Naunyn-Schmiedeberg's
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 67-72 (1976)
9 by Springer-Verlag 1976
Release of Creatine Kinase by Antigen, Histamine, and Catecholamines* W. Bernauer Pharmakologisches Institut der Universit~t Freiburg, Katharinenstrasse 29, D-7800 Freiburg i. Br., Federal Republic of Germany
Summary. In mepyramine treated, ovalbumin sensitized anaesthetized guinea pigs protracted anaphylactic shock was produced by i.p. injection of antigen, and serum creatine kinase (CK) activities were determined 4, 6, or 17 hrs thereafter. Significant increases above nonsensitized controls were obtained. In nonanaesthetized guinea pigs shock course and serum CK increase were considerably accelerated. Histamine increased the serum CK only when given in high amounts (10 mg/kg) s.c., in the presence or absence of mepyramine. I.p. injection of histamine in mepyramine treated animals had no effect. Adrenaline (1 or 10 mg/kg) given as a s.c. depot in oil produced a significant increase of serum CK, as well as noradrenaline (0.1, 1, or 10 mg/kg). Dibenamine reduced the effect of adrenaline. In isolated perfused guinea pig hearts a significant CK liberation occurred already within the first hour after eliciting anaphylaxis. Nonanaphylactic hearts released CK too, but significant amounts were obtained only in the total 4 hrs after ovalbumin administration. Isolated anaphylactic hearts incubated in Tyrode solution liberated significantly more CK than did nonsensitized control hearts. The findings are discussed in view of a possible myocardial damage in anaphylaxis. Key words." Creatine kinase - Anaphylaxis - Catecholamines - Histamine.
INTRODUCTION In man, the manifestation of anaphylaxis can mainly consist of respiratory obstruction (by laryngeal edema * A preliminary report was given on the Spring Meeting of the German Pharmacological Society at Mainz, March 4 - 7 , 1975 [Bernauer, W.: Naunyn-Schmiedeberg's Arch. Pharmacol. 287, R 62 (1975].
and/or bronchospasm), or circulatory failure may predominate. In the guinea pig, both types of shock can be clearly separated by different ways of antigen administration: I.v. injection leads to acute bronchospasm and suffocation within some few minutes. S.c., or i.p. administration of antigen evokes a protracted and obviously circulatory shock (Williamson, 1936; Stone, 1959). The latter shock type results also when the antigen is given i.v. and the bronchospasm is prevented by a Hi-receptor antagonist (Wilcox and Seegal, 1942; Green, 1953; Stone, 1959; Giertz et al., 1961). Knowledge about the protracted shock is still very limited. However, there are good reasons to assume that the heart plays an important role. Isolated guinea pig hearts react to antigen with a very intense and long lasting coronary constriction coupled with decreased contractile force and marked arrhythmias, preceded by a short stimulatory phase (Went and Lissak, 1935; Wilcox and Andrus, 1938; Andrus and Wilcox, 1939; Feigen et al., 1960; Greeff and Heeg, 1964; Hahn and Bernauer, 1970; Liebig et aL, 1975). Furthermore, in guinea pig heart-lung preparations anaphylaxis can lead to severe cardiac failure (Hahn and Bernauer, 1969). In the heart-lung preparation also a marked pulmonary vascular constriction was detected which may be involved in the genesis of the protracted shock (Bernauer et aL, 1969; Hahn et aL, 1970). Finally, hypovolemia due to increased vascular permeability has to be taken into account since a pronounced hemoconcentration was found (Bernauer and Schanz, 1974). The question is, whether besides the functional disturbances the anaphylactic coronary spasm can produce a profound damage of the myocardium. In man, it was reported that during anaphylaxis ECG alterations can be found which point to severe myocardial ischemia or even myocardial infarction (Binder et al., 1950; McManus and Lawlor, 1950;
68 Pfister a n d Plice, 1950; B e r n r e i t e r , 1959; H a n a s h i r o a n d W e i l , 1967; B o o t h a n d P a t t e r s o n , 1970; B r a s h e r a n d S a n c h e z , 1974). A s e s p e c i a l l y in t h e g u i n e a p i g t h e o c c u r r e n c e o f c o r o n a r y c o n s t r i c t i o n is w e l l e s t a b lished we investigated the serum creatine kinase (CK; ATP-creatine-transphosphorylase) activities in p r o t r a c t e d a n a p h y l a c t i c s h o c k o f t h i s species. C K is l o c a t e d in v e r y h i g h a m o u n t s in t h e h e a r t m u s c l e cells a n d r e a c h e s t h e c i r c u l a t i n g b l o o d a f t e r m y o c a r d i a l cell d a m a g e . M o r e o v e r , s u b s t a n c e s w h i c h are released during anaphylaxis were tested on their a b i l i t y to l i b e r a t e C K , n a m e l y h i s t a m i n e , a d r e n a l i n e , a n d n o r a d r e n a l i n e . A n d finally, C K r e l e a s e f r o m isolated hearts was investigated.
METHODS Guinea pigs of either sex were sensitized with ovalbumin (Albumen ovi siccum, Merck Co., Darmstadt, Germany; 5 ~ solution; 0.1 ml i.p. on days 1, 3, and 5; beginning with day 21 the animals were used for the experiments).
Intact Animals Sensitized or nonsensitized, urethane anaesthetized (1.0 g/kg i.p.) animals received mepyramine maleate (0.1 mg/kg) or only saline (1 ml/kg) s.c. in the crural region of the left hindleg, and 10 rain later ovalbumin (100 mg/kg) i.p. Blood was withdrawn from the carotid artery 4, 6 or 17 hrs after ovalbumin. As further controls served nonsensitized animals which received only uretbane. In another group of nonsensitized animals only mepyramine was given, and blood was withdrawn 4 hrs later. Anaphylaxis was also elicited in nonanaesthetized, mepyraminized guinea pigs. When a severe state of shock had developed the thorax was opened quickly by section of the sternum and the diaphragm, and blood was withdrawn from the vena cava thoracalis inf. Urethane anaesthesia was applied only for the blood withdrawal (5 min before opening the thorax). For controls, blood was taken just at the same times from nonsensitized animals from the vena cava, and in a further group from the carotid. - Finally, acute anaphylaxis was produced by i.v. injection of antigen, with or without mepyramine pretreatment, and blood was withdrawn from the carotid at 2, or 10 rain. Histamine dichloride (1, or 10 mg/kg; in saline) was injected i.p. or s.c. (hindleg) with or without mepyramine pretreatment (0.1 mg/kg, s.c.). Blood was taken 4 or 17 hrs later from the carotid. Also mere saline or mepyramine controls were performed. Adrenaline (base; tartrate; 1, or 1.0 mg/kg) was administered as a s.c. depot in peanut oil in the midline of the abdomen, and blood was taken from the carotid at 6 or 17 hrs. For controls, peanut oil only was given. In further experiments, dibenamine hydrochloride (5 mg/kg, in saline) was administered s.c. (hindleg) 1 hr before adrenaline. Controls received dibenamine and peanut-oil. Moreover, adrenaline (base; 1, or 10 mg/kg, in oil) was given s.c. 30 min after i.p. injection of ovalbumin (100 mg/kg) in sensitized, mepyraminized animals, and blood was obtained 6 brs after the antigen. Controls were nonsensitized animals treated likewise, and sensitized as well as nonsensitized animals, which received only oil instead of adrenaline. Finally, noradrenaline base was given as s.c. depot in oil (0,1, 1, or 10 mg/kg) and blood was withdrawn 17 hrs later.
