270

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Similar q u a l i t a t i v e results w e r e o b t a i n e d w h e n nitrobenzene, nitrofluorene, a n d p - n i t r o c r e s o l (or t h e i r nitroso a n d h y d r o x y l a m i n o derivatives) were carried t h r o u g h analogous i n v e s t i g a t i o n s .

Zusammen/assung. E i n e e l e k t r o c h e m i s c h e Sonde w u r d e entwickelt, u m A r y l h y d r o x y l a m i n d i r e k t in m i k r o s o m a l e n S u s p e n s i o n e n festzustellen. Dieser Sensor e r m i t t e l t e 15 This work was supported in part by NIH grant No, CA 14158-02 from the National Cancer Institute (USA).

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EXPERIENTIA3t[3

die B i l d u n g y o n A r y l h y d r o x y l a m i n wS.hrend der e n z y matischen Reduktion yon aromatischen Nitroverbind u n g e n d u r c h M i k r o s o m e n der K a n i n e h e n l e b e r . Die R e d u k t i o n der N i t r o g r u p p e w u r d e d u r c h beide E n z y m e , C y t o c h r o m P-450-abh~ingige u n d P-450-unabh/ingige, katalysiert. L. A. STERNSON 15

Department o/Medicinal Chemistry, School o/ Pharmacy, University o/Georgi G A them (Georgia 30602, USA), 73 November 197d.

Release of Hydroxyproline from Rat Hearts Perfused with Collagenase Various e n z y m e s , i.e. collagenase, t r y p s i n a n d h y a l u r o n idase, h a v e b e e n used e x t e n s i v e l y for dissociating tissues. D u r i n g p r e l i m i n a r y e x p e r i m e n t s in t h i s l a b o r a t o r y we f o u n d t h a t p e r f u s i o n of r a t h e a r t s w i t h s o m e p r e p a r a t i o n s of h y a l u r o n i d a s e r e s u l t e d in p a r t i a l i n a c t i v a t i o n of 5'n u c l e o t i d a s e ; for e x a m p l e , 20 rain of p e r f u s i o n w i t h S i g m a T y p e I h y a l u r o n i d a s e caused a g r e a t e r t h a n 1/3 loss of e n z y m e a c t i v i t y 1. T h e r e h a v e b e e n a n u m b e r of r e p o r t s of o t h e r f u n c t i o n a l losses due to similar t r e a t m e n t s of v a r i o u s tissues w i t h d i s p e r s i n g e n z y m e s ~-4. W e were t h e r e f o r e i n t e r e s t e d in i d e n t i f y i n g m e t h o d s w h i c h effect dissociation of h e a r t s while k e e p i n g f u n c t i o n a l losses to a m i n i m u m . As tissue dissociation was n o t r e a d i l y q u a n t i t a r e d b y d i r e c t o b s e r v a t i o n , a m o r e o b j e c t i v e result of e n z y m e a c t i v i t y was m o n i t o r e d . W e m e a s u r e d t h e release of h y d r o x y p r o l i n e , a n a m i n o acid w h i c h is c o n t a i n e d a l m o s t exclusively ill collagen a n d elastin. I n t h i s comm u n i c a t i o n we r e p o r t t h e r a t e of release of h y d r o x y p r o l i n e b y r a t h e a r t s e x p o s e d t o collagenase alone or in c o m b i n a tion with hyaluronidase and trypsin. Materials and methods. Sources of e n z y m e s : Collagenase T y p e I I I , h y a l u r o n i d a s e , W o r t h i n g t o n ; t r y p s i n 1:250,

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Difco. Chemicals were r e a g e n t g r a d e f r o m v a r i o u s sources; C14-hydroxyproline was p u r c h a s e d f r o m A m e r sham. U s i n g t h e L a n g e n d o r f f t e c h n i q u e , r a t h e a r t s were p e r f u s e d for 5 m i n w i t h c o m p l e t e H a n k ' s solution to w a s h o u t b l o o d a n d t h e n p e r f u s e d for 5 rain w i t h H a n k ' s solution f r o m w h i c h Ca ++ was o m i t t e d . R e c i r c u l a t i n g p e r f u s i o n w i t h Ca++-free H a n k ' s solution c o n t a i n i n g e n z y m e s was t h e n s t a r t e d (30~ a n d 1 m l s a m p l e s of t h i s p e r f u s a t e were t a k e n for h y d r o x y p r o l i n e a s s a y s as required. The h y d r o x y p r o l i n e c o n t e n t of t h e s a m p l e s was d e t e r m i n e d b y t h e m e t h o d of PROCKOP a n d UDENFRIEND ~

1 p. L. WOOD and K. NAKATSU,unpublished observations. D. B. )/rcNAMARA, Y, C. YATES, U. KROMER, N. L. STEPHANS and N. S. DHALLA, Proe. Can. Fedn biol. Soc. 75, 71 (1972). 3 C. S. GARAGLI and J. R. COOPER, Bioehem. Pharmac. 23, 911 (1974). 4 I. A. MACCHI, F. ZEYTINOOLUand S. B. BEASER, Proe. Sde. exp. Biol. Med. 145, 500 (1974). D. J. PROCKOP and S. UDENFRIEND, Analyt. Biochem. 1, 228 (1960).

