Naunyn-Schmiedeberg's Arch. Pharmacol. 291, 123--130 (1975) 9 by Springer-Verlag 1975

Effects of Reserpine and Propranolol on Anoxia-Induced Enzyme Release from the Isolated Perfused Guinea-Pig-Heart K. Sakai* a n d P. G. Spieckermann Physiologisehes Institut der Universit~t G5ttingen Received May 15 / Accepted August 19, 1975

Summary. The possibility of a protective effect by reserpine or propranoIol on anoxia-induced release of malate and lactate dehydrogenase was investigated in isolated perfused hearts of guinea-pigs. After allowing 30 min of aerobic perfusion for the development of a steady state, the hearts from the nontreated group as well as the reserpine-pretreated or propranolol-treated group were subjected to a prolonged anoxia of 5 hrs. A marked enzyme release which occurred from the anoxic nontreated hearts was significantly inhibited by reserpine or propranolol. These findings suggest that the enzyme release from the anoxic myocardium is partly related to the liberation of endogenous eatecholamines. Key words: Anoxia -- Enzyme Release -- Reserpine -- Propranolol -- ~alate Dehydrogenase -- Lactate Dehydrogenase. I t has been repeatedly reported t h a t anaerobiosis induces cellular e n z y m e release from reversibly or irreversibly d a m a g e d cells (Sobel and Shell, 1972; Hearse et al., 1973; Sakai et al., 1975). U n d e r anaerobic conditions, catecholamines are released from the myocardial s y m p a t h e t i c system (Shahab and Wollenberger, 1967). I t was assumed t h a t the release of catecholamines contributes to the elevation of myocardial oxygen d e m a n d and to further stimulation of the energy-consuming system of the hearts, resulting in a m a r k e d further increase of enzyme release from the myocardlum. The present s t u d y with reserpine a n d propranolol as pharmacological tools was u n d e r t a k e n in order to clarify the relationship between endogenous catecholamines and e n z y m e release from the anoxie hearts.

Send o/]print requests to: P. G. Spieckermann, Physiologisehes Institut der Universit~t, ])-3400 GSttingen, Humboldtallee 7, :Federal Republic of Germany. * Alexander yon Humboldt Foundation Fellow; present address: Dr. K. Sakai, Department of Pharmacology, Research Laboratories, Chugai Pharmaceutical Co., Ltd., 3-41-8, Takada, Toshima-ku, Tokyo, Japan.

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K. Sakai and P. G. Spieckermann

Methods All the experimental procedures and the perfusion solution used in these experiments were as described by Sakai et al. (1975). Guinea-pigs (300--350 g body weight) of either sex were used for the experiments. Experiments were performed in three different groups of animals: controls, reserpine-pretreated and propranololtreated. Reserpine (Serpasil | Ciba-Geigy, Switzerland) (1 mg/kg) was given i.m. twice, 48 and 24 hrs before the experiment. This dose of reserpine depletes the myocardial catecholamine stores of these animals by more than 900/0 (Muskus, 1964; Wagner, 1970). (~:)-propranolol hydrochloride (Dociten| ICI, England) was given i.p. in a dose of 1 mg/kg, 30 rain before the experiment and, moreover, added to the perfusion fluid, at a concentration of 4.1 • 10-s ~ . Animals were heparinized (5 mg i.p.) and stunned with a blow on the head. Immediately after thoracotomy, the heart was excised and immersed in ice-cold, 02-saturated Tyrode solution. The aorta was cannulated and the heart was perfused according to the Langendorff technique in a thermostatically regulated waterjacketed chamber. Perfusion was carried out by means of a non-recireulating perfusion system at a flow rate of 4 ml/min delivered by a peristaltic pump (Desaga, Heidelberg, Germany). The flow rate seemed to be adequate to maintain myocardial energy metabolism and stable mechanical activity for at least 5 hrs under aerobic perfusion (Sakai et al., ] 975). The perfusion solution was a modified Tyrode solution containing (mM) NaC1137, KC12.7, CaC12 1.9, MgC121.0, NaHC03 12, NaHI2PO4 0.5 and glucose 6 (a total concentration of about 300 mOs/1) and sufficiently equilibrated with 5~ C02--950/0 03 (pH 7.3, 35~ Anoxia was induced by equlibrating the perfusate throughout the experimental periods with a gas mixture of 50/0 C02--95~ N~, resulting in an oxygen tension below 3 mm Hg and a p H of 7.3. There were two parallel perfusion systems, either of which could be alternatively switched into the perfusion line by means of a stopcock just before the perfusion pump. During the periods of anoxia, the chamber was equilibrated with the corresponding gas mixture. The oxygen content of the perfusate was determined by analysing samples with a gas analyser, Lex-O2-Con. (Lexington Instruments Co., Waltham, ~assaehusetts, U.S.A.). Perfusion pressure was measured with Statham pressure transducers (Starham Instruments, Inc., Oxnard, California, U.S.A.) and varied between 16 and 60 mmHg. Heart rate was routinely determined with a stop watch. Malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) activities were determined kinetieally at 25~ with a filter photometer (366 nm) (Eppcndorf, Germany), as described by Bergmeyer and Bernt (1974) and Wroblewski and Ladue (1955), respectively. In the in vitro experiments, the effects of reserpine or propranolol on MDH and LDH activities were investigated as follows: for the deterruination of IVIDI-Iactivity, a solution of aspartate (42 mY) was prepared in 0.1 M phosphate buffer (pH 7.4). 3 ml of this solution, 0.05 ml of a-oxoglutarate solution (65 mM), 0.05 ml of ~TADH solution (12 raM), and 0.05 ml of 0.01 ~ GOT solution were incubated for 5 rain at 25~ At the end of the incubation, 0.05 ml ofaMDH/ NADH solution (300mUM])H, 25 ~g fl-NADI.I-Na and 150 ~g albumen) and 0.05 ml of either propranolol (3.25 ~g) or reserpine (3.25 ~g) solution were added and the optical density was immediately measured. For the determination of LDH activity, a solution of pyruvate (0.6 raM) was prepared in 50 mM phosphate buffer (pH 7.5). 3 ml of this solution, 0.05 ml of NADI~ solution (0.18 raM), 0.05 ml of LDH (from pig heart) solution (480 mU), and 0.05 ml of either propranolol (3.15 ~g) or reserpine (3.15 ~g) solution were mixed and the optical density was immediately determined. Initial velocity of enzyme reaction was measured and the difference of optical density per rain (z~E/min) was determined. All chemical agents used for the

