Naunyn-Schmiedeberg's Naunyn-Schmiedeberg's Arch. Pharmacol. 292, 271-278 (1976)

Archivesof Pharmacology 9 by Springer-Verlag 1976

Neurotoxic and Myotoxic Effects of Crotalus Phospholipase A and Its Complex with Crotapotin H. BREITHAUPT Pharmakologisches Institut der Justus Liebig-Universit/it Giessen, Frankfurter Strasse 107, D-6300 Giessen, Federal Republic of Germany

Summary. 1. The acidic peptide crotapotin potentiated the toxicity of the basic crotalus phospholipase A in all species tested. The order of sensitivity to the lethal action of the phospholipase was: chick > mouse >/ rabbit > rat. After a latency period of at least 20 rain the animals died of respiratory paralysis. Rabbits survived for more than 10 h, if they were artificially respired. The animals recovered very slowly from respiratory depression. 2. In rabbits even high doses of the basic crotalus phospholipase A or its complex with crotapotin 1 did not affect the respiratory center nor its reactivity to asphyxia. Conduction of action potentials in the phrenic nerve was not changed. 3. Phospholipase-crotapotin complexes decreased the contractile response of isolated phrenic-hemidiaphragms of rats to direct and indirect stimulation in an irreversible manner. A latency period of 2 0 - 100 min preceded the paralysis. 4. A similar block of neuromuscular transmission developed in vivo. After i.v. injection of PC-complexes the contractile response of isolated rat phrenic diaphragms to nerve stimulation was considerably lower than to direct stimulation. 5. Crotalus phospholipase A alone as well as its complex with crotapotin reduced the contractions of the isolated chick biventer cervicis muscle but did not cause a contracture, thus indicating that the phospholipase and PC-complexes act not as depolarizing blockers. 6. Blood pressure, heart rate and electrocardiogram were not substantially altered, when phospholipase A Offprint requests should be sent to : H. Breithaupt at the above address. Phospholipase-crotapotin complexes (PC-complexes) were obtained by recombination of the crotalus phospholipase A with varying amounts of crotapofin. The term "crotoxin" will be reserved for the main toxic fraction of the Crotalus durissus terrificus venom, prepared according to Slotta and Fraenkel-Conrat (1938).

or PC-complex were injected slowly i.v. into rabbits or rats. No cardiotoxic effect was observed in the Langendorff preparation of rat hearts perfused with phospholipase A alone (6 x 10 .6 M) or together with crotapotin (10 -5 M). Rapid i.v. injection of the venom produced hypotension. The degree of haemolysis correlated with the enzymatic activity, and was low for the highly toxic PC-complexes.

Key words: Snake venom -

PhospholipaseA Crotapotin - Neurotoxin - Myotoxin.

INTRODUCTION The main toxic compound of the venom of Crotalus durissus terr~'cus, crotoxin, was separated into a basic phospholipase A (M, 16294) with low toxicity, and an acidic peptide, crotapotin (Mr 6490), without known enzymatic or toxic activity (Riibsamen et al., 1971). Previous studies on the mechanism of poisoning have shown that crotoxin as well as its toxic component phospholipase A paralyze the skeletal muscle by an action on the neuromuscular junction. Brazil (1966) demonstrated that crotoxin interrupts neuromuscular transmission in the sciatic-tibialis anterior muscle preparation of dogs and cats. This block was partially antagonized by edrophonium, succinylcholine or by tetanization of the motor nerve. In the isolated, chronically denervated rat hemidiaphragm crotoxin did not produce a contracture, but decreased the response to acetylcholine. It was suggested that crotoxin impairs neuromuscular transmission by producing a non-depolarization type of block, probably by decreasing the sensitivity of the endplate to acetylcholine. The effects of the crotoxin complex and of the toxic crotalus phospholipase A on the frog neuro-

