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Positive chronotropic effect of dialysable peptides derived from plasmin digestion of bovine fibrinogen preparations
European Journal of Pharmacology, 36 (1976) 355--361 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
355
POSITIVE CHRONOTROPIC EFFECT OF DIALYSABLE PEPTIDES DERIVED FROM PLASMIN DIGESTION OF BOVINE FIBRINOGEN PREPARATIONS WLODZIMIERZ BUCZKO *, ROSETTA FRANCO, GABRIO BIANCHETTI, MARIA BENEDETTA DONATI, GIOVANNI DE GAETANO and SILVIO GARATTINI Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea, 62 --20157 Milano, Italy Received 14 July 1975, revised MS received 2 December 1975, accepted 8 December 1975
W. BUCZKO, R. FRANCO, G. BIANCHETTI, M.B. DONATI, G. DE GAETANO and S. GARATTINI, Positive chronotropic effect of dialysable peptides derived from plasmin digestion of bovine fibrinogen preparations, European J. Pharmacol. 36 (1976) 355--361. Low-molecular weight dialysable peptides, obtained by plasmin degradation of purified bovine fibrinogen preparations, have been shown to increase the chronotropic activity of isolated rat atria. This effect was dose dependent and was inhibited by inhibitors of glycolysis (NaF and 2-deoxy-D-glucose), but not by an inhilSitor of oxidative phosphorylation (2,4-dinitrophenol). Propranolol, a ~-blocking agent, was also ineffective. Fibrinogenderived peptides increased both cAMP levels and phosphorylase a activity in stimulated atria. The increase of these parameters was transitory and appeared to precede the occurrence of the positive chronotropic effect. In the test situation used, the biochemical and functional modifications induced by fibrinogen-derived peptides were similar to those induced by glucagon. Fibrinogen degradation products Isolated rat atria
Chronotropism
1. I n t r o d u c t i o n T h e cardiac s t i m u l a t o r y e f f e c t of p e p t i d e s derived f r o m t h r o m b i n digests o f f i b r i n o g e n was first described b y Laki ( 1 9 5 1 ) using t h e frog as t h e a n i m a l m o d e l . A n o t h e r s t u d y using r a t s (Malofiejew et al., 1 9 7 1 ) has s h o w n t h a t p l a s m i n f i b r i n o g e n degr a d a t i o n p r o d u c t s increase t h e s p o n t a n e o u s c o n t r a c t i l e activity o f t h e isolated right auricle o f t h e h e a r t . It has b e e n suggested t h a t these low molecular weight peptides would act d i r e c t l y o n t h e h e a r t cell m e m b r a n e , p r o b -
* Visiting Scientist, from the Department of Pharmacology, Medical School, Bialystok, Poland. ** Correspondence and reprint requests to: M.B. Donati, M.D., Ph.D., Lab. Haemostasis and Thrombosis Research, Istituto di Ricerche Farmacologiche, 'Mario Negri', Via Eritrea, 62, 20157 Milano, Italy.
Glucagon
Peptides
Cyclic AMP
a b l y b y m o d i f y i n g its m e t a b o l i c processes (Malofiejew, 1 9 7 1 ) . T h e r e l a t i o n s h i p s b e t w e e n m e c h a n i c a l , biochemical and metabolic effects of cardiotropic s u b s t a n c e s h a v e b e e n e x t e n s i v e l y investigated (Hess et al., 1 9 6 3 ; Kreisberg and Williamson, 1 9 6 4 ; Laraia et al., 1 9 6 8 ; Laraia and R e d d y , 1 9 6 9 ; A n t o n a c c i o and Lucchesi, 1970; M a y e r et al., 1 9 7 0 ; McNeill a n d V e r m a , 1 9 7 4 ; V e r m a and McNeill, 1 9 7 4 ) . It was s h o w n t h a t t h e change in c o n t r a c t i l i t y m a y o c c u r at t h e s a m e t i m e as t h e a c c e l e r a t i o n o f cardiac m e t a b o l i s m ( K u k o v e t z a n d P6ch, 1967; R o b i n s o n et al., 1 9 6 7 ; M a y e r et al., 1 9 7 0 ; R o b a k and G r y g l e w s k i , 1971). T h e p r e s e n t studies w e r e u n d e r t a k e n t o exa m i n e t h e e f f e c t s o f t h e dialysable p e p t i d e s derived f r o m p l a s m i n d e g r a d a t i o n o f b o v i n e f i b r i n o g e n o n t h e c o n t r a c t i l e activity and o n s o m e b i o c h e m i c a l and m e t a b o l i c processes o f isolated rat atria.
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W. BUCZKO ET AL.
2. Materials and methods
2.1. General Paired atria were prepared from male CD rats weighing 200--300 g (Charles River, Italy). The preparations were incubated in Krebs bicarbonate solution of the following composition (mM): NaC1 118; KC1 4.7; NaHCO3 25; glucose 11.54; CaC12 2.51; MgSO4 1.19;KH2PO4 1.19 in distilled water. The bathing solution was kept at 31.5--32°C and bubbled with a mixture of 95% O: and 5% CO2. The pH of the bathing solution remained at 7.4.
2.2. Determination of the chronotropic response Atria suspended in 20 ml of the solution were attached to isometric force-displacement transducers (FTO3C) under the minimal tension suitable for recording (150 mg); the atrial rate was recorded on a Grass-5-D-polygraph. The tissue was allowed to equilibrate until heart rate was stable (no more than a 5 beat/ min change over a 10 min period) which usually occurred after 30--45 min incubation; the preparation was washed every 10 min during this period. A dose--response curve to 1-noradrenaline was determined each time at the beginning of the experiment in order to verify possible differences in the sensitivity of the various preparations and to compare the effects of the agonists tested with those of noradrenaline. The noradrenaline effect on myocardial activity was readily washed out and the preparations did not appear to be modified in their chronotropic activity. The experiment with the test compounds was started after a further washing period of 30 min. The chronotropic responses were calculated as number of contractions/min.