125

Enzyme Release and Anoxia

analysis (Boehringer Mannheim, Germany) were dissolved in redistilled water, except LDH and MDH/NADH which were dissolved in 0.90/0 NaC1 solution. Since reserpine (Carl Roth, Germany) was dissolved using 10% N,N-dimethylaeetamide (DMAA, Fluka, Switzerland) in 0.90/0 NaC1 solution and propranolol hydrochloride was diluted with 0.9% NaC1 solution, 0.05 ml of these solutions were taken as the control. For determination of MDH and LDH in the perfusate, the effluent fluid was collected for 1 rain at different times and subjected to analysis. The concentrations were expressed as mU/(rain • g heart wet weight), as previously described (Sakai et al., 1975). Results were expressed as means • standard error of the means (SEM). The statistical significance of differences between means was calculated with the Wilcoxon, Mann and Whitney test (U-test) (Sachs, 1974).

Results

The Influence oI Reserpine or Propranolol on the Enzyme Assay T a b l e 1 shows t h a t r a t h e r high c o n c e n t r a t i o n s of reserpine a n d prop r a n o l o l did n o t influence the a c t i v i t y of m a l a t e a n d l a c t a t e dehydrogenase.

The Ellect of Reserpine or Propranolol on Enzyme Release Aerobic Per/usion. The hearts from the controls, rescrpine-pretreated a n d p r o p r a n o l o l - t r e a t e d animals, respectively, were aerobically (950/0 02:5~ COs) perfused over 5 hrs. Stable values for the release of e n z y m e s

Table 1. Effect of reserpine or propranolol on enzyme activity

Controls* (3) l~eserpine 1.6 • 10-6 M (3)

Controls** (4) Propranolol 3.3 • 10-s M (3)

MDH AE/rain

AE/min

LDtt

0.283 ~= 0.005

0.473 ~ 0.006

0.291 ~ 0.001 N.S.

0.491 • 0.004 N.S.

0.278 ~ 0.001

0.436 ~= 0.011

0.280 :J= 0.001 N.S.

0.423 ~ 0.007 N.S.

Initial velocity of enzyme (malate dehydrogenase, MDH; lactate dehydrogenase, LDH) reaction was measured kinetically at 25~ (see Methods) and the difference in optical density per rain (AE/min) was determined. Values are means ~ SEM. Numbers in parentheses give the numbers of the samples. N.S.: not significant (control vs. propranolol or reserpine). The doses of drugs are expressed in terms of a final concentration. Controls: 10~ DMAA* or 0.90/0 NaCl** solutions instead of drug solutions were used.