w. Bernauer
Isolated Hearts a) Artificially Perfused Hearts. Sensitized, or nonsensitized guinea pig hearts were perfused at constant pressure (54 cm H20) in a modified Langendorff apparatus with carbogen saturated Tyrode solution of 38~C. After a 15-min period of free perfusion a recycling circuit was closed, and 10 min later a sample was taken from the Tyrode in the reservoir. Then ovalbumin was injected intraaortically (10 rag, in 1 ml saline) and further Tyrode samples were withdrawn 1, 2 and 4 hrs thereafter. b) Incubated Hearts. In anaesthetized artificially respirated guinea pigs the venae cavae sup. and inf. were ligated, and 5 sec later the arcus aortae. The hearts were freed from lungs and vessels, except the ligated aorta, and put immediately into oxygenated Tyrode (room temperature) which was changed repeatedly. Then they were incubated for 3 hrs in 20 ml carbogen gassed Tyrode (38~ Ovalbumin (100 gg/ml Tyrode) was added after a 5 min preincubation period. Such hearts beat spontaneously, sucking the Tyrode solution into the open left atrium and left ventricle, perfusing their coronaries via the ligated stump of the aorta. In contrast to hearts in which the aorta is cut, the coronaries are free of blood already after some few contractions. As we know from unpublished observations these hearts react very strongly to antigen releasing significant amounts of histamine and SRS-A. To demonstrate that the coronaries are indeed perfnsed, 8 aorta-ligated hearts were incubated l0 min in Tyrode containing 0.25 mg Evans blue/ml. The total myocard was dark blue, containing 0.0812 + 0.00934 mg Evans blue/ml ventricular tissue, determined with the photometric method of Beach and Steinetz (1961). Hearts in which the aorta was cut (n = 8) showed only a thin layer Of dye in the endocardium and epicardium. The ventricular myocard was not stained, the coronaries were still filled with blood. The Evans blue content was significantly smaller (P < 0.01) than in the selfperfusing hearts. Extracts of identically incubated hearts, but without Evans blue, served as blanks.
Determination o f Creatine Kinase The activated backward reaction with creatine phosphate as substrate was used as kinetic test. The procedure is based on the method of Oliver (1955) and described in detail by Forster et al. (1970). Briefly, it consists of three steps: 1. Dephosphorylation of creatine phosphate and formation of ATP. 2. Formation of glucose-6-phosphate from glucose and ATP. 3. Formation of 6-phosphogluconate with simultaneous reduction of NADP. NADPH is determined spectrophotometrically at 366 nm. Incubation temperature 25~C. In all samples which gave an extinction difference of more than 0.03/min an additional determination with the 1/5 diluted serum (with saline) was performed. However, in agreement with Graig et al. (1967) we observed that diluted sera give relatively too high CK activities. Moreover, we found that the degree of error is variable; sometimes diluted sera yielded lower activities than the nondiluted ones. Therefore, in the following results only the undiluted serum values are presented. The results with the diluted sera, however, were essentially the same. All va~lues are given as means +_ standard errors of the means. Statistical evaluations were performed with the t-test.
RESULTS
A. Anaphylaxis Nonsensitized guinea pigs which received no injections b u t 1 g u r e t h a n e / k g i.p., h a d 5 m i n a f t e r i n d u c i n g
Release of Creatine Kinase anaesthesia serum creatine kinase (CK) activities of 89.6 __ 21.01 mU/ml (Fig. 1). Anaphylaxis produced marked increases of serum CK. In three different groups of sensitized guinea pigs which received ovalbumin (100 mg/kg i.p.) 10 rain after mepyramine (0.1 mg/kg s.c.), serum CK was significantly increased when compared with nonsensitized animals treated likewise at 4, 6, as well as J7hrs after ovalbumin, respectively (P < 0.001; p < 0.02; P < 0.001). From each animal blood was withdrawn only once. At 17 hrs serum CK levels were significantly higher than at 4 hrs (P < 0.02). In nonsensitized animals, no significant alterations of serum CK were found. In the 4 hrs anaphylaxis group 1 animal out of 14 died, in the 6 hrs group 3 out of 10. In the 17 hrs group, out of 22 guinea pigs 11 died in protracted shock; 2 surviving animals had strongly lipemic sera and were not used. The animals which survived, and from which CK determinations were obtained rather represent the guinea pigs with the weaker anaphylactic reactions. Serum CK increase is not produced by hemoconcentration and release of normal CK amounts into a smaller blood volume. Hematocrit values in the anaphylaxis group with the greatest C K increase (17 hrs) were 43.8 • 1.15~ (n = 8), whereas in the nonsensitized controls 44.0 i 1.67~ was found (n = 5).
Since all animals had received the antihistamine mepyramine to exclude any bronchospastic reactions, mepyramine was also tested alone. It had no effect on the serum CK activity. When 100 mg ovalbumin/kg were given i.p. into sensitized animals without mepyramine pretreatment, 12 out of 14 died before reaching 17 hrs. Therefore, only 10 mg ovalbumin/kg were used, which produced a lethality of 10~. Serum CK at 17 hrs was 312.4._+ 52.98 mU/ml (n = 9), whereas in nonsensitized animals 145.8 _+ 54.51 mU/ml was obtained (n = 1 0 ; P < 0.05). In nonanaesthetized animals a severe state of shock developed already within the first hour after antigen injection (100 mg/kg; i.p.). In this state it was not possible to obtain enough blood from the carotids. Therefore, blood was withdrawn from the vena cava thoracalis inf., at 59.4 _+_ 5.84 rain, in the mean. Serum CK was 709.2 i 52.42 mU/ml (n = 8). In 8 nonsensitized controls from which blood was taken in the same way just at the same times 135.7 _+ 22.50 mU/ml were found (P < 0.001). In a second control group blood was taken at the same times from the carotids. Here, 88.4 • 10.02 mU/ml were obtained (n = 8), so that the procedure of opening the thorax had not influenced the CK values in a relevant manner.
69 Serum Creatine Kinase mU/ml
700 -
600-
500-
400--
300-
200-
1OO-
1
23
45
Sensitization
-
+ -
+ -
+ -
Mepyramine
-
+ +
+ +
+ +
+ +
+ +
6h
Group Nr
Ovalbumin
-
+ -b
Time of blood withdrawal
5
4h
n
21
13 15
7
8
9
17 h
9
9
+
4 h
14
Fig. 1. Serumcreatine kinase activity in protracted anaphylactic shock of ovalbumin sensitized guinea pigs. Significant increase above nonsensitizedcontrol animals
During acute anaphylaxis no significant increase of serum CK was found. 8 sensitized animals received 10 mg ovalbumin/kg i.v., 5 min after i.v. injection of saline (1 ml/kg). 2 min after the antigen 93.6 _+ 24.79 mU CK/ml serum were obtained, and 119.2 _+ 47.74 in 6 animals, which received 0.1 mg mepyramine/kg instead Of saline. In further 9 sensitized, mepyraminized guinea pigs serum CK was 188.0 _+ 40.01 mU/ml, 10 rain after i.v. injection of ovalbumin; 10 nonsensitized controls had 132.5 _+ 27.34 mU/ ml. As during protracted shock adrenaline is liberated, and adrenaline itself provokes an increase of serum CK (see below), the influence of the a-receptor blocking agent, dibenamine, on anaphylactic CK release was tested. 5 mg dibenamine/kg injected s.c. 1 hr before the antigen (mepyramine treated animals) did not decrease the CK levels at 17 hrs (597.3 _+ 58.23 mU/ ml; n = 6). However, the dibenamine dose increased the CK for its own. In nonsensitized guinea pigs, which received dibenamine, mepyramine, and ovalburain, 17 hrs later 392.6 _+ 76.53 mU/ml serum were obtained (n = 7), in contrast to 160.0 • 50.42 (n = 9)
70
W. Bernauer
Table 1. Serum creatine kinase activities in guinea pigs after the i.p., or s.c. administration of histamine Group no.
