n d e s t r u c t i o n in b l o o d ? One a n s w e r m a y b e t h a t r e d cells are u n a b l e to m e t a b o l i z e it a n d t h e a c t i v e m e t a b o l i t e f o r m e d in o t h e r tissues n e v e r reaches t h e c i r c u l a t o r y s y s t e m . I n t h i s c o n t e x t it s h o u l d be m e n t i o n e d t h a t o t h e r tissues besides liver c a n p r o d u c e t h e a c t i v e m e t a b o l i t e . W e h a v e f o u n d a g r e e n p i g m e n t a t i o n in i s o l a t e d m i c r o s o m a l p r e p a r a t i o n s of l u n g a n d k i d n e y f r o m r a t s t r e a t e d w i t h A I A U 10. T h i s i n d i c a t e s a s i m i l a r p a t t e r n to t h a t i n t h e l i v e r w h e r e A I A U causes h a e m b r e a k d o w n p r o d u c t s f r o m c y t o c h r o m e P-450, c o l o u r i n g t h e microsomes. Tissues c a p a b l e of p r o d u c i n g t h e a c t i v e molecule p r o b a b l y b i n d it t i g h t l y a n d r a p i d l y to s t r u c t u r e s n e a r t h e site of its f o r m a t i o n . T h i s m e a n s t h a t t h e effective halflife of t h e n e w c o m p o u n d f o r m e d m u s t b e e x t r e m e l y short. I n t h e k i d n e y , t h e r e is t h e p o s s i b i l i t y of a n allyl g r o u p e p o x i d e f o r m i n g a n d b e i n g r a p i d l y e x c r e t e d d u e to t h e special n a t u r e of t h e t u b u l a r s y s t e m . E p o x i d e s f o u n d in r a t u r i n e a f t e r a d m i n i s t r a t i o n of A I A s u p p o r t t h i s la,1..

Zusammen/assung. W e n n R a t t e n lXngere Zeit m i t 2A l l y l - 2 - i s o p r o p y l a c e t y l h a r n s t o f f b e h a n d e l t werden, find e n sich in v e r s c h i e d e n e n O r g a n e n g r i i n - b r a u n e P i g m e n t e . Diese v o r w i e g e n d in M i c r o s o m e n l o k a l i s i e r t e n P r o d u k t e s t a m m e n v o m H~tm des C y t o c h r o m s P-450 sowie a n d e r e n H ~ m o p r o t e i n e n . A m hS~matopoetischen S y s t e m k o n n t e n d e m g e g e n t i b e r k e i n e V e r / i n d e r u n g e n g e f u n d e n werden. D a m i t i s t d e r Schluss g e s t a t t e t , dass d e r fiir die H / i m d e n a t u r i e r u n g v e r a n t w o r t l i c h e a k t i v e M e t a b o l i t eine sehr k u r z e H a l b w e r t z e i t h a b e n muss. G. RENTSCH a n d A. JOHNSTON

Animal number

Thrombocytes per ~1

K1 K2 K3 K4 K5 K average T1 T2 T3 T4 T5 3' average

836 • 10a 772 • 10a 974 • 10a 782 • 103 1102 x 103 893 • 103 9 1268 • 10a 1366 • 10a 1252 • 10a 1068 • 10a 1204 X 10a 1232 • 103b

SD : 4- 142 • 10~; b SD: 4- 109 x 103; P(0,01 See text for full experimental details.

Inhibition of the Local Hemorrhagic Inhibitor,' Pepstatin

Shwartzman

W h e n bac,terial e n d o t o x i n is i n j e c t e d i n t o r a b b i t s i n t r a c u t a n e o u s l y , followed in 24 h b y a n i.v. i n j e c t i o n of a n o t h e r endotoxin preparation, an inflammatory and necrotic r e a c t i o n is m a n i f e s t e d a t t h e site of i n t r a c u t a n e o u s i m p l a n t a t i o n of t h e m a t e r i a l 1, 2. T h e m e c h a n i s m of t h i s

Biological and Medical Research Division, Sandoz Ltd., CH-4002 Basel (Switzerland), 2 October 1974.