K. Sakai and P. G. Spieckermann

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Fig.l. Enzyme release (malate dehydrogenase, MDH; lactate dehydrogenase, LDtt) from the isolated perfused guinea-pig heart during aerobic perfusion for 5 hrs. Vertical bars indicate means :L SEM. Numbers in parentheses are numbers of

preparations

(malate and lactate dehydrogenase) were reached within 30 min after the onset of perfusion and remained constant for at least 5 hrs. In the propranolol-treated group, the release of the enzymes was slightly lower as compared to the other groups (Fig. 1, Table 2). The values for h e a ~ rate and perfusion pressure, respectively, 30 min after the onset of perfusion were as follows: non-treated, 158.3 • 4.0/rain and 28.2 • 2.4 m m H g (N -----8); reserpine-pretreated, 163.2 • 3.O/rain and 30.2 • 1.9 m m H g (N ---- 4); propranolol-treated, 155.0 -4- 5.3/rain and 24.4 -/- 4.3 m m H g (N =- 4). There were no significant differences between the corresponding values. The mechanical parameters remained rather constant throughout the course of experiments. Anaerobic Per]usion. After allowing 30 rain of aerobic perfusion for the development of a steady state of the preparations, the non-treated, reserpine-pretreated and propranolol-treated hearts were subjected to a prolonged anoxia (95% N ~ : 5 % COs) of 5 hrs. In the control hearts, an appreciable release of enzymes occurred within 60 rain of anoxic perfusion and reached a maximum level between 180--210 rain. Reserpine or propranolol treatment significantly diminished the increased release of enzymes (Fig. 2 A and B, Table 2).

Enzyme Release and Anoxia

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Table 2. Enzyme release from the hearts of controls, reserpine-pretreated and propranolol-treated guinea-pigs during anaerobiosis

Control MDtt

Maximum LDH

mU/min • g wet wt. Controls

MDtt

LDIt

mU/min • g wet wt.

(10)

47.3 • 7.1

28.2 i 4.4

586.3 • 82.9

179.3 i 23.2

Reserpine (12)

39.9 ~ 3.2 N.S.

30.0 -4- 2.4 N.S.

280.1 • 36.9 P < 0.001

89.9 • 6.5 P < 0.001

Propranolol (4)

18.8 J: 3.7 P < 0.005

12.2 -4- 3.4 P < 0.02

79.4 • 13.1 P < 0.001

35.1 :L 4.7 P < 0.001

The results were obtained from experiments according to those in Figs. 2A and B. Controls: values obtained just before anoxic perfusion. Maximum: values obtained 210 rain after the onset of anoxie pcrfusion. Values are means ~ SEM. Numbers in parentheses are numbers of preparations. P values represent differences between enzyme amounts released from the non-treated and reserpine-pretreated or propranolol-treated groups. ~q.S. : not significant. Each maximum value was significantly different from the corresponding control value (P < 0.001).

Discussion I n the present experiments, anaerobiosis induced a pronounced release of malate a n d lactate dehydrogenase f r o m the myocardium, which was significantly diminished b y the t r e a t m e n t with reserpine or propranolol. Since reserpine and propranolol h a d no influence on the enzyme assay, it is suggested t h a t the e n z y m e reIease is related to a liberation of endogenous catccholamines f r o m the adrenergic nerve endings during anaerobiosis (Shahab and Wollenberger, 1967). I t is established t h a t cardiac o x y g e n c o n s u m p t i o n is increased b y an excess o f catecholamines. U n d e r anaerobic conditions this will result in a more rapid breakdown of myocardial energy-rich phosphates. The " p r e v e n t i o n of leakiness" of enzymes (Zierler, 1958) m a y be closely coupled to myocardial energy-consuming processes. W e have previously f o u n d a close correlation (r -= - - 0.99) between the rate of e n z y m e release a n d the fall of myocardial A T P content in the anaerobically perfused heart (Spieckermann et al., 1973) ; the faster A T P breaks down, the more e n z y m e is released. This finding u n d o u b t e d l y indicates a relationship between e n z y m e release a n d myocardial energy-consuming processes. The report b y Wilkinson a n d R o b i n s o n (1974) t h a t an intracellular A T P deficiency is a m a j o r factor in p r o m o t i n g release of enzymes supports this view. I f the e n z y m e release is related to t h a t of endogenous catecholamines, as suggested b y De Leiris et al. (1972) using the aerobically perfused r a t heart, it is n o t surprising t h a t the increased e n z y m e re9 ~aunya-Schmiedeberg'sArch. Pharmacol.,Vol.291