272

Naunyn-Schmiedeberg's Arch. Pharmacol., Vol. 292, No. 3 (1976)

m u s c u l a r j u n c t i o n were studied by i n t r a c e l l u l a r recording t e c h n i q u e s (Brazil a n d Excell, 1971; Brazil et al., 1973). I n these studies neither c r o t o x i n n o r crotalus p h o s p h o l i p a s e A altered the muscle m e m b r a n e p o t e n tial, b u t b o t h toxins p r o d u c e d initially a n explosive b u r s t o f m i n i a t u r e e n d plate p o t e n t i a l s (m.e.p.p's.) associated with the onset of " l a r g e " s p o n t a n e o u s potentials. After this initial facilitation of t r a n s m i t t e r release the q u a n t a l release of t r a n s m i t t e r , calculated f r o m m . e . p . p a n d e.p.p, recordings, was i m p a i r e d a n d finally blocked. W h e n the b u r s t o f m . e . p . p ' s h a d subsided, tetanic s t i m u l a t i o n was followed b y a delayed rise in m.e.p.p frequency, whilst high potassium c o n c e n t r a t i o n s p r o d u c e d a fall in frequency. It was suggested that c r o t o x i n a n d crotalus p h o s p h o lipase A act o n the p r e j u n c t i o n a l t e r m i n a l s by i n h i b i t i o n of the m e m b r a n e d e p o l a r i z a t i o n of the m o t o r nerve terminals. F r o m preliminary observations, Breithaupt and H a b e r m a n n (1973) a s s u m e d for c r o t o x i n a n d the p h o s p h o l i p a s e a n a d d i t i o n a l m y o t o x i c a c t i o n in m a m mals. T h e p r e s e n t p a p e r reports the n e u r o t o x i c a n d m y o t o x i c effects of the basic crotalus p h o s p h o l i p a s e A a n d its c o m p l e x with c r o t a p o t i n .

volumes of Ringer solution containing Na + (147.1 reval/l), K + (4 reval/l), Ca2+ (4.5 mvat/1), and C1- (155.6 reval/l). Additionally, 1 ml of Ringer solution per h and kg body weight was continuously infused into the animals. A small amount of heparin was introduced into the catheters to prevent coagulation in the catheter tips. Lyophilized toxin, reconstituted with normal saline, was slowly infused into the jugular vein. All parameters mentioned above were permanently recorded until the animals died. Experiments in rats were done in a similar way but without recording the tidal and minute volume of respiration. Artificial Respiration. After i.v. injection of phospholipase A or PC-complex, rabbits were respired with a Starling pump when the minute volume of respiration had decreased to about 50 ~, but prior to cardiovascular failure (arrhythmia and/or pre-final hypertension). The artificial respiration was continued for up to 10 h. Recording of Phrenic Nerve Impulses. From the central end of the cut phrenic nerve, nerve activity was recorded continuously with silver wire electrodes from 30 min before to 300 min after i.v. infusion of the venom into the rabbits. The nerve impulses were amplified, monitored on a Tektronix oscilloscope and photographed. Isolated Nerve-Muscle Preparations. Rat phrenic-nerve hemidiaphragms were prepared according to Biilbring (1946). Some rat diaphragms were prepared 15, 30, and 60 min after i.v. injection of the toxin. The contractions were recorded with a force-displacement transducer, connected to a Hottinger amplifier and a Hellige Multiscriptor 9400/6. The organ bath contained 20 ml of KrebsHenseleit solution oxygenated with 95 ~ O2 and 5 ~ C Q at 37 ~ The phrenic nerve was stimulated with a single supramaximal rectangular pulse (0.7-2 V) of 100 gs duration every 20 s followed 10 s later by direct stimulation of the diaphragm (60 V, supramaximal). Isolated chick biventer cervicis nerve-muscle preparations were used according to Ginsborg and Warriner (1960). Isolated Perfused Rat Heart. Langendorff preparations of rat hearts were exposed for 10rain to crotalus phospholipaseA (6 x 10.6 M), crotapotin (10-5 M) or a mixture of phospholipase A (6 x 10.6 M) and crotapotin (10-5 M). Rate, coronary flow and contractility (dp/dt) were continuously monitored from 20 min before to 100 min after exposure to the toxin.