9 G. RENTSCn, A. JOHNSTON, CH. HODEL and A. PATAKI, in preparation. 10 G. RENTSCH and A. JOHNSTON, in preparation. 11 G. RENTSC~ and D. WITTEKIND, Toxic appl. Pharmac. 77, 8t (1967). 12 W. LEVlN, ~[. JAGOBSON, 1~. SIERNATINGERand R. KUNTZMAN, Drug Metab. Dispos. 7, 275 (1973). 13 D. J. DOEm;NS, Diss. Abstr. 32, 2951 B (1971). 14 The authors wish to thank Dr. G. R fJTTIMANNand Miss H. MAUR]ER for their technical help.

Reaction

by an Acid Proteinase

local h e m o r r h a g i c SHWARTZMAN p h e n o m e n o n is unclear. However, evidence indicates that a granulocyte mediated i n f l a m m a t o r y process a-5 is i n v o l v e d a l o n g w i t h t h e effect of e n d o t o x i n u p o n t h e b l o o d vessels a n d l y m p h o r e t i c u l a r cellsS, L C o m p l e m e n t 8 as well as t h e b l o o d c o a g u l a t i o n

210

Specialia

s y s t e m 2, 9 - n has also b e e n i m p l i c a t e d in t h i s p a t h o l o g i c p h e n o m e n o n . I t is likely t h a t t h e SHWARTZMAN r e a c t i o n is a c o n s e q u e n c e of cascade r e a c t i o n s in w h i c h e n z y m e activities could p l a y a role. T h e intracellular p r o t e o l y t i c e n z y m e s h a v e b e e n s u g g e s t e d to be essential in t h e g e n e r a t i o n of t h i s r e a c t i o n *, a n d t r y p s i n was r e p o r t e d to p o t e n t i a t e it ~. If t r y p s i n i n h i b i t o r s were i n j e c t e d i.v. s h o r t l y before t h e t i m e of t h e p r o v o c a t i v e e n d o t o x i n injection, t h e e x p r e s s i o n of t h e SHWARTZMAN p h e n o m e n o n was suppressed~a, ~. I t was s u g g e s t e d t h a t t h e tissue d a m a g e o b s e r v e d in t h e p h e n o m e n o n was c o n d i t i o n e d d i r e c t l y b y or t h r o u g h release of p r o t e i n a s e s , p a r t i c u l a r l y t h e l y s o s o m a l e n z y m e s c o n t a i n e d in granulocytes4. S o y b e a n trypsin inhibitor and bovine organ trypsin inhibitor (Trasylol) are k n o w n i n h i b i t o r s of k i n i n g e n e r a t i o n a n d of t h e t r y p s i n - l i k e p r o t e i n a s e s p a r t i c i p a t i n g in t h e b l o o d c o a g u l a t i o n processes ~5, b u t riley do n o t i n h i b i t t h e acid p r o t e i n a s e s , such as c a t h e p s i n D ~s, w h i c h is t h e m a j o r p r o t e i n a s e of r a b b i t nentrophils~L This class of acid p r o t e i n a s e s h a s b e e n i m p l i c a t e d b y DINaLE ~8 in t h e p a t h o l o g i c a l d e g r a d a t i o n of cartilage m a t r i x . If t h e proteolytic enzymes derived from the granulocytes have i n d e e d a n essential f u n c t i o n in eliciting t h e SI~WARTZ~IAN p h e n o m e n o n , t h e possible p a r t i c i p a t i o n of t h e acid p r o t e i n a s e s c a n n o t be neglected.