2.3. Determination o f (cAMP) in the rat atria
cyclic
3',5'-AMP
The concentration of cAMP was determined by the following procedure: each pair of
atria was suspended in 10 ml Krebs bicarbonate solution and allowed to equilibrate as described above. Atria were then exposed to the peptides (330 #g/ml). for various periods of time. Control experiments using atria exposed only to Krebs bicarbonate solution, were always run in parallel. At the end of the incubation period, control and treated atria were removed from the bath, rapidly blotted with filter paper and immediately placed into liquid nitrogen. The frozen atria (50--100 mg) were then quickly weighed and pulverized under liquid nitrogen, and thorougly mixed with 2 ml of cold 5% trichloroacetic acid. After homogenization, the supernatant was neutralized with 0.2 M Tris buffer pH 9.5 and placed on a Dowex 50 × 4 H ÷ column (8.5 X 0.4 cm) equilibrated with distilled water. After discarding the effluent, the columns were eluted with 2 + 6 ml of H20. The first 3 ml of the eluate were discarded and the following 5 ml (containing 85% of cAMP) were collected. An aliquot of the collected eluate was then brought to dryness under a gentle stream of nitrogen at 40°C in a thermostatic bath. The dry residue was redissolved in Tris-EDTA buffer and processed for cAMP determination according to Tovey et al. (1974) using an Amersham Radiochemical Centre cAMP test kit.
2.4. Determination of the activity o f phosphorylase a in the rat atria Phosphorylase a activity was measured according to the method described by Diamond and Brody (1965). The atria were prepared and incubated with peptides as described above. After incubation, the atria were frozen, weighed and put into ice-cooled glass homogenizers in 100 volumes of a solution containing 0.05 M Tris buffer (pH 6.8), 0.001 M EDTA, 0.02 M NaF and 0.3% serum albumin. All these procedures were carried out at 4°C. After centrifugation of the homogenate at 10,000 g for 10 min, 0.2 ml of the supernatant were incubated for 30 min at 37.5°C in test tubes containing 0.05 M Tris buffer (pH
FIBRINOGEN-DERIVED PEPTIDES AND RAT HEART 6.8), 0.4% glycogen, 0.01 M glucose-l-phosphate, 0.001 M EDTA, 0.02 M NaF and 0.3% serum albumin, in a final volume of 1.0 ml. Duplicate samples of the supernatant solutions were incubated in the same reaction mixture containing, additionally, AMP in a final concentration of 0.001 M. The reaction was stopped by the addition of 2.0 ml of 10% trichloroacetic acid. The samples were centrifuged in a cold room at 2,500 g for 10 min and the supernatants were assayed for inorganic phosphate by the method of Fiske and Row (1925).
357 mine • HCI (Farmitalia), crystalline glucagon (Eli Lilly). All solutions were prepared by dissolving these substances in distilled water.
2. 7. Statist&al evaluation Each experiment was repeated at least four times. The significance of differences was assessed by Duncan's multiple range test, after variances of the experimental groups had been found homogeneous by Bartlett--Box test.
3. Results
2. 5. Preparation of peptides Purified bovine fibrinogen (Kabi, Stockholm, Sweden), was dissolved in bi-distilled water (25 mg/ml) and desalted by dialysis against bi-distilled water at 4°C for 24 hr; the pH of the solution was adjusted to 7.4 by addition of 0.1 N NaOH. Plasmin (human, Kabi, dissolved in bi-distilled water) was then added to the solution (0.4 CTA units/ml, final concentration); the mixture was incubated at 37°C for 2 hr, then heated at 56°C for 15 min and finally centrifuged to remove the precipitate; the supernatant was collected and dialysed against 20 volumes of bi-distilled water for 24 hr at 4°C. The dialysis tubings used (A.H. Thomas Company, Philadelphia, U.S.A.) had a retention limit of about 12,000 mol. wt. according, to the manufacturer's specifications. The fluid outside the dialysis bag was evaporated at 40°C, lyophilized and stored at --20°C. The powder was reconstituted in isotonic saline just before use; the peptide concentration was determined by the method of Itzhaki and Gill (1964). Peptides constituted about 10% of the starting material.
2.6. Drugs used 1-Noradrenaline bitartrate (Recordati), 2deoxy-D-glucose (Sigma), sodium fluoride (NaF) (Carlo Erba), 2,4-dinitrophenol (2,4DNP) (Calbiochem), ouabain (Merck), hista-
3.1. Influence of peptides on the chronotropism of isolated rat atria Peptides exerted a positive chronotropic effect on isolated rat atria. This effect was concentration dependent, reached a peak within 3 min after the addition of the peptides into the isolated organ bath, lasted as a plateau during 3--4 min and then slowly disappeared (fig. 1). A slow appearance of a positive chronotropic response was also observed with glucagon (10 -7 M) the peak being reached within 5 min, while noradrenaline (3 × 10 -7 M)
A Beatslmin 40-
30~
330 pg/ml (5)
20 10,
~
220,uglm[ (6) |
0
2
L
6
8
165).~1/ml (3)
I() 1'2 I~ 1"6 Time(rain)
Fig. 1. Time course o'f the positive chronotropic effect induced by fibrinogen-derived peptides (at 3 different concentrations) on isolated rat atria. The chronotropic effect is expressed as increase in atrial frequency over the basal rate. Number of experiments in parentheses.
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W. B U C Z K O ET AL.