1 2 3 4 5 6 7 8 9
Mepyramine 0.1 mg/kg s.c.
Histamine mg/kg
Time of blood withdrawal (hrs)
Serum C K mU/mt (n)
+ + + + -
-
4 4 4 17 17 17 17 17 17
166.7 209.3 311.1 175.4 122.6 612.0 327.9 158.6 134.9
-
+ +
1 i.p. 10i.p. 10 i.p. 1 s.c. 10 s.c. 10 s.c. -
• • • • • • • • •
42.65 64.60 59.39 53.24 31.69 60.56 71.81 25.94 29.29
Serum Creatine Kinase
mU/ml
800 -
700-
(10) (6) (7) (6) (6) (5) (10) (6) (5)
600-
500-
r 400-
300-
M
200 -
100-
when no dibenamine was given (P < 0.025). Therefore, the serum CK level in the dibenamine-anaphylaxis group is partially due to bibenamine. Anaphylaxis in the dibenamine treated group did not increase significantly the CK level when compared with the respective nonsensitized dibenamine treated animals (0.05 < P < 0.1).
L_ Group Nr.
2
Mepyramine
-
Adrenaline mg/kg
n
10
3
4
1
10
10
6
6
4
+
Fig. 2.
Serum creatine kinase activity 17 hrs after injection of adrenaline in oil as subcutaneous depot
B. Histamine
1 mg histamine/kg given i.p. in mepyramine treated animals (0.1 mg/kg, s.c.) did not increase the serum CK within 4 hrs, nor did 10 mg/kg (0.05 < P < 0.1), when compared with mepyraminized guinea pigs, which received saline instead of histamine (Table 1). In a further group even 17 hrs after injection of 10 mg histamine/kg no effect was seen. 10 mg histamine/kg given s.c., however, with or without mepyramine pretreatment, increased significantly the serum CK at 17 hrs in comparison with the respective controls (groups 7/9 of Table 1; P < 0.0.5; groups 6/8 ; P < 0.001). 1 mg/kg s.c., without mepyramine, had no effect. When the 10 mg histamine were given s.c. without mepyramine, considerably forced respiration was observed. From originally 11 animals 6 died, after 22 rain in the mean, showing marked emphysema of the lungs. Even after mepyraminization 3 out of 13 did not survive the 17 hrs of observation after the s.c. injection of 10 mg histamine/kg. C. Catecholamines
Adrenaline, administered in peanut oil as s.c. depot in doses of 1, or 10 mg/kg produced after 17 hrs significant increases of serum CK, when compared with animals which received only peanut oil (P < 0.01 ; P < 0.001 ; Fig. 2).
The CK liberation by adrenaline was not inhibited by mepyramine. In further experiments, not represented in Fig. 2, adrenaline tartrate (10 mg/kg) was given s.c. in oil, instead of adrenaline base. 5 out of 9 animals died, whereas none of the adrenaline-base-group died. The surviving 4 animals had significantly increased CK levels (634.7 _+ 22.52mU/ml; P < 0.001). Dibenamine (5 mg/kg, s.c., n = 6) given 1 hr before, reduced significantly the effect of adrenaline (base, 10mg/kg; 684.2 +_ 16.51mU/ml vs. 485.9 _+ 60.79mU/ml; P < 0.02). The CK values were not significantly different from the levels found in dibenamine-peanut oil controls (325.1 +_ 41.63 mU/ ml; n = 6). Adrenaline, injected s.c. in oil 30 min after el• anaphylaxis did not alter the antigen induced CK increase within 6 hrs. Sensitized, antihistamine treated guinea pigs which received only peanut oil 30 rain after ovalbumin (100 mg/kg i.p.), had 6 hrs after antigen serum CK activities of 530.3 + 74.40 mU/ml (n = 11). Nonsensitized controls had only 190.8 + 46.30; n = 17; (P < 0.001). When I m g adrenaline/kg was given CK levels amounted to 502.9 + 89.94 mU/ml (n = 9) in sensitized, and to 229.0 _+ 69.17 (n = 10) in nonsensitized animals. Even 10 mg adrenaline/kg had no effect within the 6 hrs after el• anaphylaxis (579.9 + 87.65; n = 8).
Release of Creatine Kinase
71
Serum Creatine Kinase
(sensitized: 86.4 _+ 8.52 mU/g; n = 7; nonsensitized 41.7 _+ 6.85 mU/g; n = 7; P < 0.005).
mU/ml
700.
600.
DISCUSSION
500-
400
300-
200
7.4,
///
100-
2
GroupNr. Noradrenaline mg/kg n
-
10
3
0,1
1
8
5
4 10 5
Fig. 3. Serum creatine kinase activity 17 hrs after injection of noradrenaline in oil as subcutaneousdepot
Also noradrenaline produced an increase of serum CK 17 hrs after s.c. administration (Fig. 3). Injection of 0.1, 1, or 10 mg noradrenaline/kg in oil resulted in CK levels which were significantly higher than those of mere oil controls (P < 0.005; P < 0.005; P < 0.001). D. Isolated Hearts
It proved to be very difficult to keep isolated perfused anaphylactic hearts beating for several hours. The hearts showed the typical initial stimulation and the secondary long lasting arrhythmic phase of anaphylactic reaction. Already within the first hour after eliciting anaphylaxis a significant CK release of 2.4 + 0.79 U/g heart was found (n = 12; P < 0.02). Within 2 hrs 5.8 _+ 2.43 U/g were liberated (n = 10; P < 0.05), and within 4 hrs 15.2 _+ 4.84 U/g (n = 7; P < 0.025). However, also nonanaphylactic hearts released CK. However, the CK liberation was statistically significant only within the total time space of 4 hrs and not within the first, or the first 2 hrs (6.7 _+ 2.37 U/g; n = 6; P < 0.05). When isolated, spontaneously beating hearts were incubated in Tyrode solution for 3 hrs after the addition of 100 gg ovalbumin/ml, sensitized hearts released significantly more CK than nonsensitized hearts
The results have shown that indeed a significant increase of serum creatine kinase occurs in protracted anaphylactic shock of guinea pigs. Probably, the increased serum CK stems, at least in part, from the heart. This assumption is supported by the fact that isolated anaphylactic hearts released considerable amounts of the enzyme. The quantities which were liberated from the artificially perfused hearts could easily account for the total increase of serum CK. That also nonanaphylactic hearts released CK is not surprising. The circumstances under which isolated organs are kept are not physiological. Some degree of hypoxia is inevitable, and this could account for the CK liberation from '"normal" hearts. The differences between the CK amounts released from artificially perfused, and incubated hearts may be due to the different methodical conditions. However, the ratio of CK amounts released from sensitized, and nonsensitized hearts, was the same in both preparations (2.3, and 2.1, respectively). Besides the possibility that the wellknown anaphylactic coronary spasm is responsible for a myocardial damage, a direct effect of the antigen-antibody reaction on the membrane permeability of the heart muscle cells could be discussed. In the intact animals, a hypoxic injury as a result of bronchospasm is not to be considered since all animals were pretreated with the antihistamine mepyramine to prevent any bronchospastic reaction. All guinea pigs were subjected to autopsy. Emphysema of the lungs was lacking. It must be taken into account, however, that also the skeletal muscles contain creatine kinase. Skeletal muscle CK may be liberated during protracted anaphylaxis by hypoxic damage due to disturbances of the microcirculation. Differential determinations of the CK isoenzymes in the serum should allow to clarify this problem. Theoretically, a decreased inactivation rate of the enzyme could be involved in its increase in the serum of anaphylactic animals. No data exist in the literature on the metabolism of CK in guinea pigs. However, it seems very unlikely that such a mechanism would be crucial. Especially, the serum CK increase in nonanaesthetized animals, amounting to about 800 of the control values within the first hour after the antigen injection, speaks against this assumption. Extremely high histamine doses were necessary to liberate significant amounts of CK. To imitate the course of its release in anaphylaxis, histamine was not
72
administered as a depot in oil, but in a highly diffusible form. Probably, a considerable part of the i.p. injected histamine was inactivated in the liver. However, also by the s.c. route histamine acted only when given in excess amounts. From these findings it can be concluded that histamine plays only a minor role in the anaphylactic CK release. It is, perhaps, of importance that histamine dilates the coronaries, in contrast to the action of antigen. The impressive effect of adrenaline and noradrenaline of the serum CK levels can be ascribed to their myocardial damaging effect. It is wellknown that high doses of catecholamines produce cardiac necroses. As in protracted anaphylaxis catecholamines are released throughout the course of the shock (Bernauer et al., 1971, 1975), adrenaline and noradrenaline were administered as a s.c. depot in oil. It is likely, that released catecholamines play a role in the anaphylactic CK liberation. This is also supported by the effect which dibenamine had in anaphylactic animals. Although protracted administration of high catecholamine doses produces myocardial cell necroses, also a release of CK from the skeletal muscles must be taken into consideration in our experiments. High doses of adrenaline, and of course noradrenaline, contract the vessels of the striated muscles and may thus produce a hypoxic damage of the muscle cells. The inhibitory effect of dibenamine would support
this opinion. Acknowledgements. The support of this work by the Deutsche Forschungsgemeinschaft is gratefully acknowledged, as well as the skillful technical assistance of Miss Irmgard Ernenputsch.