25

I

i

I

20

o

x E

15

~,

I0

g n

4

6

8

I0

Reaction pH

Fig. 1. pH profile of the proteolytic activity of rabbit peritoneal polymorphonuelear leukocytes. The cells (about 4 x 105), prepared according to COHI~and HIRSCH~s were homogenized with a Polytron homogenizer after freezing and thawing in 10 ml of saline solution. The material was centrifuged for 5 rain at 400 x g, and 0.2 ml aliquots of the supernatant fraction were used for the assay. The general proteolytie activity on methyl-14C-glycinated hemoglobin29 was determined after a 2 h-incubation at 37~ (9169 0.4 M sodium citrate buffer and 0.5 M Tris-HC1 buffer were used in the pH range of 2 to 5.5 and 6 to 9, respectively. The effects of the following proteinase inhibitors were also examined in the entire pH range tested : hydroxystilbamidine at 1.9• -5 M (A--A), soybean trypsin inhibitor at 0.8• .5 M (10 ~xg/ml) (A--&), and pepstatin at ] .5 • 10-s M (0.01 ~zg/ml) (@--@).

EXPERIENTIA31/2

I n t h i s c o m m u n i c a t i o n , we p r e s e n t e v i d e n c e i n d i c a t i n g that by proper administration route and amount, a specific i n h i b i t o r of acid p r o t e i n a s e , p e p s t a t i n ~9, can s u p p r e s s t h e SltWARTZMAN p h e n o m e n o n . This s u g g e s t s t h e i n v o l v e m e n t of s t e p s c a t a l y z e d b y acid p r o t e i n a s e s in t h e reaction. Materials and methods. A d u l t f e m a l e w h i t e r a b b i t s (New Zealand), w e i g h i n g 1.5-2.5 kg, were p a r t i a l l y d e p i l a t e d of t h e a b d o m i n a l h a i r w i t h t h e aid of clippers a n d a d e p i l a t o r y agent. T h e p r o t e i n a s e i n h i b i t o r s e m p l o y e d were c r y s t a l l i n e s o y b e a n t r y p s i n i n h i b i t o r (Calbiochem, California), p e p s t a t i n (a generous gift of Dr. H. U~EZAWA, I n s t i t u t e of Microbial C h e m i s t r y , Tokyo), h y d r o x y s t i l b a m i d i n e i s e t h i o n a t e (May & Baker, N e w York), a n d Compound A Esodium-2-(N-methyl-4-octylbenzenesulfonic) e t h a n e s u l f o n a t e ] (General Aniline & Film, N e w York). The illhibitors were e i t h e r m a d e up in a s o l u t i o n or a fine s u s p e n s i o n in p y r o g e n free saline or in 50% sterile d i m e t h y l s u l f o x i d e (DMSO) in saline, a n d were a d m i n i s t e r e d i n t r a d e r m a l l y 30 rain before t h e p r o v o c a t i v e i n j e c t i o n of e n d o t o x i n . F o r e v a l u a t i o n of i n h i b i t o r s a p p l i e d i n t r a d e r m a l l y , 4 t e s t sites on e a c h a n i m a l were used for v a r i o u s i n h i b i t o r s a n d a control. I n t h e e x p e r i m e n t using i.v. i n j e c t i o n of i n h i b i t o r s in 20% D M S O - s t e r i l e saline, 2 r e a c t i o n sites were p r e p a r e d o n e a c h animal. Ill all cases t h e p r e p a r a t i v e dose of e n d o t o x i c lipop o l y s a c c h a r i d e ( L i p o p o l y s a c c h a r i d e W E. colt 026:B6, Difco L a b o r a t o r i e s , Michigan) was 50 ~g, given i n t r a d e r m a l l y in a t o t a l v o l u m e of 0.5 m l of saline solution. T h e p r o v o c a t i v e dose w a s 100 or 250 ~g of e n d o t o x i n in 1 m l of p y r o g e n free saline solution, given i.v. 22 t o 24 h following t h e p r e p a r a t i v e dose. The e x t e n t of h e m o r r h a g i c lesions was d e t e r m i n e d b y t h e m e a s u r e m e n t of a h e m o r r h a g i c area size in millimeters, b y scoring of t h e a p p a r e n t i n t e n s i t y of h e m o r r h a g e a n d b y e s t i m a t i o n of t h e abs o r b a n c e s a t 430 n m of t h e 2% s o d i u m c a r b o n a t e e x t r a c t of a 40 • 40 m m excized t e s t area of skins. T h e s e d e t e r m i n a t i o n s were m a d e e i t h e r a t 5 or 20 h following t h e p r o v o c a t i v e i n j e c t i o n of e n d o t o x i n . I n a p o s i t i v e response, t h e skin sites p r e p a r e d d e v e l o p e d t y p i c a l h e m o r r h a g i c lesions w i t h i n 5 h.