TABLE 1 E f f e c t o f p r o p r a n o l o l and s o m e m e t a b o l i c i n h i b i t o r s o n t h e m a x i m a l c h r o n o t r o p i c activity o f isolated rat atria induced by fibrinogen-derived p e p t i d e s , n o r a d r e n a l i n e and glucagon. Atria were p r e i n c u b a t e d for 5 rain w i t h the i n h i b i t o r y s u b s t a n c e s b e f o r e the a d d i t i o n o f the stimulus. Each value r e p r e s e n t s the m e a n o f t h e n u m b e r o f b e a t s / r a i n ± S.E. o f at least 4 e x p e r i m e n t s . The m a x i m a l increase in atrial rate i n d u c e d by the 3 stimuli was statistically significant (p < 0.01) w h e n c o m p a r e d w i t h saline. Substances
Final concentration
Saline
Saline Propranolol 2,4-DNPt NaF ~-t 2-Deoxy-Dglucose
-10 -7 M 5 × 10 -6 M 2 x 10 -2 M
191.7 192.2 187.8 189.1
20 m g / m l
190.5 ± 9.5
± ± ± ±
3.2 5.6 4.1 6.6
Peptides 330 p g / m l
Noradrenaline 3 × 10 -7 M
Glugagon 10 -7 M
241.6 225.0 242.5 192.5
256.5 210.7 277.5 227.5
271.4 278.7 276.6 193.7
± ± ± ±
4.7 5.0 4.8 7.5 **
190.0 ± 21.2 **
± 4.4 ± 5.2 ** ± 14.9 ± 13.6 *
209.0 ± 18.6 **
± 6.3 ± 13.7 ± 6.6 ± 13.4 **
181.2 ± 27.5 **
* p < 0.05; * * p < 0.01, as c o m p a r e d to t h e value o b t a i n e d w i t h each i n d u c e r w h e n i s o t o n i c saline was used instead o f the inhibitor. 2,4-dinitrophenol. J ' t S o d i u m fluoride.
induced its maximal effect more rapidly, usually within 1--2 min. In contrast with that of peptides, the activity of the latter two agonists was long-lasting.
3.2. Effect o f some cardio-inhibitors on the chronotropic effect of peptides and other stimulants
nificantly inhibited the activation induced by noradrenaline but did not influence the action of glucagon and of the peptides. The action of the latter agonists was abolished by glycolytic inhibitors such as NaF or 2-deoxy-D-glucose b u t not by 2,4-DNP, an inhibitor of oxidative phosphorylation.
3.3. Effect o f peptides on cAMP level and phosphorylase a activity o f isolated rat atria The chronotropic effect of peptides on isolated rat atria was compared with that of glucagon, noradrenaline, histamine and ouabain in the absence and presence of a/~-blocking agent and some metabolic inhibitors. At the concentrations used, noradrenaline, glucagon and peptides had a significant positive chronotropic effect (table 1). Neither ouabain (10-6--10 -4 M) nor histamine (I0-7--I0 -4 M) significantly modified the chronotropism of isolated rat atria. Arrhythmias were frequently observed with the highest concentrations of ouabain. Propranolol, a fl-blocking agent, sig-
The time course of biochemical events was measured after the addition of 330 gg/ml of peptides to the atria (table 2). cAMP in the atria increased significantly after 30 sec, whereas the maximal increase of phosphorylase a activity was observed 90 sec after incubation of atria with peptides. Glucagon induced a marked increase of both cAMP levels (162.5 + 6.8 pmol/g tissue) and phosphorylase a (22.6 + 3.9%) after 30 and 90 sec, respectively.
FIBRINOGEN-DERIVED PEPTIDES AND RAT HEART
359
TABLE 2 Time c o u r s e o f t h e variations o f cyclic AMP level and p h o s p h o r y l a s e a activity o f isolated rat atria a f t e r t h e a d d i t i o n o f f i b r i n o g e n - d e r i v e d p e p t i d e s (330 ~ug/ml). Each value r e p r e s e n t s the m e a n + S.E. o f at least 4 experiments. T i m e (sec)
0 15 20 30 60 9O 120 150 180 240 300
Cyclic A M P (pmol/g)
P h o s p h o r y l a s e a (%)
Control
Peptides
Control
Peptides
123.8 ± 6.7 115.4 ± 10.1 1 2 1 . 5 ± 6.5 1 1 7 . 6 ± 4.8 122.6 ± 8.6
121.6-+ 5.9 115.3 -+ 14.9 1 4 3 . 1 ± 13.6 138.1-+ 4 . 6 " 139.6 ± 11.5
12.4 ± 2.8
10.0 ± 2.1
119.5±
122.4-+ 10.3
7.4
11.1 11.0 10.0 12.8 11.3 14.2
± 2.0 -+ 1.3 ± 3.1 + 1.3 + 2.8 ± 3.2
12.0 -+3.5
7.0 10.0 24.6 22.0 20.2 17.0 10.0 9.7
-+ 2.0 -+ 2.0 ± 2.8 ** ± 3.6 * ± 6.3 ± 3.0 -+ 2.4 -+ 2.1
* p < 0.05; ** p < 0.01, as c o m p a r e d t o t h e c o n t r o l s t e s t e d s i m u l t a n e o u s l y .
4. Discussion The present study confirms and extends previous observations on the cardio-stimulatory action of peptides derived from fibrinogen digestion (Laki, 1951; Malofiejew et al., 1971). We have shown that fibrinogen
berg and Williamson, 1964). In our experiments, the chronotropic activity induced by glucagon and peptides was blocked by inhibitors of glycolysis such as NaF and 2-deoxyD-glucose. These results suggest that the mechanism of action of peptides may be similar to that of glucagon. It may be worth mentioning here that'the most active peptides in our preparations have lately been shown to have a molecular weight lower than 5,000 (Wisniewski et al., 1975; Buczko et al., 1976). Glucagon has a mol. wt. of 3,485. Glucagon has been reported to increase intracellular cAMP concentrations (Murad and Vaughan, 1969) and to activate phosphorylase a (Mayer et al., 1970) in the rat myocardium, two findings confirmed in the present study. In our experimental conditions, fibrinogen-derived peptides also increased both cAMP levels and phosphorylase a activity. Both glucagon and peptides showed their maximal effects on cAMP and on phosphorylase a after 30 and 90 sec incubation respectively. Although the increase of these biochemical parameters preceded the occurrence of a significant positive chronotropic effect,
360
the possibility that these events are causally related should be considered with caution. Indeed, even the role of cAMP in determining the cardiostimulatory effects of glucagon, as well as of catecholamines, is still debatable (Laraia, 1968; Laraia and Reddy, 1969; Sobel and Mayer, 1973; Kukovetz et al., 1973). It may be of interest, however, that fibrinogen-derived peptides similar to those used in the present study, were able to inhibit ADPinduced platelet aggregation, to increase skin vascular permeability (Buczko et al., 1976) and to stimulate the chemotactic activity of leucocytes (Kay et al., 1973). These phenomena have been reported to be mediated by an increase of cAMP (Salzman, 1972; Leahy et al., 1970). Whatever is the mechanism of action o f fibrinogen-derived peptides, their positive interference with heart contractility may have some clinical relevance. Indeed, the amounts of peptides used in this study are well within the range of the concentrations of fibrinogen degradation products (0.6 mg/ml, according to Solum et al., 1973) which can be obtained in man following administration of fibrinolytic agents such as streptokinase. Streptokinase has been reported to be of benefit for patients with recent myocardial infarction (Verstraete, 1971). Whether the products of fibrinogen digestion contribute in some way to the therapeutic efficacy of fibrinolytic drugs is still an unproven but stimulating hypothesis.