REFERENCES Andrus, E. G., Wilcox, H. B. : The effects of anaphylaxis, and of histamine, upon the coronary arteries in the isolated heart. J. exp. Med. 69, 545-553 (1939) Beach, V. L., Steinetz, B. G. : Quantitative measurements of Evans blue space in the tissues of the rat: Influence of 5-hydroxytryptamine antagonists and phenelzine on experimental inflammation. J. Pharmacol. exp. Ther. 131, 400-406 (1961) Bernauer, W., Bollhagen, S., Mahlstedt, J., Hahn, F.: Die anaphylaktischen Reaktionen am Herzlungenpr~iparat des Meerschweinchens. Naunyn-Schmiedebergs Arch. Pharmak. 264, 215-216 (1969) Bernauer, W., Hagedorn, M., Filipowski, P. : Catecholamine release during anaphylactic shock in guinea pigs. Naunyn-Schmiedebergs Arch. Pharmak. 270, 326-334 (1971) Bernauer, W., Schanz, H.-M.: Factors involved in the protracted anaphylactic shock of guinea pigs. Int. Arch. Allergy 46, 3 0 0 309 (1974) Bernreiter, M. : Electrocardiogram of patient in anaphylactic shock. J. Amer. reed. Ass. 170, 1628-1630 (1959) Binder, M. J., Gunderson, H. J., Cannon, J., Rosove, L. : Electrocardiographic changes associated with allergic reactions to penicillin. Amer. Heart J. 40, 9 4 0 - 944 (1950) Booth, B. H., Patterson, R. : Electrocardiographic changes during human anaphylaxis. J. Amer. med. Ass. 211, 627--631 (1970)
W. Bernauer Brasher, G.W., Sanchez, S. A. : Reversible electrocardiographic changes associated with wasp sting anaphylaxis. J. Amer. reed. Ass. 229, 1210-1211 (1974) Feigen, G. A., Vaughan Williams, E. M. V., Peterson, J. K., Nielsen, C. B. : Histamine release and intracellular potentials during anaphylaxis in the isolated heart. Circulat. Res. VIII, 713-723 (1960) Forster, G., Bernt, E., Bergmeyer, H. U. : Creatin-Kinase. In: Methoden der enzymatischen Analyse, Vol. I, H. U. Bergmeyer, ed., pp. 750-759. Weinheim: Verlag Chemic 1970 Giertz, H., Hahn, F., Jurna, J., Schmutzler, W.: Vergleichende Untersuchungen fiber den anaphylaktischen und den Anaphylatoxinschock am intakten Meerschweinchen. NaunynSchmiedebergs Arch. exp. Path. Pharmak. 242, 6 5 - 7 5 (1961) Graig, F. A., Smith, J. C., Foldes, F. F. : Effect of dilution on the activity of serum creatine phosphokinase. Clin. chim. Acta 15, 107-111 (1967) Greeff, K., Heeg, E. : Die anaphylaktische Reaktion perfundierter Herzprfiparate, isolierter Herzvorhofprgparate und isolierter Papillarmuskeln. Arch. int. Pharmacodyn. 149, 136-152 (1964) Green, H. F. : The antagonism of histamine and the anaphylactic response by phenylpyridylallylamines. Brit. J. Pharmacol. 8, 171 - 176 (1953) Hahn, F., Bernauer, W. : Studies on heart anaphylaxis. I. Effect of antigen and histamine on the guinea pig heart-lung preparation. Int. Arch. Allergy 35, 476-494 (1969) Hahn, F., Bernauer, W. : Studies on heart anaphylaxis. III. Effect of antigen and histamine on perfused guinea-pig heart. Arch. int. Pharmacodyn. 184, 129-157 (1970) Hahn, F., Bernauer, W., Mahlstedt, J., Resch-Bollhagen, S., Beck, E. : Synergistic effects of adrenaline and mepyramine on the anaphylactic reactions of the bronchi and pulmonary vessels in the guinea pig heart-lung-preparation. Naunyn-Schmiedebergs Arch. Pharmak. 267, 224-240 (1970) Hanashiro, P. K., Well, M. H. : Anaphylactic shock in man. Report of two cases with detailed hemodynamic and metabolic studies. Arch. intern. Med. 119, 129-140 (1967) Liebig, R., Bernauer, W., Peskar, B. A. : Prostaglandin, slow-reacting substance, and histamine release from anaphylactic guinea pig hearts, and its pharmacological modification. NaunynSchmiedeberg's Arch. Pharmacol. 289, 6 5 - 7 6 (1975) McManus, J. F., Lawlor, J. J. : Myocardial infarction following the administration of tetanus antitoxin. New Engl. J. Med. 242, 1 7 - 1 9 (1950) Oliver, I. T. : A spectrophotometric method for the determination of creatine phosphokinase and myokinase. Biochem. J. 61, 116-122 (1955) Pfister, Ch. W., Plice, S. G. : Acute myocardial infarction during a prolonged allergic reaction to penicillin. Amer. Heart J. 40, 9 4 5 - 947 (1950) Stone, S. H. : Acute and protracted anaphylactic shock in guinea pigs after subcutaneous eliciting injection of antigen. J. Immunol. 82, 1 3 8 - 145 (1959) Went, S., Lissak, K. : Histamin- und Proteinwirkung am normalen und sensibilisierten Meerschweinchenherz. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 179, 609-615 (1935) Wilcox, H. B., Andrus, E. C. : Anaphylaxis in the isolated heart. J. exp. Med. 67, 169-180 (1938) Wilcox, H. B., Seegal, B. G. : Influence of an ethylendiamine derivative on histamine intoxication and anaphylactic shock in the intact guinea pig and the isolated guinea pig heart. J. Immunol. 44, 219-229 (1942) Williamson, R. : Late death occurring in active anaphylaxis. J. Hyg. (Lond.) 36, 5 8 8 - 593 (1936)
Received June 23/Accepted September 19, 1975
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 67-72 (1976)
9 by Springer-Verlag 1976
Release of Creatine Kinase by Antigen, Histamine, and Catecholamines* W. Bernauer Pharmakologisches Institut der Universit~t Freiburg, Katharinenstrasse 29, D-7800 Freiburg i. Br., Federal Republic of Germany
Summary. In mepyramine treated, ovalbumin sensitized anaesthetized guinea pigs protracted anaphylactic shock was produced by i.p. injection of antigen, and serum creatine kinase (CK) activities were determined 4, 6, or 17 hrs thereafter. Significant increases above nonsensitized controls were obtained. In nonanaesthetized guinea pigs shock course and serum CK increase were considerably accelerated. Histamine increased the serum CK only when given in high amounts (10 mg/kg) s.c., in the presence or absence of mepyramine. I.p. injection of histamine in mepyramine treated animals had no effect. Adrenaline (1 or 10 mg/kg) given as a s.c. depot in oil produced a significant increase of serum CK, as well as noradrenaline (0.1, 1, or 10 mg/kg). Dibenamine reduced the effect of adrenaline. In isolated perfused guinea pig hearts a significant CK liberation occurred already within the first hour after eliciting anaphylaxis. Nonanaphylactic hearts released CK too, but significant amounts were obtained only in the total 4 hrs after ovalbumin administration. Isolated anaphylactic hearts incubated in Tyrode solution liberated significantly more CK than did nonsensitized control hearts. The findings are discussed in view of a possible myocardial damage in anaphylaxis. Key words." Creatine kinase - Anaphylaxis - Catecholamines - Histamine.