1 G. SHWARTZMAN,J. exp. Ned. 48, 247 (1928). C. A. STETSON, J. exp. Med. 93, 489 (1951). a C. A. STETSON and R. A. GOOD, J. exp. Med. 93, 49 (1951). 4 L. THOMAS,Proc. Soe. exp. Biol. Med. 115, 235 (1964). 5 R. G. HoRY, J. infect. Dis. 128, S134 (1973). 5 L. THomAs, Arch. int. Med. 101,452 (1958). 7 A. C. ALLISOI%P. DAVIESand R. C. PAGE,J. infect. Dis. 128, $212 (1973). s j. S. C. FONG and R. A. GooD, J. exp. Med. 134, 642 (1971). s R. M. DEsPR~z, H. I. HOROWITZand E. W. HooK, J. exp. Med. 114, 857 (1961). 10 D. G. McKAu and S. S. SHAPIRO, J. exp. IVfed. 107, 353 (1958). i1 j. LEvIN and L. E. CLUF~, J. exp. Med. 121, 235 (1965). 12 W. ANTOPOL and C. CHRYSSANTHOU, Proe. Soe. exp. Biol. Med. 103, 725 (1960). 13 C. CHRYSSANTKOUand W. ANTOPOL, Proc. Soc. exp. Biol. Med. 108, 587 (1961). 14 B. N. HALPERN, Proe. Soc. exp. Biol. Ned. 115, 273 (1964). 15 R. VOGEL, I. TRAIJTSCHOLD and E. WERLE, Natural Proteinase Inhibitors (Academic Press, New York 1968), p. 26-29 and 76-95. is A. J. BARRETT, in Lysosomes (Ed. J. T. DINGLE; American Elsevier, New York 1972), p. 98. 1~ C. G. COCHRANEand B. S. AIKIN, J. exp, Med. 124, 733 (1966). 15 j. T. DINGLE, in Rheumatoid Diseases (Eds. J. J. A. DUTHIE and W. R. M. ALEXANDER;Edinburgh University Press 1969), p. 80. IS H . UMEZAWA, T. AOYAGI, H . MORISHIMA, M, MATSUZAKI, iVY. HAMADAand T. TAKEUCHI,J. Antibiot. 23, 259 (1970).

15.2. 1975

211

Speeialia

Results and discussion. T h e results oI t h e i n t r a d e r m a l a p p l i c a t i o n of p r o t e i n a s e i n h i b i t o r s are s h o w n in T a b l e I. E a c h of tile i n h i b i t o r s e x a m i n e d caused s u p p r e s s i o n of t h e SI~WARTZ~AN r e a c t i o n t o v a r y i n g degrees w h e n c o m p a r e d t o t h e controls. H o w e v e r , a dose r e s p o n s e r e l a t i o n s h i p was difficult t o e s t a b l i s h in t h e dose r a n g e t e s t e d . F o r e x a m p l e , s o y b e a n t r y p s i n i n h i b i t o r g i v e n in a m o u n t s of 0.5, 1.0 a n d 2.0 m g p e r site was s u p p r e s s i v e in e a c h case, b u t no c o r r e l a t i o n could be o b s e r v e d b e t w e e n t h e dose g i v e n a n d t h e e x t e n t of lesions. This is due p a r t i a l l y t o t h e i n s e n s i t i v i t y of q u a n t i t a t i v e e v a l u a t i o n of t h e p h e n o m e n o n , b u t m a y also be due t o t h e s t e p w h i c h is s e n s i t i v e t o t h e p r o t e i n a s e intlibitors occurring a t tile e a r l y s t a g e of t h e series of c a s c a d e reactions.