Acknowledgements The authors are grateful to Dr. L. Mussoni who performed the statistical analysis of the data. The technical assistance of Miss M.G. Castelli is greatly appreciated. Purified bovine fibrinogen was generously provided by AB Kabi, Stockholm, Sweden, through the courtesy of Dr. B. Strindberg. Plasmin was a gift from Dr. Lang, Kabi-Lang, Milano, Italy.
References Antonaccio, M.J. and T. Cavaliere, 1974, A comparison of the effects of some inotropic and chrono-
W. BUCZKO ET AL. tropic agents on isolated atria from normotensive (NTR) and spontaneously hypotensive (SHR) rats, Arch. Intern. Pharmacodyn. 209, 273. Antonaccio, M.J. and B.R. Lucchesi, 1970, The interaction of glucagon with theophylline, PGE1, isoproterenol, ouabain, and CaC12 on the dog isolated papillary muscle, Life Sci. 9, Part I, 1081. Bartlet, A.L., 1963, The action of histamine on the isolated heart, Brit. J. Pharmacol. 21,450. Buczko, W., R. Franco, G. De Gaetano and M.B. Donati, 1976, Biological properties of dialysable peptides derived from plasmin digestion of bovine fibrinogen preparations, Thromb. Diath. Haemorrh. (in press). Diamond, J. and T.M. Brody, 1965, Phosphorylase activity in rat uterus after catecholamine administration, Biochem. Pharmacol. 14, 7. Fiske, C.H. and Y.S. Row, 1925, The colorimetric determination of phosphorus, J. Biol. Chem. 66, 375. Hess, M.E., D. Hottenstein, J. Shanfeld and N. Haugaard, 1963, Metabolic effects of theophylline in cardiac and skeletal muscle, J. Pharmacol. Exptl. Therap. 141,274. Itzhaki, R.F. and D.M. Gill, 1964, A micro-biuret method for estimating proteins, Anal. Biochem. 9, 401. Kay, A.B., D.S. Pepper and M.R. Ewart, 1973, Generation of chemotactic activity for leucocytes by the action of thrombin on human fibrinogen, Nature (London) New Biol. 243, 56. Kreisberg, R.A. and J.R. Williamson, 1964, Metabolic effects of glucagon in the perfused rat heart, Amer. J. Physiol. 207,721. Kukovetz, W.R. and G. PSch, 1967, The action of imidazole on the effects of methyl-xanthines and catecholamines on cardiac contraction and phosphorylase activity, J. Pharmacol. Exptl. Therap. 156, 514. Kukovetz, W.R., G. PSch and A. Wurm, 1973, Effect of catecholamines, histamine and oxyfedrine on isotonic contraction and cyclic AMP in the guineapig heart, Naunyn-Schmiedeb. Arch. Pharmacol. 278, 403. Laki, K., 1951, Action of thrombin on fibrinogen, Science 114, 435. Laraia, P.J. and W.J. Reddy, 1969, Hormonal regulation of myocardial adenosine 3',5'-monophosphate, Biochim. Biophys. Acta 177,189. Laraia, P.J., R.J. Craig and W.J. Reddy, 1968, Glucagon: effect of adenosine 3',5'-monophosphate in the rat heart, Amer. J. Physiol. 215,968. Leahy, D.R., E.R. McLean, Jr. and J.T. Bonner, 1970, Evidence for cyclic-3',5'-adenosine monophosphate as chemotactic agent for polymorphonuclear leukocytes, Blood 36, 52. Malofiejew, M., 1971, The biological and pharmaco-
FIBRINOGEN-DERIVED PEPTIDES AND RAT HEART logical properties of some fibrinogen degradation products, Scand. J. Haematol. Suppl. 13, 303. Malofiejew, M., A. Kostrzewska and K. Buluk, 1971, The influence of fibrinogen degradation products on the electrobiological activity of the rat heart, Experientia 27,139. Mayer, S.E., D.H. Namm and L. Rice, 1970, Effect of glucagon on cyclic 3',5'-AMP phosphorylase activity and contractility of heart muscle of the rat, Circulation Res. 26, 225. McNeill, J.H. and S.C. Verma, 1974, Blockade by burimamide of the effects of histamine and histamine analogs on cardiac contractility, phosphorylase activation and cyclic adenosine monophosphate, J. Pharmacol. Exptl. Therap. 188, 180. Murad, F. and M. Vaughan, 1969, Effect of glucagon on rat heart adenyl cyclase, Biochem. Pharmacol. 18, 1053. Repke, K., 1963, Metabolism of cardiac glycosides, in: Proceedings of the First International Pharmacological Meeting, Stockholm, Vol. III, New Aspects of Cardiac Glycosides, ed. W. Wilbrandt, (Pergamon Press, Oxford) p. 47. Robak, J. and R. Gryglewski, 1971, Influence of INPEA, pindolol and propranolol on the chronotropic and metabolic responses to ~-adrenergic stimulation in intact rats, Biochem. Pharmacol. 20, 2749. Robinson, G.A., R.W. Butcher and E.W. Sutherland,
361 1967, Adenyl cyclase as an adrenergic receptor, Ann. N.Y. Acad. Sci. 139, 703. Salzman, E.W., 1972, Cyclic AMP and platelet function, N. Engl. J. Med. 286, 358. Sobel, B.E. and S.E. Mayer, 1973, Cyclic adenosine monophosphate and cardiac contractility, Circulation Res. 32, 407. Solum, N.O., C. Rigoltot, A.Z. Budzynski and V.J. Marder, 1973, A quantitative evaluation of the inhibition of platelet aggregation by low molecular weight degradation products of fibrinogen, Brit. J. Haematol. 24, 419. Tovey, K.C., K.G. Oldham and J.A.M. Whelan, 1974, A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding technique, Clin. Chim. Acta 56, 221. Verma, S.C. and J.H. McNeill, 1974, Action of imidazole on the cardiac inotropic, phosphorylase activating and cyclic AMP producing effects of nqrepinephrine and histamine, Res. Commun. Chem. Pathol. Pharmacol. 7, 305. Verstraete, M., 1971, The difficulty of appraisal of streptokinase treatment of myocardial infarction, Angiologica 8, 43. Wisniewski, K., W. Buczko and J. Moniuszko-Jakoniuk, 1975, The effects of products of fibrinogen digestion by plasmin [P-FDP] on the central nervous system, Acta Neurobiol. Exp. 35, 137.