INTRODUCTION In man, the manifestation of anaphylaxis can mainly consist of respiratory obstruction (by laryngeal edema * A preliminary report was given on the Spring Meeting of the German Pharmacological Society at Mainz, March 4 - 7 , 1975 [Bernauer, W.: Naunyn-Schmiedeberg's Arch. Pharmacol. 287, R 62 (1975].
and/or bronchospasm), or circulatory failure may predominate. In the guinea pig, both types of shock can be clearly separated by different ways of antigen administration: I.v. injection leads to acute bronchospasm and suffocation within some few minutes. S.c., or i.p. administration of antigen evokes a protracted and obviously circulatory shock (Williamson, 1936; Stone, 1959). The latter shock type results also when the antigen is given i.v. and the bronchospasm is prevented by a Hi-receptor antagonist (Wilcox and Seegal, 1942; Green, 1953; Stone, 1959; Giertz et al., 1961). Knowledge about the protracted shock is still very limited. However, there are good reasons to assume that the heart plays an important role. Isolated guinea pig hearts react to antigen with a very intense and long lasting coronary constriction coupled with decreased contractile force and marked arrhythmias, preceded by a short stimulatory phase (Went and Lissak, 1935; Wilcox and Andrus, 1938; Andrus and Wilcox, 1939; Feigen et al., 1960; Greeff and Heeg, 1964; Hahn and Bernauer, 1970; Liebig et aL, 1975). Furthermore, in guinea pig heart-lung preparations anaphylaxis can lead to severe cardiac failure (Hahn and Bernauer, 1969). In the heart-lung preparation also a marked pulmonary vascular constriction was detected which may be involved in the genesis of the protracted shock (Bernauer et aL, 1969; Hahn et aL, 1970). Finally, hypovolemia due to increased vascular permeability has to be taken into account since a pronounced hemoconcentration was found (Bernauer and Schanz, 1974). The question is, whether besides the functional disturbances the anaphylactic coronary spasm can produce a profound damage of the myocardium. In man, it was reported that during anaphylaxis ECG alterations can be found which point to severe myocardial ischemia or even myocardial infarction (Binder et al., 1950; McManus and Lawlor, 1950;
68 Pfister a n d Plice, 1950; B e r n r e i t e r , 1959; H a n a s h i r o a n d W e i l , 1967; B o o t h a n d P a t t e r s o n , 1970; B r a s h e r a n d S a n c h e z , 1974). A s e s p e c i a l l y in t h e g u i n e a p i g t h e o c c u r r e n c e o f c o r o n a r y c o n s t r i c t i o n is w e l l e s t a b lished we investigated the serum creatine kinase (CK; ATP-creatine-transphosphorylase) activities in p r o t r a c t e d a n a p h y l a c t i c s h o c k o f t h i s species. C K is l o c a t e d in v e r y h i g h a m o u n t s in t h e h e a r t m u s c l e cells a n d r e a c h e s t h e c i r c u l a t i n g b l o o d a f t e r m y o c a r d i a l cell d a m a g e . M o r e o v e r , s u b s t a n c e s w h i c h are released during anaphylaxis were tested on their a b i l i t y to l i b e r a t e C K , n a m e l y h i s t a m i n e , a d r e n a l i n e , a n d n o r a d r e n a l i n e . A n d finally, C K r e l e a s e f r o m isolated hearts was investigated.
METHODS Guinea pigs of either sex were sensitized with ovalbumin (Albumen ovi siccum, Merck Co., Darmstadt, Germany; 5 ~ solution; 0.1 ml i.p. on days 1, 3, and 5; beginning with day 21 the animals were used for the experiments).
Intact Animals Sensitized or nonsensitized, urethane anaesthetized (1.0 g/kg i.p.) animals received mepyramine maleate (0.1 mg/kg) or only saline (1 ml/kg) s.c. in the crural region of the left hindleg, and 10 rain later ovalbumin (100 mg/kg) i.p. Blood was withdrawn from the carotid artery 4, 6 or 17 hrs after ovalbumin. As further controls served nonsensitized animals which received only uretbane. In another group of nonsensitized animals only mepyramine was given, and blood was withdrawn 4 hrs later. Anaphylaxis was also elicited in nonanaesthetized, mepyraminized guinea pigs. When a severe state of shock had developed the thorax was opened quickly by section of the sternum and the diaphragm, and blood was withdrawn from the vena cava thoracalis inf. Urethane anaesthesia was applied only for the blood withdrawal (5 min before opening the thorax). For controls, blood was taken just at the same times from nonsensitized animals from the vena cava, and in a further group from the carotid. - Finally, acute anaphylaxis was produced by i.v. injection of antigen, with or without mepyramine pretreatment, and blood was withdrawn from the carotid at 2, or 10 rain. Histamine dichloride (1, or 10 mg/kg; in saline) was injected i.p. or s.c. (hindleg) with or without mepyramine pretreatment (0.1 mg/kg, s.c.). Blood was taken 4 or 17 hrs later from the carotid. Also mere saline or mepyramine controls were performed. Adrenaline (base; tartrate; 1, or 1.0 mg/kg) was administered as a s.c. depot in peanut oil in the midline of the abdomen, and blood was taken from the carotid at 6 or 17 hrs. For controls, peanut oil only was given. In further experiments, dibenamine hydrochloride (5 mg/kg, in saline) was administered s.c. (hindleg) 1 hr before adrenaline. Controls received dibenamine and peanut-oil. Moreover, adrenaline (base; 1, or 10 mg/kg, in oil) was given s.c. 30 min after i.p. injection of ovalbumin (100 mg/kg) in sensitized, mepyraminized animals, and blood was obtained 6 brs after the antigen. Controls were nonsensitized animals treated likewise, and sensitized as well as nonsensitized animals, which received only oil instead of adrenaline. Finally, noradrenaline base was given as s.c. depot in oil (0,1, 1, or 10 mg/kg) and blood was withdrawn 17 hrs later.