Fig. 2. Control. The excized skins (the dermis side) from the rabbits given intradermal injection of 50 [zg of endotoxin followed 24 h later by 250 ~g of endotoxin in 1 ml of saline i.v. 30 rain prior to the provocative endotoxin injection animals were given 4 ml of 20% DMSO in saline i.v. Soybean trypsin inhibitor. The excized skins from rabbits subjected to the same treatment as the control group but given 2.4 mg of soybean trypsin inhibitor in 20% DMSO-saline i.v. prior to the provocative injection. Pepstatin. The exeized skins from the animals with the same treatment of the controls but given 4 mg of pepstatin in 20% D~SO-saline, i.v. prior to the provocative endotoxin injection.

U n d e r t h e e x p e r i m e n t a l conditions, s o y b e a n t r y p s i n i n h i b i t o r r e d u c e d t h e h e m o r r h a g i c lesions due t o t h e SI~WARTZMAN p h e n o m e n o n on a n a v e r a g e of a b o u t 65% w i t h r e s p e c t t o t h e control. T h e acid p r o t e i n a s e i n h i b i t o r , p e p s t a t i n (mol. wt. 685.9), a d m i n i s t e r e d in a s u s p e n s i o n of saline or 50% D M S O a t 0.5, 1.0, a n d 2.0 m g dose p e r site, p a r t i a l l y s u p p r e s s e d t h e m a n i f e s t a t i o n of t h e localized SHWARTZMAN reaction. H o w e v e r , t h e e x t e n t of i n h i b i t i o n b y p e p s t a t i n , on an overall average, w a s n o t as r e m a r k a b l e as tile p r o t e c t i v e effect of s o y b e a n t r y p s i n i n h i b i t o r (tool. wt. 21,500), w h i c h was c o m p a r e d a t less t h a n 1120 t h e m o l a r q u a n t i t y of p e p s t a t i n . Since t h e dissociation c o n s t a n t ( K d for cathepsirt D - p e p s t a t i n c o m p l e x is in t h e o r d e r of 10 -s to 10 -9 M ~~ 2 m g (about 3 ~zmoles) of p e p s t a t i n p e r site could be e x p e c t e d t o e f f e c t i v e l y i n h i b i t t h e acid p r o t e i n a s e s p o t e n t i a l l y s e q u e s t e r e d a t t h e r e a c t i o n loci. Thus, t h e s t e p s w h i c h p o s s i b l y i n v o l v e acid p r o t e i n uses a p p e a r e d to be less critical to t h e d e v e l o p m e n t of t h e localized SHWARTZMAN r e a c t i o n w h e n c o m p a r e d w i t h those involving the soybean trypsin inhibitor-sensitive proteinases. N e v e r t h e l e s s , t h e i n h i b i t o r y effect of p e p s t a t i n was reproducible, a n d m o r e o v e r , o t h e r o l i g o p e p t i d e s , w h i c h i n h i b i t c a t h e p s i n D a n d p e p s i n w i t h K~ values of 10 -~ t o 10 -s M , s h o w e d a s i g n i f i c a n t r e d u c t i o n in b o t h t h e s e v e r i t y a n d incidence of h e m o r r h a g i c necrosis, while t h e n o n - i n h i b i t o r p e p t i d e s w i t h similar a m i n o acid s e q u e n c e s did n o t ( u n p u b l i s h e d results). C o m p o u n d A, w h i c h i n h i b i t s in v i t r o h e m o g l o b i n o l y s i s b y c a t h e p s i n D a t p H 4 a n d b y t r y p s i n a t p H 7, did n o t give a conclusive result, m a i n l y b e c a u s e t h i s r e a g e n t itself c a u s e d a d i r e c t i n f l a m m a t o r y r e a c t i o n of t h e skin. I n one e x p e r i m e n t , h y d r o x y s t i l b alnidine, a p o t e n t t r y p s i n i n h i b i t o r in v i t r o (K,, 6.2 • 10 -e M) 22, failed to s u p p r e s s t h e SHWARTZ~IAN reaction. T h e a c t i v i t y of p r o t e i n a s e i n h i b i t o r s t o r e d u c e tile localized h e m o r r h a g i c SHWARTZ~IAN r e a c t i o n w a s also d e m o n s t r a t e d b y i.v. a d m i n i s t r a t i o n of i n h i b i t o r s p r i o r t o t h e p r o v o k i n g ~ntravenous e n d o t o x i n i n j e c t i o n (Table II, F i g u r e 2). B o t h p e p s t a t i n a n d s o y b e a n t r y p s i n