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Fig. 1. Enzymatic release of hydroxyproline from perfused rat hearts. Rat hearts were perfused with eollagenase (1 mg/ml CLS Worthington, Type III) and[or hyaluronidase (2 mg/ml HSE, Worthington) in Hank's solution from which Ca++ was omitted. The effect of 1.25 mM Ca++ is shown by the closed circles. A recireulating perfusion system was used and aliquots of the perfusate were taken for hydroxyproline determination at the times indicated, The data are expressed as ~zmoles of hydroxyproline released per heart at the perfusion times indicated and each point with a vertical bar represents the mean of 2 to 6 hearts q- S.D.

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Fig. 2. Hydroxyproline relaese from perfused rat hearts after preperfusion with trypsin. Hearts were preperfused with trypsin (1 mg/ml) in Ca++-free Hank's solution for 10 min and then the trypsin was washed out prior to perfnsion with eollagenase. Control hearts were preperfused with Ca++-free Hank's solution. Other conditions same as for Figure 1.

15.3. 1975

271

Specialia

w i t h t h e following modification. The b o r a t e buffer of I~IVIRIKKO, LAITINEN a n d PROCKOP ~ w a s used instead of p y r o p h o s p h a t e and C14-hydroxyproline was added at the start of the assay to allow calculation of yield. The l a t t e r was necessary because our yield (usually 80%) was lower t h a n p r e v i o u s l y r e p o r t e d (about 90%)6. Results and discussion. W h e n hearts of 200-250 g rats were perfused w i t h collagenase alone, 1 mg/ml, t h e hydrolysis of collagen was c o n s t a n t for t h e first 120 rain. During this t i m e t h e rate of a p p e a r a n c e of h y d r o x y p r o l i n e in the perfusate was a b o u t 1.34 ~zmoles/h and after 120 m i n this rate decreased b y a b o u t 30% (Figure 1). A f t e r establishing t h e rate of collagen hydrolysis b y collagenase alone, we studied t h e effects of t r e a t i n g hearts w i t h hyaluronidase, Ca ++ and t r y p s i n in conjunction w i t h collagenase. S e v e r a l years ago MATEYKO and KOPAC ~ r e p o r t e d t h a t hyaluronidase increased the effectiveness of t r y p s i n in dissociating fibrous o v a r i a n tumors. T h e y a t t r i b u t e d this to one e n z y m e aiding the p e r m e a t i o n of the other b y u n m a s k i n g r e a c t i v e groups. B y analogy a similar relationship would explain the basis for the widely accepted use of collagenase in c o m b i n a t i o n w i t h hyaluronidase. Collagen is considered to be in i n t i m a t e c o n t a c t w i t h acidic mucopolysaccharides which are substrates for hyaluronidase s. I t seems reasonable t h e n t h a t r e m o v a l of acidic mucopolysaccharide b y this e n z y m e should increase t h e effectiveness of collagenase. Our d a t a h o w e v e r do not support such a prediction. I n our e x p e r i m e n t s there was little or no effect on t h e rate of h y d r o x y p r o l i n e release f r o m r a t hearts b y collagenase w h e n hyaluronidase (2 mg/ml) was added to the perfusing solution (Figure 1). After 60 rain of perfusion 1.34 -t- 0.32 tzmoles of h y d r o x y proline were released b y collagenase alone c o m p a r e d to 1.58 • 0.50 ~zmoles w h e n the perfusion solution contained b o t h collagenase (1 mg/ml) and hyaluronidase (2 mg/ml). F r o m Figure 1 it is also e v i d e n t t h a t hyaluronidase alone did not cause a n y release of h y d r o x y p r o l i n e . Our observation t h a t h y a l u r o n i d a s e did n o t alter the r a t e of release of h y d r o x y p r o l i n e was consistent over a range of a n i m a l sizes. The a m o u n t of h y d r o x y p r o l i n e released in 60 m i n by hearts of 135-175 g rats was 0.86 -t- 0.25 ~xmoles w i t h collagenase alone and 1.04 • 0.13 ~moles w i t h collagenase plus hyaluronidase. W h e n 340-510 g rats were used the values were 1.34 • 0.82 and 1.92 zk 0.42 respectively. Thus, hearts from large rats release h y d r o x y p r o l i n e faster t h a n those of small rats b u t the effect of hyaluronidase was n o t statistically significant, Ca++ is an a c t i v a t o r of collagenase 9, therefore we c o m p a r e d the rate of h y d r o x y p r o l i n e release from hearts perfused w i t h H a n k ' s solution which contained collagenase