w. Bernauer
Isolated Hearts a) Artificially Perfused Hearts. Sensitized, or nonsensitized guinea pig hearts were perfused at constant pressure (54 cm H20) in a modified Langendorff apparatus with carbogen saturated Tyrode solution of 38~C. After a 15-min period of free perfusion a recycling circuit was closed, and 10 min later a sample was taken from the Tyrode in the reservoir. Then ovalbumin was injected intraaortically (10 rag, in 1 ml saline) and further Tyrode samples were withdrawn 1, 2 and 4 hrs thereafter. b) Incubated Hearts. In anaesthetized artificially respirated guinea pigs the venae cavae sup. and inf. were ligated, and 5 sec later the arcus aortae. The hearts were freed from lungs and vessels, except the ligated aorta, and put immediately into oxygenated Tyrode (room temperature) which was changed repeatedly. Then they were incubated for 3 hrs in 20 ml carbogen gassed Tyrode (38~ Ovalbumin (100 gg/ml Tyrode) was added after a 5 min preincubation period. Such hearts beat spontaneously, sucking the Tyrode solution into the open left atrium and left ventricle, perfusing their coronaries via the ligated stump of the aorta. In contrast to hearts in which the aorta is cut, the coronaries are free of blood already after some few contractions. As we know from unpublished observations these hearts react very strongly to antigen releasing significant amounts of histamine and SRS-A. To demonstrate that the coronaries are indeed perfnsed, 8 aorta-ligated hearts were incubated l0 min in Tyrode containing 0.25 mg Evans blue/ml. The total myocard was dark blue, containing 0.0812 + 0.00934 mg Evans blue/ml ventricular tissue, determined with the photometric method of Beach and Steinetz (1961). Hearts in which the aorta was cut (n = 8) showed only a thin layer Of dye in the endocardium and epicardium. The ventricular myocard was not stained, the coronaries were still filled with blood. The Evans blue content was significantly smaller (P < 0.01) than in the selfperfusing hearts. Extracts of identically incubated hearts, but without Evans blue, served as blanks.
Determination o f Creatine Kinase The activated backward reaction with creatine phosphate as substrate was used as kinetic test. The procedure is based on the method of Oliver (1955) and described in detail by Forster et al. (1970). Briefly, it consists of three steps: 1. Dephosphorylation of creatine phosphate and formation of ATP. 2. Formation of glucose-6-phosphate from glucose and ATP. 3. Formation of 6-phosphogluconate with simultaneous reduction of NADP. NADPH is determined spectrophotometrically at 366 nm. Incubation temperature 25~C. In all samples which gave an extinction difference of more than 0.03/min an additional determination with the 1/5 diluted serum (with saline) was performed. However, in agreement with Graig et al. (1967) we observed that diluted sera give relatively too high CK activities. Moreover, we found that the degree of error is variable; sometimes diluted sera yielded lower activities than the nondiluted ones. Therefore, in the following results only the undiluted serum values are presented. The results with the diluted sera, however, were essentially the same. All va~lues are given as means +_ standard errors of the means. Statistical evaluations were performed with the t-test.
RESULTS
A. Anaphylaxis Nonsensitized guinea pigs which received no injections b u t 1 g u r e t h a n e / k g i.p., h a d 5 m i n a f t e r i n d u c i n g
Release of Creatine Kinase anaesthesia serum creatine kinase (CK) activities of 89.6 __ 21.01 mU/ml (Fig. 1). Anaphylaxis produced marked increases of serum CK. In three different groups of sensitized guinea pigs which received ovalbumin (100 mg/kg i.p.) 10 rain after mepyramine (0.1 mg/kg s.c.), serum CK was significantly increased when compared with nonsensitized animals treated likewise at 4, 6, as well as J7hrs after ovalbumin, respectively (P < 0.001; p < 0.02; P < 0.001). From each animal blood was withdrawn only once. At 17 hrs serum CK levels were significantly higher than at 4 hrs (P < 0.02). In nonsensitized animals, no significant alterations of serum CK were found. In the 4 hrs anaphylaxis group 1 animal out of 14 died, in the 6 hrs group 3 out of 10. In the 17 hrs group, out of 22 guinea pigs 11 died in protracted shock; 2 surviving animals had strongly lipemic sera and were not used. The animals which survived, and from which CK determinations were obtained rather represent the guinea pigs with the weaker anaphylactic reactions. Serum CK increase is not produced by hemoconcentration and release of normal CK amounts into a smaller blood volume. Hematocrit values in the anaphylaxis group with the greatest C K increase (17 hrs) were 43.8 • 1.15~ (n = 8), whereas in the nonsensitized controls 44.0 i 1.67~ was found (n = 5).
Since all animals had received the antihistamine mepyramine to exclude any bronchospastic reactions, mepyramine was also tested alone. It had no effect on the serum CK activity. When 100 mg ovalbumin/kg were given i.p. into sensitized animals without mepyramine pretreatment, 12 out of 14 died before reaching 17 hrs. Therefore, only 10 mg ovalbumin/kg were used, which produced a lethality of 10~. Serum CK at 17 hrs was 312.4._+ 52.98 mU/ml (n = 9), whereas in nonsensitized animals 145.8 _+ 54.51 mU/ml was obtained (n = 1 0 ; P < 0.05). In nonanaesthetized animals a severe state of shock developed already within the first hour after antigen injection (100 mg/kg; i.p.). In this state it was not possible to obtain enough blood from the carotids. Therefore, blood was withdrawn from the vena cava thoracalis inf., at 59.4 _+_ 5.84 rain, in the mean. Serum CK was 709.2 i 52.42 mU/ml (n = 8). In 8 nonsensitized controls from which blood was taken in the same way just at the same times 135.7 _+ 22.50 mU/ml were found (P < 0.001). In a second control group blood was taken at the same times from the carotids. Here, 88.4 • 10.02 mU/ml were obtained (n = 8), so that the procedure of opening the thorax had not influenced the CK values in a relevant manner.
69 Serum Creatine Kinase mU/ml
700 -
600-
500-
400--
300-
200-
1OO-
1
23
45
Sensitization
-
+ -
+ -
+ -
Mepyramine
-
+ +
+ +
+ +
+ +
+ +
6h
Group Nr
Ovalbumin
-
+ -b
Time of blood withdrawal
5
4h
n
21
13 15
7
8
9
17 h
9
9
+
4 h
14
Fig. 1. Serumcreatine kinase activity in protracted anaphylactic shock of ovalbumin sensitized guinea pigs. Significant increase above nonsensitizedcontrol animals
During acute anaphylaxis no significant increase of serum CK was found. 8 sensitized animals received 10 mg ovalbumin/kg i.v., 5 min after i.v. injection of saline (1 ml/kg). 2 min after the antigen 93.6 _+ 24.79 mU CK/ml serum were obtained, and 119.2 _+ 47.74 in 6 animals, which received 0.1 mg mepyramine/kg instead Of saline. In further 9 sensitized, mepyraminized guinea pigs serum CK was 188.0 _+ 40.01 mU/ml, 10 rain after i.v. injection of ovalbumin; 10 nonsensitized controls had 132.5 _+ 27.34 mU/ ml. As during protracted shock adrenaline is liberated, and adrenaline itself provokes an increase of serum CK (see below), the influence of the a-receptor blocking agent, dibenamine, on anaphylactic CK release was tested. 5 mg dibenamine/kg injected s.c. 1 hr before the antigen (mepyramine treated animals) did not decrease the CK levels at 17 hrs (597.3 _+ 58.23 mU/ ml; n = 6). However, the dibenamine dose increased the CK for its own. In nonsensitized guinea pigs, which received dibenamine, mepyramine, and ovalburain, 17 hrs later 392.6 _+ 76.53 mU/ml serum were obtained (n = 7), in contrast to 160.0 • 50.42 (n = 9)
70
W. Bernauer
Table 1. Serum creatine kinase activities in guinea pigs after the i.p., or s.c. administration of histamine Group no.