20 S. I{UNIMOTO, T. AOYAGI, I{. 1VfORISHIMA, T. TAKEUCHI a n d

H. UM~ZAW&J. Antibiot. 25, 251 (1972). ~ T.-Y. LIN and D. S. FLt~CHTEII,9th Int. Congress of Biochemistry Abstract, 2024 (1973), p. 97. 22 j. D. GERATZ, Experientia 25, 1254 (1969).

Table I. Suppression of the SH~VARTZMANreaction by proteinase inhibitors in skirt prepared with endotoxin: intradermal administration of inhibitors Hemorrhagic lesions Agent (dose range)

Area (mm2) • intensity score (number of animals)

Suppression (%)

2% Na2CO3 extract of reaction loci b A4~onm(number of animals)

Suppression (%)

Saline control Soybean trypsin inhibitor (0.5-1 mg) Pepstatin (0.5-2 mg) Compound A (0.6-15 rag)

764 • 29.6 c (23) a 266 ~= 23.9 (17) 410 i 29.3 (17) 670 • 106 (18)

-65 46 12

10 :t0.8 ~ (10) 6.1 ~ 0.2 (9) 5.7 ,4- 0.9 (12) 5.9 ~ 0.5 (10)

-39 43 41

The results of 5 separate experiments with different amounts of inhibitors are summarized by normalization of alI other experimental data to the control level in the first experiment. The normalization factors ranged from 1.5 to 5.5. b A 16 cm2 area of the reaction loci was extracted with 10 ml of 2 % Na2CQ. The extract after filtration through glass wool was subjected to determination of A430,mand protein concentration. e Standard error of mean. a About 74% of the animals showed a positive SKWARTZ~IANreaction with an intensity score (1 to 4) of 3. The mortality rate in the control group was about 7% in 5 experiments.

212

Specialia

EXPERIENTIA 31[2

Table II. Effect of proteinase inhibitors on the localized hemorrhagic SI~WARTZMANphenomenon in skin prepared with endotoxin : intravenous administration of inhibitors Hemorrhagic lesions Agent (dose range)

Area (ram~) x intensity score (number of test areas)

Suppression (%)

2 % Na2COa extract of reaction loci A430nm(number of test areas)

Suppression (%)

Saline control Soybean trypsin inhibitor (2.4 rag) Pepstatin (4.0 rag)

1626 ~ 235 (6) ~ 334 4- 93 (8) 591 ~ 240 (8)

-80 64

11.2 4- 2.2 (6)~ 4.0 4- 0.7 (8) 3.8 4- 0.7 (8)

-64 66

S t a n d a r d error of m e a n .