355
POSITIVE CHRONOTROPIC EFFECT OF DIALYSABLE PEPTIDES DERIVED FROM PLASMIN DIGESTION OF BOVINE FIBRINOGEN PREPARATIONS WLODZIMIERZ BUCZKO *, ROSETTA FRANCO, GABRIO BIANCHETTI, MARIA BENEDETTA DONATI, GIOVANNI DE GAETANO and SILVIO GARATTINI Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea, 62 --20157 Milano, Italy Received 14 July 1975, revised MS received 2 December 1975, accepted 8 December 1975
W. BUCZKO, R. FRANCO, G. BIANCHETTI, M.B. DONATI, G. DE GAETANO and S. GARATTINI, Positive chronotropic effect of dialysable peptides derived from plasmin digestion of bovine fibrinogen preparations, European J. Pharmacol. 36 (1976) 355--361. Low-molecular weight dialysable peptides, obtained by plasmin degradation of purified bovine fibrinogen preparations, have been shown to increase the chronotropic activity of isolated rat atria. This effect was dose dependent and was inhibited by inhibitors of glycolysis (NaF and 2-deoxy-D-glucose), but not by an inhilSitor of oxidative phosphorylation (2,4-dinitrophenol). Propranolol, a ~-blocking agent, was also ineffective. Fibrinogenderived peptides increased both cAMP levels and phosphorylase a activity in stimulated atria. The increase of these parameters was transitory and appeared to precede the occurrence of the positive chronotropic effect. In the test situation used, the biochemical and functional modifications induced by fibrinogen-derived peptides were similar to those induced by glucagon. Fibrinogen degradation products Isolated rat atria
Chronotropism
1. I n t r o d u c t i o n T h e cardiac s t i m u l a t o r y e f f e c t of p e p t i d e s derived f r o m t h r o m b i n digests o f f i b r i n o g e n was first described b y Laki ( 1 9 5 1 ) using t h e frog as t h e a n i m a l m o d e l . A n o t h e r s t u d y using r a t s (Malofiejew et al., 1 9 7 1 ) has s h o w n t h a t p l a s m i n f i b r i n o g e n degr a d a t i o n p r o d u c t s increase t h e s p o n t a n e o u s c o n t r a c t i l e activity o f t h e isolated right auricle o f t h e h e a r t . It has b e e n suggested t h a t these low molecular weight peptides would act d i r e c t l y o n t h e h e a r t cell m e m b r a n e , p r o b -
* Visiting Scientist, from the Department of Pharmacology, Medical School, Bialystok, Poland. ** Correspondence and reprint requests to: M.B. Donati, M.D., Ph.D., Lab. Haemostasis and Thrombosis Research, Istituto di Ricerche Farmacologiche, 'Mario Negri', Via Eritrea, 62, 20157 Milano, Italy.
Glucagon
Peptides
Cyclic AMP
a b l y b y m o d i f y i n g its m e t a b o l i c processes (Malofiejew, 1 9 7 1 ) . T h e r e l a t i o n s h i p s b e t w e e n m e c h a n i c a l , biochemical and metabolic effects of cardiotropic s u b s t a n c e s h a v e b e e n e x t e n s i v e l y investigated (Hess et al., 1 9 6 3 ; Kreisberg and Williamson, 1 9 6 4 ; Laraia et al., 1 9 6 8 ; Laraia and R e d d y , 1 9 6 9 ; A n t o n a c c i o and Lucchesi, 1970; M a y e r et al., 1 9 7 0 ; McNeill a n d V e r m a , 1 9 7 4 ; V e r m a and McNeill, 1 9 7 4 ) . It was s h o w n t h a t t h e change in c o n t r a c t i l i t y m a y o c c u r at t h e s a m e t i m e as t h e a c c e l e r a t i o n o f cardiac m e t a b o l i s m ( K u k o v e t z a n d P6ch, 1967; R o b i n s o n et al., 1 9 6 7 ; M a y e r et al., 1 9 7 0 ; R o b a k and G r y g l e w s k i , 1971). T h e p r e s e n t studies w e r e u n d e r t a k e n t o exa m i n e t h e e f f e c t s o f t h e dialysable p e p t i d e s derived f r o m p l a s m i n d e g r a d a t i o n o f b o v i n e f i b r i n o g e n o n t h e c o n t r a c t i l e activity and o n s o m e b i o c h e m i c a l and m e t a b o l i c processes o f isolated rat atria.