1 2 3 4 5 6 7 8 9
Mepyramine 0.1 mg/kg s.c.
Histamine mg/kg
Time of blood withdrawal (hrs)
Serum C K mU/mt (n)
+ + + + -
-
4 4 4 17 17 17 17 17 17
166.7 209.3 311.1 175.4 122.6 612.0 327.9 158.6 134.9
-
+ +
1 i.p. 10i.p. 10 i.p. 1 s.c. 10 s.c. 10 s.c. -
• • • • • • • • •
42.65 64.60 59.39 53.24 31.69 60.56 71.81 25.94 29.29
Serum Creatine Kinase
mU/ml
800 -
700-
(10) (6) (7) (6) (6) (5) (10) (6) (5)
600-
500-
r 400-
300-
M
200 -
100-
when no dibenamine was given (P < 0.025). Therefore, the serum CK level in the dibenamine-anaphylaxis group is partially due to bibenamine. Anaphylaxis in the dibenamine treated group did not increase significantly the CK level when compared with the respective nonsensitized dibenamine treated animals (0.05 < P < 0.1).
L_ Group Nr.
2
Mepyramine
-
Adrenaline mg/kg
n
10
3
4
1
10
10
6
6
4
+
Fig. 2.
Serum creatine kinase activity 17 hrs after injection of adrenaline in oil as subcutaneous depot
B. Histamine
1 mg histamine/kg given i.p. in mepyramine treated animals (0.1 mg/kg, s.c.) did not increase the serum CK within 4 hrs, nor did 10 mg/kg (0.05 < P < 0.1), when compared with mepyraminized guinea pigs, which received saline instead of histamine (Table 1). In a further group even 17 hrs after injection of 10 mg histamine/kg no effect was seen. 10 mg histamine/kg given s.c., however, with or without mepyramine pretreatment, increased significantly the serum CK at 17 hrs in comparison with the respective controls (groups 7/9 of Table 1; P < 0.0.5; groups 6/8 ; P < 0.001). 1 mg/kg s.c., without mepyramine, had no effect. When the 10 mg histamine were given s.c. without mepyramine, considerably forced respiration was observed. From originally 11 animals 6 died, after 22 rain in the mean, showing marked emphysema of the lungs. Even after mepyraminization 3 out of 13 did not survive the 17 hrs of observation after the s.c. injection of 10 mg histamine/kg. C. Catecholamines
Adrenaline, administered in peanut oil as s.c. depot in doses of 1, or 10 mg/kg produced after 17 hrs significant increases of serum CK, when compared with animals which received only peanut oil (P < 0.01 ; P < 0.001 ; Fig. 2).
The CK liberation by adrenaline was not inhibited by mepyramine. In further experiments, not represented in Fig. 2, adrenaline tartrate (10 mg/kg) was given s.c. in oil, instead of adrenaline base. 5 out of 9 animals died, whereas none of the adrenaline-base-group died. The surviving 4 animals had significantly increased CK levels (634.7 _+ 22.52mU/ml; P < 0.001). Dibenamine (5 mg/kg, s.c., n = 6) given 1 hr before, reduced significantly the effect of adrenaline (base, 10mg/kg; 684.2 +_ 16.51mU/ml vs. 485.9 _+ 60.79mU/ml; P < 0.02). The CK values were not significantly different from the levels found in dibenamine-peanut oil controls (325.1 +_ 41.63 mU/ ml; n = 6). Adrenaline, injected s.c. in oil 30 min after el• anaphylaxis did not alter the antigen induced CK increase within 6 hrs. Sensitized, antihistamine treated guinea pigs which received only peanut oil 30 rain after ovalbumin (100 mg/kg i.p.), had 6 hrs after antigen serum CK activities of 530.3 + 74.40 mU/ml (n = 11). Nonsensitized controls had only 190.8 + 46.30; n = 17; (P < 0.001). When I m g adrenaline/kg was given CK levels amounted to 502.9 + 89.94 mU/ml (n = 9) in sensitized, and to 229.0 _+ 69.17 (n = 10) in nonsensitized animals. Even 10 mg adrenaline/kg had no effect within the 6 hrs after el• anaphylaxis (579.9 + 87.65; n = 8).
Release of Creatine Kinase
71
Serum Creatine Kinase
(sensitized: 86.4 _+ 8.52 mU/g; n = 7; nonsensitized 41.7 _+ 6.85 mU/g; n = 7; P < 0.005).
mU/ml
700.
600.
DISCUSSION
500-
400
300-
200
7.4,
///
100-
2
GroupNr. Noradrenaline mg/kg n
-
10
3
0,1
1
8
5
4 10 5
Fig. 3. Serum creatine kinase activity 17 hrs after injection of noradrenaline in oil as subcutaneousdepot
Also noradrenaline produced an increase of serum CK 17 hrs after s.c. administration (Fig. 3). Injection of 0.1, 1, or 10 mg noradrenaline/kg in oil resulted in CK levels which were significantly higher than those of mere oil controls (P < 0.005; P < 0.005; P < 0.001). D. Isolated Hearts
It proved to be very difficult to keep isolated perfused anaphylactic hearts beating for several hours. The hearts showed the typical initial stimulation and the secondary long lasting arrhythmic phase of anaphylactic reaction. Already within the first hour after eliciting anaphylaxis a significant CK release of 2.4 + 0.79 U/g heart was found (n = 12; P < 0.02). Within 2 hrs 5.8 _+ 2.43 U/g were liberated (n = 10; P < 0.05), and within 4 hrs 15.2 _+ 4.84 U/g (n = 7; P < 0.025). However, also nonanaphylactic hearts released CK. However, the CK liberation was statistically significant only within the total time space of 4 hrs and not within the first, or the first 2 hrs (6.7 _+ 2.37 U/g; n = 6; P < 0.05). When isolated, spontaneously beating hearts were incubated in Tyrode solution for 3 hrs after the addition of 100 gg ovalbumin/ml, sensitized hearts released significantly more CK than nonsensitized hearts
The results have shown that indeed a significant increase of serum creatine kinase occurs in protracted anaphylactic shock of guinea pigs. Probably, the increased serum CK stems, at least in part, from the heart. This assumption is supported by the fact that isolated anaphylactic hearts released considerable amounts of the enzyme. The quantities which were liberated from the artificially perfused hearts could easily account for the total increase of serum CK. That also nonanaphylactic hearts released CK is not surprising. The circumstances under which isolated organs are kept are not physiological. Some degree of hypoxia is inevitable, and this could account for the CK liberation from '"normal" hearts. The differences between the CK amounts released from artificially perfused, and incubated hearts may be due to the different methodical conditions. However, the ratio of CK amounts released from sensitized, and nonsensitized hearts, was the same in both preparations (2.3, and 2.1, respectively). Besides the possibility that the wellknown anaphylactic coronary spasm is responsible for a myocardial damage, a direct effect of the antigen-antibody reaction on the membrane permeability of the heart muscle cells could be discussed. In the intact animals, a hypoxic injury as a result of bronchospasm is not to be considered since all animals were pretreated with the antihistamine mepyramine to prevent any bronchospastic reaction. All guinea pigs were subjected to autopsy. Emphysema of the lungs was lacking. It must be taken into account, however, that also the skeletal muscles contain creatine kinase. Skeletal muscle CK may be liberated during protracted anaphylaxis by hypoxic damage due to disturbances of the microcirculation. Differential determinations of the CK isoenzymes in the serum should allow to clarify this problem. Theoretically, a decreased inactivation rate of the enzyme could be involved in its increase in the serum of anaphylactic animals. No data exist in the literature on the metabolism of CK in guinea pigs. However, it seems very unlikely that such a mechanism would be crucial. Especially, the serum CK increase in nonanaesthetized animals, amounting to about 800 of the control values within the first hour after the antigen injection, speaks against this assumption. Extremely high histamine doses were necessary to liberate significant amounts of CK. To imitate the course of its release in anaphylaxis, histamine was not
72
administered as a depot in oil, but in a highly diffusible form. Probably, a considerable part of the i.p. injected histamine was inactivated in the liver. However, also by the s.c. route histamine acted only when given in excess amounts. From these findings it can be concluded that histamine plays only a minor role in the anaphylactic CK release. It is, perhaps, of importance that histamine dilates the coronaries, in contrast to the action of antigen. The impressive effect of adrenaline and noradrenaline of the serum CK levels can be ascribed to their myocardial damaging effect. It is wellknown that high doses of catecholamines produce cardiac necroses. As in protracted anaphylaxis catecholamines are released throughout the course of the shock (Bernauer et al., 1971, 1975), adrenaline and noradrenaline were administered as a s.c. depot in oil. It is likely, that released catecholamines play a role in the anaphylactic CK liberation. This is also supported by the effect which dibenamine had in anaphylactic animals. Although protracted administration of high catecholamine doses produces myocardial cell necroses, also a release of CK from the skeletal muscles must be taken into consideration in our experiments. High doses of adrenaline, and of course noradrenaline, contract the vessels of the striated muscles and may thus produce a hypoxic damage of the muscle cells. The inhibitory effect of dibenamine would support
this opinion. Acknowledgements. The support of this work by the Deutsche Forschungsgemeinschaft is gratefully acknowledged, as well as the skillful technical assistance of Miss Irmgard Ernenputsch.