METHODS

Substances. The basic crotalus phospholipase A and the acidic crotapotin were prepared from the Crotalus durissus terrificus venom (Dr. Bficherl, Instituto Butantan, S~o Paulo) according to Rtibsamen et al. (1971). Toxicity. Toxicity tests were carried out in chicks (80-100 g), mice (NMRI, Hannover; 20-- 30 g) and rats (Wistar AR-Hannover, 150-200 g) by i.v. injection of 0.05 ml test solution per 10 g body weight. Rabbits (1.5-2.8 kg) received up to 2.8 ml via an ear vein. Ten minutes after a first injection of 0.05 mg of phospholipase or PC-complex/kg (1 min duration of injection) the rabbits received within 1 min the residual amount of the toxin. All solutions were made in saline. Deaths occurring during the next 24 h were recorded. For mice the LDs0 was calculated by the method of Litchfield and Wilcoxon (1949). The toxicity of recent preparations of crotalus phospholipase A was the same as reported earlier (Rfibsamen et al., 1971). Monitoring of Respiration. The arterial blood pressure of rabbits ( 2 - 3 kg), anaesthetized with sodium pentobarbital (50 mg/ kg i.v.) was continuously monitored from the carotid artery using a Statham strain gauge, a Hellige Ma-88K as Wheatstone's bridge and a Hellige Multiscriptor 9400/6. Minute and tidal respiratory volumes were recorded with a Hellige Pneumotachometer connected to a tracheal cannula and a Multi-Pen-Recorder (Hellige). The electrocardiogram was continuously recorded, especially to check the heart rate and for hypoxic signs. The frequency of respiration was recorded by the use of a pulse recorder fixed to the thorax and connected to the Multiscriptor. pH, pO 2 and pCO2 of arterial blood samples were measured according to Astrup (1957). [Na +] and [K +] in the serum were determined by flame photometry. Free haemoglobin was measured by the method of Ecklebe (1959). Body temperature was checked and kept at 39.4 -4- 0.5~C. Volume losses (blood, urine) were compensated by the administration of equivalent

RESULTS

A. Toxicity T h e sensitivity to the basic crotalus p h o s p h o l i p a s e A as well as to p h o s p h o l i p a s e - c r o t a p o t i n complex (1 : 1 ; w/w) varied with the a n i m a l species. As s h o w n in T a b l e 1 chicks p r o v e d to be highly sensitive to i.v. injection of the toxins, while rats were extremely resistant. Toxicity tests in mice d e m o n s t r a t e d the p o t e n t i a t i o n by c r o t a p o t i n o f the toxicity of phospholipase. The slope f u n c t i o n S of Litchfield a n d W i l c o x o n (1949) was f o u n d to be 1.37 for p h o s p h o l i p a s e a n d 1.19 for P C - c o m p l e x (1 : 1, w/w). T h e p h o s p h o l i p a s e a n d the P C - c o m p l e x elicited flaccid paralysis in all species tested. Muscle fibrillat i o n a n d spastic c o n t r a c t i o n s occurred i n t e r m i t t e n t l y before the a n i m a l s died of respiratory paralysis.

H. Breithaupt: Toxicity of Crotalus Phospholipase A and Its Complex with Crotapotin Table 1.

273

Death-rate of different species produced by the basic crotalus phospholipase A and crotapotin, injected i.v. separately or together

Substance

Chick

Mouse

Rabbit

Rat

Crotalus phospholipase A

0.08a (4/4)b 0.04 (4/4) 0.02 (4/4)

LDs0: 0.54 _+ 0.16 (P = 0.05; n = 50)

2 (1/1) 1 (1/1) 0.5 (0/1)

40 20

(0/2) (0/2)

Crotapotin

5 2.5

280 (0/2) 140 (0/2)

3 (0/1) 1.5 (0/1)

10 5

(0/1) (0/1)

PC-complex ~

0.016 (4/4) 0,008 (4/4) 0.004 (4/4)

LD50: 0.14 _+ 0.04 (P = 0.05; n -- 50)

0.5 (1/1) 0.3 (1/1) 0.15 (0/1)

4 2 1

(1/1) (1/1) (0/i)

0.5

(0/1)

b ~

(0/2) (0/2)

Doses in mg per kg. Deaths per total number of animals. Phospholipase-crotapotin complex (i : 1, w/w, coresponding to a molar ratio of I : 1.7).