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2. Materials and methods
2.1. General Paired atria were prepared from male CD rats weighing 200--300 g (Charles River, Italy). The preparations were incubated in Krebs bicarbonate solution of the following composition (mM): NaC1 118; KC1 4.7; NaHCO3 25; glucose 11.54; CaC12 2.51; MgSO4 1.19;KH2PO4 1.19 in distilled water. The bathing solution was kept at 31.5--32°C and bubbled with a mixture of 95% O: and 5% CO2. The pH of the bathing solution remained at 7.4.
2.2. Determination of the chronotropic response Atria suspended in 20 ml of the solution were attached to isometric force-displacement transducers (FTO3C) under the minimal tension suitable for recording (150 mg); the atrial rate was recorded on a Grass-5-D-polygraph. The tissue was allowed to equilibrate until heart rate was stable (no more than a 5 beat/ min change over a 10 min period) which usually occurred after 30--45 min incubation; the preparation was washed every 10 min during this period. A dose--response curve to 1-noradrenaline was determined each time at the beginning of the experiment in order to verify possible differences in the sensitivity of the various preparations and to compare the effects of the agonists tested with those of noradrenaline. The noradrenaline effect on myocardial activity was readily washed out and the preparations did not appear to be modified in their chronotropic activity. The experiment with the test compounds was started after a further washing period of 30 min. The chronotropic responses were calculated as number of contractions/min.
2.3. Determination o f (cAMP) in the rat atria
cyclic
3',5'-AMP
The concentration of cAMP was determined by the following procedure: each pair of
atria was suspended in 10 ml Krebs bicarbonate solution and allowed to equilibrate as described above. Atria were then exposed to the peptides (330 #g/ml). for various periods of time. Control experiments using atria exposed only to Krebs bicarbonate solution, were always run in parallel. At the end of the incubation period, control and treated atria were removed from the bath, rapidly blotted with filter paper and immediately placed into liquid nitrogen. The frozen atria (50--100 mg) were then quickly weighed and pulverized under liquid nitrogen, and thorougly mixed with 2 ml of cold 5% trichloroacetic acid. After homogenization, the supernatant was neutralized with 0.2 M Tris buffer pH 9.5 and placed on a Dowex 50 × 4 H ÷ column (8.5 X 0.4 cm) equilibrated with distilled water. After discarding the effluent, the columns were eluted with 2 + 6 ml of H20. The first 3 ml of the eluate were discarded and the following 5 ml (containing 85% of cAMP) were collected. An aliquot of the collected eluate was then brought to dryness under a gentle stream of nitrogen at 40°C in a thermostatic bath. The dry residue was redissolved in Tris-EDTA buffer and processed for cAMP determination according to Tovey et al. (1974) using an Amersham Radiochemical Centre cAMP test kit.
2.4. Determination of the activity o f phosphorylase a in the rat atria Phosphorylase a activity was measured according to the method described by Diamond and Brody (1965). The atria were prepared and incubated with peptides as described above. After incubation, the atria were frozen, weighed and put into ice-cooled glass homogenizers in 100 volumes of a solution containing 0.05 M Tris buffer (pH 6.8), 0.001 M EDTA, 0.02 M NaF and 0.3% serum albumin. All these procedures were carried out at 4°C. After centrifugation of the homogenate at 10,000 g for 10 min, 0.2 ml of the supernatant were incubated for 30 min at 37.5°C in test tubes containing 0.05 M Tris buffer (pH
FIBRINOGEN-DERIVED PEPTIDES AND RAT HEART 6.8), 0.4% glycogen, 0.01 M glucose-l-phosphate, 0.001 M EDTA, 0.02 M NaF and 0.3% serum albumin, in a final volume of 1.0 ml. Duplicate samples of the supernatant solutions were incubated in the same reaction mixture containing, additionally, AMP in a final concentration of 0.001 M. The reaction was stopped by the addition of 2.0 ml of 10% trichloroacetic acid. The samples were centrifuged in a cold room at 2,500 g for 10 min and the supernatants were assayed for inorganic phosphate by the method of Fiske and Row (1925).
357 mine • HCI (Farmitalia), crystalline glucagon (Eli Lilly). All solutions were prepared by dissolving these substances in distilled water.
2. 7. Statist&al evaluation Each experiment was repeated at least four times. The significance of differences was assessed by Duncan's multiple range test, after variances of the experimental groups had been found homogeneous by Bartlett--Box test.
3. Results
2. 5. Preparation of peptides Purified bovine fibrinogen (Kabi, Stockholm, Sweden), was dissolved in bi-distilled water (25 mg/ml) and desalted by dialysis against bi-distilled water at 4°C for 24 hr; the pH of the solution was adjusted to 7.4 by addition of 0.1 N NaOH. Plasmin (human, Kabi, dissolved in bi-distilled water) was then added to the solution (0.4 CTA units/ml, final concentration); the mixture was incubated at 37°C for 2 hr, then heated at 56°C for 15 min and finally centrifuged to remove the precipitate; the supernatant was collected and dialysed against 20 volumes of bi-distilled water for 24 hr at 4°C. The dialysis tubings used (A.H. Thomas Company, Philadelphia, U.S.A.) had a retention limit of about 12,000 mol. wt. according, to the manufacturer's specifications. The fluid outside the dialysis bag was evaporated at 40°C, lyophilized and stored at --20°C. The powder was reconstituted in isotonic saline just before use; the peptide concentration was determined by the method of Itzhaki and Gill (1964). Peptides constituted about 10% of the starting material.