and 1.26 m M Ca ++ w i t h t h a t from hearts perfused w i t h H a n k ' s solution which contained collagenase b u t no added Ca ++ . This cation had essentially no effect on collagen hydrolysis during the first 60 min of perfusion; the r a t e of h y d r o x y p r o l i n e release in its presence was not different f r o m t h a t in Ca++-free H a n k ' s solution (Figure 1). E x p e r i m e n t s w i t h trypsin could not be reasonably c o n d u c t e d in the same m a n n e r as those w i t h hyaluronidase because prolonged exposure to t r y p s i n causes considerable functional damage2. Therefore we perfused hearts w i t h t r y p s i n (1 mg/ml) for 10 min, washed out the t r y p s i n and t h e n started t r e a t m e n t w i t h collagenase, instead of exposing t h e m to b o t h enzymes simultaneously. I n Figure 2 it can be seen t h a t trypsin p r e t r e a t m e n t results in a small increase in h y d r o x y p r o l i n e release; this difference was not statistically significant for the 30 min samples. I n s u m m a r y , the hydrolysis of rat heart collagen b y perfusion w i t h collagenase was e n h a n c e d to a small degree by p r e t r e a t m e n t w i t h trypsin b u t was not signific a n t l y enhanced b y hyaluronidase or Ca++. This m a y indicate t h a t in cases in which hyaluronidase has been shown to enhance tissue dissociation by collagenase, the m e c h a n i s m s of e n h a n c e m e n t m a y not involve greater accessibility of substrate to collagenase.

Rdsumd. La lib6ration de l ' h y d r o x y p r o l i n e dans le caput du rat a 6t6 d6termin6e en p e r f u s a n t le coeur avec la solution de H a n k ' s c o n t e n a n t de la collag6nase. Le t a u x de lib6ration par la collag6nase n ' a pas 6t6 a u g m e n t 6 s i g n i f i c a t i v e m e n t q u a n d la hyaluronidase on le Ca++ ont 6t6 ajout6s 5, la solution utilis6e p o u r la perfusion. Une pr6perfusion avec de la trypsine a a u g m e n t 6 16g~rement le t a u x de lib6ration de l ' h y d r o x y p r o l i n e . K. NAKATSU a n d J. H . WARR 10

Department o/ Pharmacology, Faculty o/Medicine, Queens University, Kingston (Ontario, Canada), 23 September 197d. 6 K. I. KIVlRIKKO, O. LAITINEN and D. J. PROCKOP, Analyt.

Biochem. 19, 249 (1967). 7 G. M. MATEYKOand M. J. KOPAC,Ann. N.Y. Acad. Sci. 105, 183 (1963). a R. A. GELMAN and J. BLACKW~LL, Connect. Tissue Res. 2, 31

(1973). 9 S. TAKAHASHIand S. SEIFTER,Biochim. biophys. Acta 214, 556 (1970). lo This work was supported by a grant from the Medical Research Council of Canada.

A n in v i t r o D e m o n s t r a t i o n of P r o t e o l y s i s by M a c r o p h a g e s and its I n c r e a s e w i t h C o u m a r i n i C o u m a r i n and benZo-pyrones in general h a v e been shown to be v e r y effective in reducing high protein oedemas, and especially l y m p h o e d e m a 3-7. F i n e s t r u c t u r a l studies of l y m p h o e d e m a t o u s tissues h a v e shown t h a t c o u m a r i n s, and t h e related c o m p o u n d t r o x e r u t i n 9, reduce the a m o u n t of protein in b o t h the c o n n e c t i v e tissues and the lymphatics% D u e to the r e d u c t i o n in the protein's osmotic effect, the fluid and hence the o e d e m a are also reduced. T h a t the drugs do n o t reduce the protein by increasing l y m p h flow is shown b o t h b y t h e fact t h a t t h e y are effective in e x p e r i m e n t a l l y m p h o e d e m a , w h e n the

l y m p h a t i c s h a v e been tigated 7,1% and by the fact t h a t e o u m a r i n does not increase either l y m p h flow or its r e m o v a l of protein in the thoracic d u c t of rats w i t h a b u r n t leg n. I t is also unlikely t h a t the drugs w o r k b y decreasing the protein outflow from the blood vessels, since it has been shown t h a t a n u m b e r of t h e m slightly injure the blood vascular intercellular junctions in n o r m a l conditions, l y m p h o e d e m a , and t h e r m a l i n j u r y ~, ~, 10. T h a t the r e m o v a l of the protein is p r o b a b l y b r o u g h t a b o u t b y its increased catabolism in the tissues is suggested b y the fact t h a t c o u m a r i n greatly increases the r e m o v a l of proteins, b u t not t h a t of n o n - m e t a b o l i z a b l e

Release of hydroxyproline from rat hearts perfused with collagenase.

270 Specialia Similar q u a l i t a t i v e results w e r e o b t a i n e d w h e n nitrobenzene, nitrofluorene, a n d p - n i t r o c r e s o l (or...
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