Fig. 1. Arterial blood pressure, tidal and minute volume of respiration of a rabbit (2.8 kg) under pentobarbital anaesthesia, w h i c h w a s given i.v. crotalus phospholipase A (0.25 mg/kg) together with crotapotin (0.5 mg/kg). Death occurred 75 min later. Part Arterial blood pressure in m m Hg. Vr Tidal volume of respiration in ml. V,,~, Minute volume of respiration in ml/min. f ~ p Frequency of respiration (min-~). poe (mm Hg), pC02 (mm Hg), pH, Na + (mval/l serum) and K + (mval/1 serum) w e r e measured 0, 15, 45 and 60 min after injection of the toxin

mm Hg 120-

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70

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39

30

24 7. 50

30 7 30

39 7. 26

75

75

(mmHg)

71 21 7.51 138 3.'0

148 3.2

pO2 pCO2 {mmHg ) pH ( mvalq-1 ) Na ( mvalxi 1 ) K

Even at high doses a lag period of at least 20 min was noticed before the musculature weakened. Consciousness and reactivity to noise and touching were not visibly altered. The effects of phospholipase and PC-complex on the respiratory system and arterial blood pressure were recorded in nine anaesthetized rabbits which received 6, 3, and 2 mg phospholipase alone or 1, 0.5, and 0.25 mg phospholipase together with 0.5 or 0.25 mg crotapotin/kg. One of these experiments is shown in Figure 1. In all experiments a lag period of 2 0 - 1 0 0 min precBded the progressive decrease of the tidal volume and/respiratory rate. Complete arrest of ventilation followed within 4 0 - 2 4 0 min. Shortly before death

ab

t

45

{min) ( min-I )

70

70

50

0

140 3.2

the blood pressure increased to high values simultaneously with the occurrence of bradyarrhythmia. The respiratory failure was also reflected by a severe acidosis and hypoxia, as demonstrated by the blood pH, pCO2 and pOz. With 5 control rabbits ( 2 . 6 - 2 . 8 kg) the following values w e r e constantly measured for a 5-h period : mean carotid blood pressure 100 _+ 11 m m Hg; heart rate 2 5 0 - 3 6 0 / m i n ; respiratory frequency 7 0 - 1 0 0 / m i n ; tidal volume 6 - 8 mI corresponding to a minute volume of about 600 ml;pO2 art 70--85 mm Hg; pHar ~ 7.50 _+ 0.06; pCO2art 24 • 3 mm Hg; plasma Hb 10 -4 g/ml.

Rats also died of a slowly developing respiratory paralysis after the i.v. injection of 5, 2 or 1 mg of phospholipase together with 2 or 1 mg ofcrotapotin/kg

274

Naunyn-Schmiedeberg'sArch. Pharmacol., Vol. 292, No. 3 (1976)

R~s~iration ~ L / V A q W W b . -

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~r162162162 t l.,!ll!

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6O 2O 0

i Resp. off

i Resp. on

, r 0

1'0 sec.

