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Significance of kinins in the coronary circulation
136
Pharmacological Research Communications, VoL I, No.2, 1969
the v a s c u l a r s y s t e m w a s p e r f u s e d w i t h s a l i n e f o r 30 m i n u t e s by r e t r o g r a d e t e c h n i q u e into the a b d o m i n a l a o r t a . The t u m o r s w e r e g r o u p e d a c c o r d i n g to w e i g h t , e x t r a c t e d w i t h s a l i n e , and s u b j e c t e d to the a p p r o p r i a t e p r o c e d u r a l m e t h o d s f o r b i o a a s a y of k l n i n , k i n i n o g e n , k a l l i k r e i n , p r e - k a l l i k r e i n , and k i n i n a s e . F i v e t u r n e r s w e r e a s s a y e d in e a c h w e i g h t g r o u p r a n g i n g f r o m Z-4Z g r a r n s . T h e t u m o r e x t r a c t s did n o t show any k t n i n o r k i n i n - f o r r n i n g a c t i v i t y . K i n i n o g e n l e v e l s i n c r e a s e d in t u m o r s up to 14 g r a m s of w e i g h t w h e r e a s k i n i n a s e a c t ~ v l t y i n c r e a s e d s h a r p l y d u r i n g the e a r l y growt;h p h a s e and t h e n r e d u c e d to i n i t i a l l e v e l s s u b s e q u e n t l y , t k c e t o n e - p r e c i p i t a t e d e x t r a c t s c o n t a i n e d k i n i n - £ o r m i n g a c t i v i t y t h a t i n c e e a s e d d u r i n g t u m o r g r o w t h and t h e n d i m i n i s h e d to a l m o s t z e r o l e v e l s in t u m o r s w e i g h i n g m o r e t h a n Z0 grarras. T h e k i n i n - £ o r m i n g a c t i v i t y of the a c e t o n e - p r e c i p i t a t e d t u m o r e x t r a c t s w a s c o m p a r e d to t h a t of cat p l a s m a k a l l i k r e i n p r e p a r e d by s i m i l a r t e c h n i q u e s . T h e a c t i v i t y f r o m the M.SLS and r a t p l a s m a w a s i d e n t i c a l w i t h r e g a r d to the pH p r o f i l e , a c t i o n of p l a n t , s y n t h e t i c , and m a m m a l i a n p r e t e n s e i n h i b i t o r s and h e p a r i n , k i n e t i c s of k i n i n f o r m a t i o n , and a c t i o n on s y n t h e t i c s u b s t r a t e s . T h e in viv...__2oe f f e c t o£ p r o t e a s e i n h i b i t o r s a n d h e p a r i n on t h e g r o w t h of the MS.LS as w e l l as the b i o c h e m i c a l p a r a m e t e r s of the ] < a l l i k r e i n - k i n i n s y s t e m a l s o w a s s t u d i e d . E p s i l o n a m i n o c a p r o i c a c i d (EACA), TtLASYLOL, and h e p a r i n w a s i n j e c t c d t~cice d a i l y i n t r a p e r i t o n e a l l y into ~ o d e n t s , 8 p e r g r o u p , be"arzng ' ~ f r e s h l y - ~ r a n s p l a n t e d t~amors. At e s t ; a b h s h e d t i m e i n t e r v a l s aniz~a'ls w e r e p e r f u s e d and t u m o r s e x c i s e d f o r w e i g h t d e t e r m i n a t i o n and e s t i m a t i o n of b i o c h e m i c a l a c t i v i t i e s . The e f f e c t on t u m o r g r o w t h and on the b i o c h e m i c a l p a r a r n o t e r s w i l l be d i s c u s s e d in d e t a i l . T h e t u r n e r - i n h i b i t o r y e f f e c t of E A C A on t'he g r o w t h of E h r ] . i c h a s c i t e s t u I n o r s a l s o w i l l be d e t a i l e d . T h e s i g n i f i c a n c e of t b e s e d a t a as r e l a t e d to v a s o m o t o r r e g u l a t o r s in t u r n e r t i s s u e w i l l be c o n sidered. ( S u p p o r t e d by USPHS g r a n t s H E - 1 1 4 9 Z and H E - 0 3 7 8 4 . )
SIGNIFICANCE OF KININS IN THE CORONARY CIRCULATION
E. Bassenge, E. Werle, J. Holtz and H. Fritz Physiologi,sehes Institut und Institut fur Kiini~ehe Chemie und KlinischeBiochemieder Univer~it/it, Miinehen
Expcrirmnts were carried out in open chest dogs in order to investigate the role of kinins in tile regulation of coronary blood flow. Small areas of tile beating clog heart in_ ~ were perfused under constant pressure with tile animal's own blood, following pentobarbital anesthesia. Mean and phasic coronary inflow were recorded by an electromagnetic flowmeter attached to the extracorporeal perfusion system. Carboxypeptidase B, or several inhibitors of this enzyme were administered intracoronarilly with a constant infusion pump Isometric tension was measured by a Walton-Brodie strain gauge arch attached to the perfused area of the left ventricle. Four series of experiments were carried out in order to study the role of kinins in coronary regulation.