128

K. SakM and P. G. Spieekermann

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Fig.2A and ]3. Enzyme release (malate dehydrogenase, MDtt; lactate dehydrogenase, LDH) from the isolated perfused guinea-pig heart during anaerobic perfusion for 5 hrs. Vertical bars indicate means 4- SEM. Numbers in parentheses are numbers of preparations. Differences in the values of enzyme release between the controls and reserpine-pretreated (A) or propranolol-treated (B) groups were signifikant (P < 0.01), except for those indicated by asterisks

lease is significantly reduced by the treatment with reserpine or propranolol. Reserpine depletes the cardiac catecholamine stores and propranolol prevents the stimulation of the cardiac fl-adrenergic receptors b y the liberated cateeholamines. The resulting decrease in myocardial oxygen consumption leads to the conservation of energy-rich phosphates and the observed diminution of enzyme release. Whether the membrane stabilizing effect of propranolol (Davis, 1970; Fitzgerald et al., 1972) contributes to the decrease in enzyme release cannot be decided. I t is concluded that the elevation of enzyme release from the heart during anoxia is in part related to the liberation of endogenous catecholamines.

Aclzaowledgemen~s. We are thankful to Miss A. Holtmeyer and Miss D. Jeschke for their excellent assistance, and to Mrs. A. K15ppner for typing the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 89, Kardiologie G6ttingen), and the Alexander yon Humboldt Foundation.

Enzyme Release and Anoxia

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References Bergmeyer, It. U., Bernt, E. : Malate-Dehydrogenase. In: Methoden der enzymatischen Analyse, pp. 649--653. Edited by It. U. Bergmeyer. Weinheim: Verlag Chemie 1974 Davis, W. G. : A comparison of the local anaesthetic, quinidine-like and adrenergic fl-blocking activities of five beta receptor antagonists. J. Pharm. Pharmacol. 22, 284--290 (1970) De Leiris, J., Freuvray, D., Come, C. : Acetyleholine-induced release of lactate dehydrogenase from isolated perfused rat heart. J. tool. Cell. Cardiol. 4, 357--365 (1972) Fitzgerald, J. D., Wale, J. L., Austin, M.: The haemodynamic effects of (• pranolol, dexpropranolol, oxprenolol, practolol and sotalol in anaesthetised dogs. Europ. J. Pharmacol. 17, 123--134 (1972) Hearse, D. J., Humphery, S. M., Chain, E. B. : Abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: a study of myocardial enzyme release. J. tool. Cell. Cardiol. 5, 395--407 (1973) Muskus, A. J. : Evidence for different sites of action of reserpine and guanethidine. Naunyn-Sehmiedeberg's Arch. exp. Path. Pharmak. 24S, 498--513 (1964) Sachs, L.: Angewandte Statistik, pp. 230--238. Berlin-Heidelberg-New York: Springer 1974 Sakai, K., Gebhard, M.M., Spieckermann, P.G., Bretschneider, H . J . : Enzyme release resulting from total ischemia and reperfusion in the isolated perfused guinea pig heart. J. tool. Cell. Cardiol. (in press, 1975) 9*

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Shahab, L., Wollenberger, A.: Freisetzung yon Noradrenalin aus dem isolierten durchstr6mten Herzen bei akuter Anoxie und nach Gabe yon St~)ffwechsetgiften. Acta biol. reed. germ. 19, 939--959 (1967) Sobel, B. E., Shell, W. E. : Serum enzyme in the diagnosis and assessment of myocardial infarction. Circulation 45, 471--482 (1972) Spieckermann, P. G., Gebhard, M. M., Kalbow, K., Knoll, D., Kohl, F., Nordbeck, H., Sakai, K., Bretschneider, H. J.: Freisetzung yon Enzymen aus der Herzmuskelzelte w~hrend Sauerstoffmangel. Verh. dtsch. Ges. Kreisl.-Forsch. 89, 193--198 (1973) Wagner, J. : Effect of phentolamine on isolated guinea-pig atria. Europ. J. Pharmacol. 9, 276--280 (1970) Wilkinson, J. H., Robinson, J. M. : Effect of ATP on release of intracellular enzymes from damaged cells. Nature (Lond.) 249, 662--663 (1974) Wroblewski, F., Ladue, J. S. : Lactic dehydrogenase activity in blood. Proc. Soc. exp. Biol. (N. u 90, 210--213 (1955) Zierler, K. L. : Muscle membrane as a dynamic structure and its permeability to aldolase. Ann. N. u Acad. Sci. 75, 227--234 (1958)

Effects of reserpine and propranolol on anoxia-induced enzyme release from the isolated perfused guinea-pig-heart.

The possibility of a protective effect by reserpine or propranolol on anoxia-induced released of malate and lactate dehydrogenase was investigated in ...
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