Fig. 2. Respiratory m o v e m e n t s , phrenic nerve discharges and arterial blood pressure recording f r o m a rabbit under pentobarbital anaesthesia 4 h after i.v. injection of crotalus p h o s p h o l i p a s e A (1.3 mg/kg) together with crotapotin (1.3 mg/kg). Artificial respiration was interrupted for 30 s f r o m "Resp. off" to "Reap. on", as indicated on the abscissa. Respiration: T h o r a x m o v e m e n t s elicited by the artificial respiration. Imp. N. phren. : Impulses recorded f r o m the proximal end of the cut phrenic nerve. P~,.~: Arterial blood pressure

(one experiment for each of these six dose combinations). However, phospholipase A alone (5 to 10 mg/kg i.v., 4 experiments), did not decrease respiration within 3 h. Strong intravital haemolysis and occurrence of high serum potassium and serum haemoglobin (up to 0.04 g/ml) were observed in rabbits (n = 3) and rats (n = 3) when phospholipase was injected alone (0.5 mg/kg). In rabbits (n = 3) and rats (n = 3) 0.5 mg of phospholipase together with 0.5 mg crotapotin/kg elevated haemoglobin levels up to 0.004 g/ml serum. After reduction of spontaneous respiration to about 50 ~ of the initial minute volume, 8 rabbits were artificially ventilated. The rabbits received 5 and 2 mg of phospholipase alone or 1.3, 0.5, and 0.25 mg of phospholipase together with 1.3 or 0.5 mg of crotapotin/kg i.v. All rabbits survived as long as they were respired ( 4 - 1 0 h). After 10 h of artificial respiration those animals, which had obtained low doses, showed spontaneous but deficient respiration. Figure 2 demonstrates phrenic nerve action potentials which had not changed after the i.v. injection of a high dose of phospholipase A (1.3 mg/kg) together with crotapotin (1.3 mg/kg rabbit). The central part of the nerve continued to fire, even when the minute volume of respiration had decreased to a critical point and artificial ventilation was started. An increase in both rate and amplitude of the mass action potentials was noted, when artificial respiration was interrupted. In all experiments (5 rabbits) there was no evidence for an alteration of the respiratory center and the conduction of action potentials in the phrenic nerve after injection of high doses of phospholipase (6 and 3 mg/kg) or PC-complex (1.3 mg of phospholipase

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.......

'--i. . . ....... .... 1

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Effect of crotalus phospholipase A alone (6 x 10 o M) or

together with crotapotin (10-5 M) on the twitch response of the isolated phrenic-hemidiaphragmof the rat. The ordinate represents the ~o tension developed relative to the initial tension to direct (d, - - - - ) and indirect stimulation (i, ), respectively. The abscissa indicates the time (h) after addition of the toxin to the organ bath. 2 _+ sz (n = 3)

together with 1.3, 0.5 or 0.25 mg of crotapotin/kg i.v.).

B. Toxic Effects on Nerve-Muscle Preparations In vitro experiments with isolated phrenic-hemidiaphragms of rats demonstrated the paralyzing action of PC-complex (6 x 1 0 - 6 M phospholipase together with 10 -5 M crotapotin; n = 3) on this nerve-muscle preparation (Fig. 3). An almost complete block of the twitch response to direct and indirect stimulation was observed. No contractures were found in any experiment. Phospholipase alone (6 x 10 -6 M; n = 3) or crotapotin alone (10 -4 M, n = 3) were inactive.

H. Breithaupt: Toxicity of Crotalus PhospholipaseA and Its Complex with Crotapotin ! Fig. 4. Contractile response of phrenic-hemidiaphragms of rats prepared 15, 30 and 60 min after i.v. \ injection ofcrotalus phospholipase A (1 mg/kg) together with crotapotin (l mg/kg). Abscissa: Time (rain) from 15 injection of toxin to decapitation of animal. Ordinate: J'~ Tension (g) developed during twitch response to direct v (d, - - - - ) and indirect stimulation (i, ), respectively. 2 ++sx (n = 7) CO

275

X\Xkk

lO

~I\\\\\

CO L_ r(P -=? 5

\\\N crotap.