Pharmacological Research Communicat/ons, Vo/. 1, No.2, 1969
137
1 - Application of specific kinin antagonists was not satisfactory because all tl~t: substances tested riffled to suppress the effect of kinins on coronary flow. 2 - Experimental increase in concentration of the kininase, carboxypeptidase B in blood resulted in a cessation of the hyperemie response following intracoronary kinin administration in doses ranging from 10-7 to 10 "2 rag, whereas the hyperemic responst~ due to 25 secomls of lscht~rnia remained unatTected. 3 - During constant intraeoronary infilsion of CdSO4, CaE!YfA, 8-hydroxyquinoltn sulpltonie aqid or tl~ioglycolic acid, which inhibit kininases~ the dilating effect of kinins administered intracoronarilly was significantly prolonged; reactive hyper¢mia, however, remained essentially unchanged under these conditiorls, Kininase inhibitors in concentrations ranging from 10-4 to ~0"2 molesper litre coronary blood, had no effect on coronary flow. 4 . When the myocardium was perfused with ~i suspension! containing washed red eel|s, bt~t no kininogen, a hyperemic response following 25 seconds of iscliemia was still observed. Since tile control resistance changed, however, due to the perfiasion witll the red cell suspension, this series was not conclusive. Kinins administered intraeoronarilly did not increase isometric tension dtwelopment, The dilating effect of kinins on the coronary bed depends on the pll. Lowering the plf to 7.2 results in a more pronounced effect,; increasing pll to 7.5 lessens the effect, From the above results, i~ is concluded tlmt kinins do not play a major role in coronary regulation. LOCAL ISCHEM!A AS A KININOGEN DEPRESSANT: EFFECT OF CORTISONE, TAURINE AND KALLIKREIN INltlBITOR A. Bavazzano, N. Sidell, S. Michelaccj and F. Sicuteri Departnlent of Medicine, Unlvernity of Florence, Viale Morgagnt, Florence, Italy It w~ts postulated that the capillary bed has a dual action on tile kjninogen-kinin system: (a) release of a small quantity of kinins from their plasmatic precursor and (b) kini~t inactivation. "I'l~e cotadition (b) was demonstrated during arterial infiJsion with synthetic bradykinin, iLeeovery in reflux venous blood is minimal (Sicuteri e t ~i), 1963, Allwood el aJ, 1963); the measurement <.:f arterial-venous kininogen demonstrated a non-statistically significant diftk.rence (SicuterL~t ~J~, 1966). The aim of the present study was to observe if, with an increase in time of contact will~ capillary walls (microvessels in general), a variztion in kininogen is observable in plasma. When circulation is arrested in the superior limb of man with a sphygmomano~leter cuff" i
'J
p
~,
inflated to a pressure substantially higher than systohc, a "circulatory arrest" is erroneously sa~d to occur. In effect, a gradual refilling of the veins during ischemia is noted. Blood probably passe,~ from the general circulation to the ischemia limb tllrough m~Jrrow arteries. Bata from the present study demonstrate: a) In 2 cases, injecting tagged albumin intravenously, a certain quantity is always demonstrable in venous blood taken From the antecubital vein during 5 rain of local ischcmia.