2.6. Drugs used 1-Noradrenaline bitartrate (Recordati), 2deoxy-D-glucose (Sigma), sodium fluoride (NaF) (Carlo Erba), 2,4-dinitrophenol (2,4DNP) (Calbiochem), ouabain (Merck), hista-
3.1. Influence of peptides on the chronotropism of isolated rat atria Peptides exerted a positive chronotropic effect on isolated rat atria. This effect was concentration dependent, reached a peak within 3 min after the addition of the peptides into the isolated organ bath, lasted as a plateau during 3--4 min and then slowly disappeared (fig. 1). A slow appearance of a positive chronotropic response was also observed with glucagon (10 -7 M) the peak being reached within 5 min, while noradrenaline (3 × 10 -7 M)
A Beatslmin 40-
30~
330 pg/ml (5)
20 10,
~
220,uglm[ (6) |
0
2
L
6
8
165).~1/ml (3)
I() 1'2 I~ 1"6 Time(rain)
Fig. 1. Time course o'f the positive chronotropic effect induced by fibrinogen-derived peptides (at 3 different concentrations) on isolated rat atria. The chronotropic effect is expressed as increase in atrial frequency over the basal rate. Number of experiments in parentheses.
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W. B U C Z K O ET AL.
TABLE 1 E f f e c t o f p r o p r a n o l o l and s o m e m e t a b o l i c i n h i b i t o r s o n t h e m a x i m a l c h r o n o t r o p i c activity o f isolated rat atria induced by fibrinogen-derived p e p t i d e s , n o r a d r e n a l i n e and glucagon. Atria were p r e i n c u b a t e d for 5 rain w i t h the i n h i b i t o r y s u b s t a n c e s b e f o r e the a d d i t i o n o f the stimulus. Each value r e p r e s e n t s the m e a n o f t h e n u m b e r o f b e a t s / r a i n ± S.E. o f at least 4 e x p e r i m e n t s . The m a x i m a l increase in atrial rate i n d u c e d by the 3 stimuli was statistically significant (p < 0.01) w h e n c o m p a r e d w i t h saline. Substances
Final concentration
Saline
Saline Propranolol 2,4-DNPt NaF ~-t 2-Deoxy-Dglucose
-10 -7 M 5 × 10 -6 M 2 x 10 -2 M
191.7 192.2 187.8 189.1
20 m g / m l
190.5 ± 9.5
± ± ± ±
3.2 5.6 4.1 6.6
Peptides 330 p g / m l
Noradrenaline 3 × 10 -7 M
Glugagon 10 -7 M
241.6 225.0 242.5 192.5
256.5 210.7 277.5 227.5
271.4 278.7 276.6 193.7
± ± ± ±
4.7 5.0 4.8 7.5 **
190.0 ± 21.2 **
± 4.4 ± 5.2 ** ± 14.9 ± 13.6 *
209.0 ± 18.6 **
± 6.3 ± 13.7 ± 6.6 ± 13.4 **
181.2 ± 27.5 **
* p < 0.05; * * p < 0.01, as c o m p a r e d to t h e value o b t a i n e d w i t h each i n d u c e r w h e n i s o t o n i c saline was used instead o f the inhibitor. 2,4-dinitrophenol. J ' t S o d i u m fluoride.
induced its maximal effect more rapidly, usually within 1--2 min. In contrast with that of peptides, the activity of the latter two agonists was long-lasting.
3.2. Effect o f some cardio-inhibitors on the chronotropic effect of peptides and other stimulants
nificantly inhibited the activation induced by noradrenaline but did not influence the action of glucagon and of the peptides. The action of the latter agonists was abolished by glycolytic inhibitors such as NaF or 2-deoxy-D-glucose b u t not by 2,4-DNP, an inhibitor of oxidative phosphorylation.
3.3. Effect o f peptides on cAMP level and phosphorylase a activity o f isolated rat atria The chronotropic effect of peptides on isolated rat atria was compared with that of glucagon, noradrenaline, histamine and ouabain in the absence and presence of a/~-blocking agent and some metabolic inhibitors. At the concentrations used, noradrenaline, glucagon and peptides had a significant positive chronotropic effect (table 1). Neither ouabain (10-6--10 -4 M) nor histamine (I0-7--I0 -4 M) significantly modified the chronotropism of isolated rat atria. Arrhythmias were frequently observed with the highest concentrations of ouabain. Propranolol, a fl-blocking agent, sig-
The time course of biochemical events was measured after the addition of 330 gg/ml of peptides to the atria (table 2). cAMP in the atria increased significantly after 30 sec, whereas the maximal increase of phosphorylase a activity was observed 90 sec after incubation of atria with peptides. Glucagon induced a marked increase of both cAMP levels (162.5 + 6.8 pmol/g tissue) and phosphorylase a (22.6 + 3.9%) after 30 and 90 sec, respectively.
FIBRINOGEN-DERIVED PEPTIDES AND RAT HEART
359
TABLE 2 Time c o u r s e o f t h e variations o f cyclic AMP level and p h o s p h o r y l a s e a activity o f isolated rat atria a f t e r t h e a d d i t i o n o f f i b r i n o g e n - d e r i v e d p e p t i d e s (330 ~ug/ml). Each value r e p r e s e n t s the m e a n + S.E. o f at least 4 experiments. T i m e (sec)
0 15 20 30 60 9O 120 150 180 240 300
Cyclic A M P (pmol/g)
P h o s p h o r y l a s e a (%)
Control
Peptides
Control
Peptides
123.8 ± 6.7 115.4 ± 10.1 1 2 1 . 5 ± 6.5 1 1 7 . 6 ± 4.8 122.6 ± 8.6
121.6-+ 5.9 115.3 -+ 14.9 1 4 3 . 1 ± 13.6 138.1-+ 4 . 6 " 139.6 ± 11.5
12.4 ± 2.8
10.0 ± 2.1
119.5±
122.4-+ 10.3
7.4
11.1 11.0 10.0 12.8 11.3 14.2
± 2.0 -+ 1.3 ± 3.1 + 1.3 + 2.8 ± 3.2
12.0 -+3.5
7.0 10.0 24.6 22.0 20.2 17.0 10.0 9.7
-+ 2.0 -+ 2.0 ± 2.8 ** ± 3.6 * ± 6.3 ± 3.0 -+ 2.4 -+ 2.1
* p < 0.05; ** p < 0.01, as c o m p a r e d t o t h e c o n t r o l s t e s t e d s i m u l t a n e o u s l y .