i n h i b i t o r a t t h e doses t e s t e d effectively r e d u c e d t h e m a n i f e s t a t i o n of t h e localized SHWARTZ~A~ p h e n o m e n o n in r a b b i t s . T h e s u p p r e s s i v e effectiveness of p e p s t a t i n was less t h a n t h a t of s o y b e a a i n h i b i t o r if c o m p a r e d a t t h e s a m e m o l a r c o n c e n t r a t i o n . T h i s is c o n s i s t e n t w i t h t h e r e s u l t of t h e e x p e r i m e n t s w i t h i n t r a d e r m a l a p p l i c a t i o n of inhibitors. Prior studies have established that both platelets and g r a n u l o c y t e s are e s s e n t i a l t o t h e e x p r e s s i o n of b o t h localized a n d g e n e r a l i z e d SHWARTZ~AN reactions3,10. THOMAS 4 was able t o d e m o n s t r a t e t h a t p r e p a r a t i o n of t h e skin of r a b b i t s b y i n t r a d e r m a l i n j e c t i o n of t h e granule fraction obtained from peritoneal granulocytes, followed b y i.v. i n j e c t i o n of e n d o t o x i n , r e s u l t e d in a SHWA~TZSIAN t y p e h e m o r r h a g i c lesion. O u r a n a l y s i s of t h e g e n e r a l p r o t e o l y t i c a c t i v i t y in r a b b i t p o l y m o r p h o n u c l e a r leukocytes, as s h o w n in F i g u r e 1, r e v e a l e d t h a t t h e m a j o r a c t i v i t y is in t h e acidic r a n g e a n d little t r y p s i n i n h i b i t o r - s e n s i t i v e general p r o t e o l y s i s could b e d e t e c t e d . T h e n e u t r a l a n d a l k a l i n e proteillases of l e u k o c y t e s m a y therefore have strict substrate requirements with relatively l i m i t e d h y d r o l y s i s , a n d t h e d i r e c t effect of s u c h e n z y m e s in c a u s i n g tissue d a m a g e would b e i n s i g n i f i c a n t unless t h e a c t i v i t i e s of t h e s e e n z y m e s could b e l i n k e d to some a c t i v a t i o n m e c h a n i s m s l e a d i n g t o tissue i n j u r y . T h e c a t h e p s i n D t y p e acid p r o t e i n a s e a c t i v i t i e s of l e u k o c y t e s was s h o w n to b e s i g n i f i c a n t l y i n h i b i t e d b y p e p s t a t i n

23 H. IKEZAWA, T. AOYAC-t T. TAKEUCHI and H. UMEZAWA, J Anfibiot. 2d, 488 (1971). 24 ~-. GROSS, J. LAZARand H. ORTH, Science 775, 656 (1972). 25 R. P. MILLER, C. J. POPER, C. W. WILsON and E. DEVITO, Biochem. Pharmac. 21, 2941 (1972). 26 K. K. F. NG, in Prostaglandins, Peptides and Amines (Eds. P. MANTEGAZZAand E. W. HORTON; Academic Press, New York 1969) p. 9. 27 M. OVRRTURF, IV[. LEONARD a n d W. M.