Pharmacological Research Communications, VoL I, No.2, 1969
the v a s c u l a r s y s t e m w a s p e r f u s e d w i t h s a l i n e f o r 30 m i n u t e s by r e t r o g r a d e t e c h n i q u e into the a b d o m i n a l a o r t a . The t u m o r s w e r e g r o u p e d a c c o r d i n g to w e i g h t , e x t r a c t e d w i t h s a l i n e , and s u b j e c t e d to the a p p r o p r i a t e p r o c e d u r a l m e t h o d s f o r b i o a a s a y of k l n i n , k i n i n o g e n , k a l l i k r e i n , p r e - k a l l i k r e i n , and k i n i n a s e . F i v e t u r n e r s w e r e a s s a y e d in e a c h w e i g h t g r o u p r a n g i n g f r o m Z-4Z g r a r n s . T h e t u m o r e x t r a c t s did n o t show any k t n i n o r k i n i n - f o r r n i n g a c t i v i t y . K i n i n o g e n l e v e l s i n c r e a s e d in t u m o r s up to 14 g r a m s of w e i g h t w h e r e a s k i n i n a s e a c t ~ v l t y i n c r e a s e d s h a r p l y d u r i n g the e a r l y growt;h p h a s e and t h e n r e d u c e d to i n i t i a l l e v e l s s u b s e q u e n t l y , t k c e t o n e - p r e c i p i t a t e d e x t r a c t s c o n t a i n e d k i n i n - £ o r m i n g a c t i v i t y t h a t i n c e e a s e d d u r i n g t u m o r g r o w t h and t h e n d i m i n i s h e d to a l m o s t z e r o l e v e l s in t u m o r s w e i g h i n g m o r e t h a n Z0 grarras. T h e k i n i n - £ o r m i n g a c t i v i t y of the a c e t o n e - p r e c i p i t a t e d t u m o r e x t r a c t s w a s c o m p a r e d to t h a t of cat p l a s m a k a l l i k r e i n p r e p a r e d by s i m i l a r t e c h n i q u e s . T h e a c t i v i t y f r o m the M.SLS and r a t p l a s m a w a s i d e n t i c a l w i t h r e g a r d to the pH p r o f i l e , a c t i o n of p l a n t , s y n t h e t i c , and m a m m a l i a n p r e t e n s e i n h i b i t o r s and h e p a r i n , k i n e t i c s of k i n i n f o r m a t i o n , and a c t i o n on s y n t h e t i c s u b s t r a t e s . T h e in viv...__2oe f f e c t o£ p r o t e a s e i n h i b i t o r s a n d h e p a r i n on t h e g r o w t h of the MS.LS as w e l l as the b i o c h e m i c a l p a r a m e t e r s of the ] < a l l i k r e i n - k i n i n s y s t e m a l s o w a s s t u d i e d . E p s i l o n a m i n o c a p r o i c a c i d (EACA), TtLASYLOL, and h e p a r i n w a s i n j e c t c d t~cice d a i l y i n t r a p e r i t o n e a l l y into ~ o d e n t s , 8 p e r g r o u p , be"arzng ' ~ f r e s h l y - ~ r a n s p l a n t e d t~amors. At e s t ; a b h s h e d t i m e i n t e r v a l s aniz~a'ls w e r e p e r f u s e d and t u m o r s e x c i s e d f o r w e i g h t d e t e r m i n a t i o n and e s t i m a t i o n of b i o c h e m i c a l a c t i v i t i e s . The e f f e c t on t u m o r g r o w t h and on the b i o c h e m i c a l p a r a r n o t e r s w i l l be d i s c u s s e d in d e t a i l . T h e t u r n e r - i n h i b i t o r y e f f e c t of E A C A on t'he g r o w t h of E h r ] . i c h a s c i t e s t u I n o r s a l s o w i l l be d e t a i l e d . T h e s i g n i f i c a n c e of t b e s e d a t a as r e l a t e d to v a s o m o t o r r e g u l a t o r s in t u r n e r t i s s u e w i l l be c o n sidered. ( S u p p o r t e d by USPHS g r a n t s H E - 1 1 4 9 Z and H E - 0 3 7 8 4 . )
SIGNIFICANCE OF KININS IN THE CORONARY CIRCULATION
E. Bassenge, E. Werle, J. Holtz and H. Fritz Physiologi,sehes Institut und Institut fur Kiini~ehe Chemie und KlinischeBiochemieder Univer~it/it, Miinehen
Expcrirmnts were carried out in open chest dogs in order to investigate the role of kinins in tile regulation of coronary blood flow. Small areas of tile beating clog heart in_ ~ were perfused under constant pressure with tile animal's own blood, following pentobarbital anesthesia. Mean and phasic coronary inflow were recorded by an electromagnetic flowmeter attached to the extracorporeal perfusion system. Carboxypeptidase B, or several inhibitors of this enzyme were administered intracoronarilly with a constant infusion pump Isometric tension was measured by a Walton-Brodie strain gauge arch attached to the perfused area of the left ventricle. Four series of experiments were carried out in order to study the role of kinins in coronary regulation.