4. Discussion The present study confirms and extends previous observations on the cardio-stimulatory action of peptides derived from fibrinogen digestion (Laki, 1951; Malofiejew et al., 1971). We have shown that fibrinogen
berg and Williamson, 1964). In our experiments, the chronotropic activity induced by glucagon and peptides was blocked by inhibitors of glycolysis such as NaF and 2-deoxyD-glucose. These results suggest that the mechanism of action of peptides may be similar to that of glucagon. It may be worth mentioning here that'the most active peptides in our preparations have lately been shown to have a molecular weight lower than 5,000 (Wisniewski et al., 1975; Buczko et al., 1976). Glucagon has a mol. wt. of 3,485. Glucagon has been reported to increase intracellular cAMP concentrations (Murad and Vaughan, 1969) and to activate phosphorylase a (Mayer et al., 1970) in the rat myocardium, two findings confirmed in the present study. In our experimental conditions, fibrinogen-derived peptides also increased both cAMP levels and phosphorylase a activity. Both glucagon and peptides showed their maximal effects on cAMP and on phosphorylase a after 30 and 90 sec incubation respectively. Although the increase of these biochemical parameters preceded the occurrence of a significant positive chronotropic effect,
360
the possibility that these events are causally related should be considered with caution. Indeed, even the role of cAMP in determining the cardiostimulatory effects of glucagon, as well as of catecholamines, is still debatable (Laraia, 1968; Laraia and Reddy, 1969; Sobel and Mayer, 1973; Kukovetz et al., 1973). It may be of interest, however, that fibrinogen-derived peptides similar to those used in the present study, were able to inhibit ADPinduced platelet aggregation, to increase skin vascular permeability (Buczko et al., 1976) and to stimulate the chemotactic activity of leucocytes (Kay et al., 1973). These phenomena have been reported to be mediated by an increase of cAMP (Salzman, 1972; Leahy et al., 1970). Whatever is the mechanism of action o f fibrinogen-derived peptides, their positive interference with heart contractility may have some clinical relevance. Indeed, the amounts of peptides used in this study are well within the range of the concentrations of fibrinogen degradation products (0.6 mg/ml, according to Solum et al., 1973) which can be obtained in man following administration of fibrinolytic agents such as streptokinase. Streptokinase has been reported to be of benefit for patients with recent myocardial infarction (Verstraete, 1971). Whether the products of fibrinogen digestion contribute in some way to the therapeutic efficacy of fibrinolytic drugs is still an unproven but stimulating hypothesis.
Acknowledgements The authors are grateful to Dr. L. Mussoni who performed the statistical analysis of the data. The technical assistance of Miss M.G. Castelli is greatly appreciated. Purified bovine fibrinogen was generously provided by AB Kabi, Stockholm, Sweden, through the courtesy of Dr. B. Strindberg. Plasmin was a gift from Dr. Lang, Kabi-Lang, Milano, Italy.
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FIBRINOGEN-DERIVED PEPTIDES AND RAT HEART logical properties of some fibrinogen degradation products, Scand. J. Haematol. Suppl. 13, 303. Malofiejew, M., A. Kostrzewska and K. Buluk, 1971, The influence of fibrinogen degradation products on the electrobiological activity of the rat heart, Experientia 27,139. Mayer, S.E., D.H. Namm and L. Rice, 1970, Effect of glucagon on cyclic 3',5'-AMP phosphorylase activity and contractility of heart muscle of the rat, Circulation Res. 26, 225. McNeill, J.H. and S.C. Verma, 1974, Blockade by burimamide of the effects of histamine and histamine analogs on cardiac contractility, phosphorylase activation and cyclic adenosine monophosphate, J. Pharmacol. Exptl. Therap. 188, 180. Murad, F. and M. Vaughan, 1969, Effect of glucagon on rat heart adenyl cyclase, Biochem. Pharmacol. 18, 1053. Repke, K., 1963, Metabolism of cardiac glycosides, in: Proceedings of the First International Pharmacological Meeting, Stockholm, Vol. III, New Aspects of Cardiac Glycosides, ed. W. Wilbrandt, (Pergamon Press, Oxford) p. 47. Robak, J. and R. Gryglewski, 1971, Influence of INPEA, pindolol and propranolol on the chronotropic and metabolic responses to ~-adrenergic stimulation in intact rats, Biochem. Pharmacol. 20, 2749. Robinson, G.A., R.W. Butcher and E.W. Sutherland,
361 1967, Adenyl cyclase as an adrenergic receptor, Ann. N.Y. Acad. Sci. 139, 703. Salzman, E.W., 1972, Cyclic AMP and platelet function, N. Engl. J. Med. 286, 358. Sobel, B.E. and S.E. Mayer, 1973, Cyclic adenosine monophosphate and cardiac contractility, Circulation Res. 32, 407. Solum, N.O., C. Rigoltot, A.Z. Budzynski and V.J. Marder, 1973, A quantitative evaluation of the inhibition of platelet aggregation by low molecular weight degradation products of fibrinogen, Brit. J. Haematol. 24, 419. Tovey, K.C., K.G. Oldham and J.A.M. Whelan, 1974, A simple direct assay for cyclic AMP in plasma and other biological samples using an improved competitive protein binding technique, Clin. Chim. Acta 56, 221. Verma, S.C. and J.H. McNeill, 1974, Action of imidazole on the cardiac inotropic, phosphorylase activating and cyclic AMP producing effects of nqrepinephrine and histamine, Res. Commun. Chem. Pathol. Pharmacol. 7, 305. Verstraete, M., 1971, The difficulty of appraisal of streptokinase treatment of myocardial infarction, Angiologica 8, 43. Wisniewski, K., W. Buczko and J. Moniuszko-Jakoniuk, 1975, The effects of products of fibrinogen digestion by plasmin [P-FDP] on the central nervous system, Acta Neurobiol. Exp. 35, 137.