REFERENCES Andrus, E. G., Wilcox, H. B. : The effects of anaphylaxis, and of histamine, upon the coronary arteries in the isolated heart. J. exp. Med. 69, 545-553 (1939) Beach, V. L., Steinetz, B. G. : Quantitative measurements of Evans blue space in the tissues of the rat: Influence of 5-hydroxytryptamine antagonists and phenelzine on experimental inflammation. J. Pharmacol. exp. Ther. 131, 400-406 (1961) Bernauer, W., Bollhagen, S., Mahlstedt, J., Hahn, F.: Die anaphylaktischen Reaktionen am Herzlungenpr~iparat des Meerschweinchens. Naunyn-Schmiedebergs Arch. Pharmak. 264, 215-216 (1969) Bernauer, W., Hagedorn, M., Filipowski, P. : Catecholamine release during anaphylactic shock in guinea pigs. Naunyn-Schmiedebergs Arch. Pharmak. 270, 326-334 (1971) Bernauer, W., Schanz, H.-M.: Factors involved in the protracted anaphylactic shock of guinea pigs. Int. Arch. Allergy 46, 3 0 0 309 (1974) Bernreiter, M. : Electrocardiogram of patient in anaphylactic shock. J. Amer. reed. Ass. 170, 1628-1630 (1959) Binder, M. J., Gunderson, H. J., Cannon, J., Rosove, L. : Electrocardiographic changes associated with allergic reactions to penicillin. Amer. Heart J. 40, 9 4 0 - 944 (1950) Booth, B. H., Patterson, R. : Electrocardiographic changes during human anaphylaxis. J. Amer. med. Ass. 211, 627--631 (1970)
W. Bernauer Brasher, G.W., Sanchez, S. A. : Reversible electrocardiographic changes associated with wasp sting anaphylaxis. J. Amer. reed. Ass. 229, 1210-1211 (1974) Feigen, G. A., Vaughan Williams, E. M. V., Peterson, J. K., Nielsen, C. B. : Histamine release and intracellular potentials during anaphylaxis in the isolated heart. Circulat. Res. VIII, 713-723 (1960) Forster, G., Bernt, E., Bergmeyer, H. U. : Creatin-Kinase. In: Methoden der enzymatischen Analyse, Vol. I, H. U. Bergmeyer, ed., pp. 750-759. Weinheim: Verlag Chemic 1970 Giertz, H., Hahn, F., Jurna, J., Schmutzler, W.: Vergleichende Untersuchungen fiber den anaphylaktischen und den Anaphylatoxinschock am intakten Meerschweinchen. NaunynSchmiedebergs Arch. exp. Path. Pharmak. 242, 6 5 - 7 5 (1961) Graig, F. A., Smith, J. C., Foldes, F. F. : Effect of dilution on the activity of serum creatine phosphokinase. Clin. chim. Acta 15, 107-111 (1967) Greeff, K., Heeg, E. : Die anaphylaktische Reaktion perfundierter Herzprfiparate, isolierter Herzvorhofprgparate und isolierter Papillarmuskeln. Arch. int. Pharmacodyn. 149, 136-152 (1964) Green, H. F. : The antagonism of histamine and the anaphylactic response by phenylpyridylallylamines. Brit. J. Pharmacol. 8, 171 - 176 (1953) Hahn, F., Bernauer, W. : Studies on heart anaphylaxis. I. Effect of antigen and histamine on the guinea pig heart-lung preparation. Int. Arch. Allergy 35, 476-494 (1969) Hahn, F., Bernauer, W. : Studies on heart anaphylaxis. III. Effect of antigen and histamine on perfused guinea-pig heart. Arch. int. Pharmacodyn. 184, 129-157 (1970) Hahn, F., Bernauer, W., Mahlstedt, J., Resch-Bollhagen, S., Beck, E. : Synergistic effects of adrenaline and mepyramine on the anaphylactic reactions of the bronchi and pulmonary vessels in the guinea pig heart-lung-preparation. Naunyn-Schmiedebergs Arch. Pharmak. 267, 224-240 (1970) Hanashiro, P. K., Well, M. H. : Anaphylactic shock in man. Report of two cases with detailed hemodynamic and metabolic studies. Arch. intern. Med. 119, 129-140 (1967) Liebig, R., Bernauer, W., Peskar, B. A. : Prostaglandin, slow-reacting substance, and histamine release from anaphylactic guinea pig hearts, and its pharmacological modification. NaunynSchmiedeberg's Arch. Pharmacol. 289, 6 5 - 7 6 (1975) McManus, J. F., Lawlor, J. J. : Myocardial infarction following the administration of tetanus antitoxin. New Engl. J. Med. 242, 1 7 - 1 9 (1950) Oliver, I. T. : A spectrophotometric method for the determination of creatine phosphokinase and myokinase. Biochem. J. 61, 116-122 (1955) Pfister, Ch. W., Plice, S. G. : Acute myocardial infarction during a prolonged allergic reaction to penicillin. Amer. Heart J. 40, 9 4 5 - 947 (1950) Stone, S. H. : Acute and protracted anaphylactic shock in guinea pigs after subcutaneous eliciting injection of antigen. J. Immunol. 82, 1 3 8 - 145 (1959) Went, S., Lissak, K. : Histamin- und Proteinwirkung am normalen und sensibilisierten Meerschweinchenherz. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 179, 609-615 (1935) Wilcox, H. B., Andrus, E. C. : Anaphylaxis in the isolated heart. J. exp. Med. 67, 169-180 (1938) Wilcox, H. B., Seegal, B. G. : Influence of an ethylendiamine derivative on histamine intoxication and anaphylactic shock in the intact guinea pig and the isolated guinea pig heart. J. Immunol. 44, 219-229 (1942) Williamson, R. : Late death occurring in active anaphylaxis. J. Hyg. (Lond.) 36, 5 8 8 - 593 (1936)
Received June 23/Accepted September 19, 1975