Pharmacological Research Communicat/ons, Vo/. 1, No.2, 1969
137
1 - Application of specific kinin antagonists was not satisfactory because all tl~t: substances tested riffled to suppress the effect of kinins on coronary flow. 2 - Experimental increase in concentration of the kininase, carboxypeptidase B in blood resulted in a cessation of the hyperemie response following intracoronary kinin administration in doses ranging from 10-7 to 10 "2 rag, whereas the hyperemic responst~ due to 25 secomls of lscht~rnia remained unatTected. 3 - During constant intraeoronary infilsion of CdSO4, CaE!YfA, 8-hydroxyquinoltn sulpltonie aqid or tl~ioglycolic acid, which inhibit kininases~ the dilating effect of kinins administered intracoronarilly was significantly prolonged; reactive hyper¢mia, however, remained essentially unchanged under these conditiorls, Kininase inhibitors in concentrations ranging from 10-4 to ~0"2 molesper litre coronary blood, had no effect on coronary flow. 4 . When the myocardium was perfused with ~i suspension! containing washed red eel|s, bt~t no kininogen, a hyperemic response following 25 seconds of iscliemia was still observed. Since tile control resistance changed, however, due to the perfiasion witll the red cell suspension, this series was not conclusive. Kinins administered intraeoronarilly did not increase isometric tension dtwelopment, The dilating effect of kinins on the coronary bed depends on the pll. Lowering the plf to 7.2 results in a more pronounced effect,; increasing pll to 7.5 lessens the effect, From the above results, i~ is concluded tlmt kinins do not play a major role in coronary regulation. LOCAL ISCHEM!A AS A KININOGEN DEPRESSANT: EFFECT OF CORTISONE, TAURINE AND KALLIKREIN INltlBITOR A. Bavazzano, N. Sidell, S. Michelaccj and F. Sicuteri Departnlent of Medicine, Unlvernity of Florence, Viale Morgagnt, Florence, Italy It w~ts postulated that the capillary bed has a dual action on tile kjninogen-kinin system: (a) release of a small quantity of kinins from their plasmatic precursor and (b) kini~t inactivation. "I'l~e cotadition (b) was demonstrated during arterial infiJsion with synthetic bradykinin, iLeeovery in reflux venous blood is minimal (Sicuteri e t ~i), 1963, Allwood el aJ, 1963); the measurement <.:f arterial-venous kininogen demonstrated a non-statistically significant diftk.rence (SicuterL~t ~J~, 1966). The aim of the present study was to observe if, with an increase in time of contact will~ capillary walls (microvessels in general), a variztion in kininogen is observable in plasma. When circulation is arrested in the superior limb of man with a sphygmomano~leter cuff" i
'J
p
~,
inflated to a pressure substantially higher than systohc, a "circulatory arrest" is erroneously sa~d to occur. In effect, a gradual refilling of the veins during ischemia is noted. Blood probably passe,~ from the general circulation to the ischemia limb tllrough m~Jrrow arteries. Bata from the present study demonstrate: a) In 2 cases, injecting tagged albumin intravenously, a certain quantity is always demonstrable in venous blood taken From the antecubital vein during 5 rain of local ischcmia.