b u t was n o t affected b y t h e t r y p s i n i n h i b i t o r s a t t h e concentrations tested. Since t h e specificity of p e p s t a t i n as t h e i n h i b i t o r of p e p s i n - c a t h e p s i n D t y p e acid p r o t e i n a s e s h a s b e e n e s t a b l i s h e d b y n u m e r o u s studies2~ ~3, t h e r e s u l t s of t h e p r e s e n t s t u d y m a y b e r e a s o n a b l y i n t e r p r e t e d as e v i d e n c e of p~ssible p a r t i c i p a t i o n of c a t h e p s i n D f r o m g r a n u l o c y t e s in t h e e x p r e s s i o n of t h e localized SHWARTZMAN r e a c t i o n . The soybean trypsin inhibitor-sensitive proteinases may well b e t h o s e i n v o l v e d ill t h e b l o o d c o a g u l a t i o n s y s t e m a n d c o m p l e m e n t a c t i v a t i o n . P a t h o l o g i c a l t e r a t i o n s of t h e c o a g u l a t i o n s y s t e m followed b y t h e i n t e r a c t i o n b e t w e e n p l a t e l e t s a n d e n d o t o x i c l i p o p o l y s a c c h a r i d e were d e s c r i b e d before 7,10 E v i d e n c e for i n v o l v e m e n t of t h e c o m p l e m e n t s y s t e m in t h e p a t h o g e n e s i s of t h e SHWARTZMA~ r e a c t i o n h a s b e e n o b t a i n e d < P e p s t a t i n h a s also b e e n r e p o r t e d to i n h i b i t tile r e n i n - a n g i o t e n s i n o g e n s y s t e m ~4, 25. T h e k i n i n like s u b s t a n c e s p o t e n t i a l l y g e n e r a t e d b y t h e a c t i o n of r e n i n i n b l o o d ~ could p l a y a role in t h e e x p r e s s i o n of t h e SI~WARTZ~AN p h e n o m e n o n . T h e e n z y m e renin, w h i c h c a t a l y z e s t h e s e reactiolls a t n e u t r a l p H , e x h i b i t s t h e c h a r a c t e r i s t i c s of acid p r o t e i n a s e s i n c l u d i n g p e p s i n a n d is i n h i b i t e d b y p e p s t a t i n ~. E x p l o r a t i o n of t h e m e c h a n i s m of t i s s u e i n j u r y b y use of specific e n z y m e i n h i b i t o r s m a y aid i n u n d e r s t a n d i n g a n d c o n t r o I of t h i s p a t h o l o g i c p h e n o m e n o n . T h e s y s t e m d e s c r i b e d ill t h i s c o m m u n i c a t i o n w o u l d offer a n i n t e r e s t i n g a n d v a l u a b l e in v i v o t e s t s y s t e m for e v a l u a t i o n of p h a r m a c o l o g i c a l l y useful p r o t e i n a s e i n h i b i t o r s .

Zusammen/assung. Nachweis, dass das lokale SftWARTZrdAN-Ph~Lnomen in K a n i n c h e n d u t c h i n t r a d e r m a l e o d e r i.v. G a b e y o n P e p s t a t i n , e i n e m s a u r e n P r o t e i n a s e Hemmer, oder yon Sojabohnen-Trypsin-Hemmer kurz v o r der a u s l 6 s e n d e n I n j e k t i o n y o n b a k t e r i e l l e m E n d o toxin unterdriickt werden kann.

KmKENDALL, Bioehem.

Pharmac. 23, 671 (1974). 2s Z. A. Co~N and J. G. HIRSeH, J. exp. Med. 712, 983 (1960). 29 H. R. WILLIAMSand T.-Y. LI~, Bioehim. biophys. Acts 250, 603 (1971).

TSAU-YEN LIN a n d I-I. I~. WILLIAMS

Merck Institute for Therapeutic Research, Rahway (New Jersey 07065, USA), 70 September 7974.

Effect of the V e n o m Sac C o n t e n t of the O r i e n t a l H o r n e t (Vespa M e t a m o r p h o s i s of t h e T o a d T a d p o l e (Bufo viridis) T h e v e n o m of t h e O r i e n t a l h o r n e t (Vespa orientalis) is k n o w n t o c o n t a i n several f a c t o r s c a p a b l e of p r e v e n t i n g f u r t h e r d e v e l o p m e n t of i n s e c t l a r v a e i n t o w h i c h it h a s b e e n i n j e c t e d 1. I t is r e a s o n a b l e t o a s s u m e t h a t t h e l a c k of d e v e l o p m e n t is d u e to i m p a i r m e n t of t h e h o r m o n a l balance.

orientalis)

on the

I g n o r i n g t h e m e c h a n i s m of a c t i o n of t h e v e n o m , we i n v e s t i g a t e d t h e m e t a m o r p h o s i s of t h e t o a d t a d p o l e (Bujo v~ridis) u n d e r t h e i n f l u e n c e of t h e w h o l e c o n t e n t of t h e v e n o m sac of t h e O r i e n t a l h o r n e t , as a p r e l i m i n a r y s t u d y . T h e a d v a n t a g e of u s i n g t a d p o l e s is t h a t t h e i r d e v e l o p m e n t is easily followed a n d t h a t t h e v e n o m c a n