(1 mglkg) and phosp.A (lmg/kg) 6

1 3'0

1'5 time (h)

These experiments demonstrate that the paralytic action of the PC-complex was due to a myotoxic effect. In addition, a block of neuromuscular transmission may have occurred. Lag periods of 20 to 100 min preceded the progressive decrease of the twitch response of phrenic-hemidiaphragms of rats, when the organ bath contained the following concentrations of phospholipase together with crotapotin: 1.8x 1 0 - S M phospholipase together with 3 x 10 -s M crotapotin, 6 x 10 - 6 M together with 10 -5 M, 3 • 10 -6 M together with 10 -5 M, and 2.5 x 10 7 M together with 2.5 x 10- 6 M (2 experiments for each combination). The diaphragms were almost completely paralyzed by 6 x 10 .6 M phospholipase combined with 10 .5 M crotapotin, but not by 3 • 10 - 6 M phospholipase combined with 10 .5 M crotapotin. Phrenic-hemidiaphragms of rats were exposed to 6 x 10-6 M phospholipase together with 10-5 M crotapotin for 1, 2, 3, 5, 10 or 30 rain, respectively, before the bath fluid was replaced (1 experiment for each incubation period). An exposure for 5 rain was long enough to induce maximal paralysis after a latency period of 20 min and with a time course of paralysis similar to that of Figure 3. An exposure for 1, 2 or 3 rain elicited after a lag period of more than 20 min paralysis which remained incomplete during the whole experiment (4 h). Phrenic-hemidiaphragms prepared 15, 30, and 60 min after the i.v. injection of a phospholipasecrotapotin complex (1 mg of each/kg; n = 7) to rats exhibited a marked block of neuromuscular transmission (Fig. 4). In phrenic diaphragms prepared 30 min after the i.v. injection a nearly complete neuromuscular block was found. The response to direct stimulation

41i

6'o

was decreased too, albeit to a lesser degree. The twitch response to direct stimulation was greater for diaphragms of rats decapitated 60 min after injection than for diaphragms which were prepared after 30 min. Isolated chick biventer cervicis nerve-muscle preparations were exposed to 3 x 10 - 6 and 6 x 10 .6 M of phospholipase alone or to 5 x 10 -v and 10 -6 M of a PC-complex (molar ratio of J : l ; 3 experiments for each concentration). Typical time courses of the effects of the toxins on the twitch response are illustated in Figure 5. The minimal concentration to produce paralysis was 6 x 10 - 6 M for phospholipase and j 0 - 6 M for the PC-complex. No paralysis was observed within 200 rain with 3 x 1 0 - 6 M phospholipase or with 5 • J 0 - 7 M of the PC-complex. It should be emphasized that in contrast to the effect of acetylcholine (Fig. 5) no contracture was elicited by 1.2x 10 -5 and 6• 1 0 - 6 M of phospholipase or by 2 x 10 -6 and 1 0 - 6 M of PC-complex with a molar ratio of 1:1 (n = 3 for each concentration). When 0.05 mg of phospholipase were injected i.v. together with 0.01 to 0.05 mg of crotapotin/kg chick, 30 min later the biventer-muscles were found to be paralyzed (Fig. 5, lowest pannel; 3 experiments). A similar paralysis was observed after the i.v. injection of phospholipase alone (0.02 to 0.1 mg/kg; 3 experiments). C. Cardiovascular Effects

Figure 6 represents the typical response of the arterial blood pressure of rabbits to the i.v. injection ofcrotalus phospholipase and PC-complex. After a l-rain ad-

2-]~

Naunyn-Schmiedeberg'sArch. Pharmacol., Vol. 292, No. 3 (1976) tion of 0.007 mg phospholipase/kg (n = 2) and after a 7-min administration of 0.05 mg phospholipase/kg (n = 3). After a l-rain administration of 0.1 to 0.3 mg of PC-complex (1:1, w/w)/kg rabbit, the arterial blood pressure decreased very slowly (Fig. 6, lower pannel; 3 experiments). As shown by Figure 6 complete tachyphylaxis was found for the hypotensive effects of phospholipase and PC-complex. N o hypotensive reaction was observed in rats after a 1-min administration of 0.01 mg phospholipase or 0.03 mg PC-complex (1:1, w/w)/kg. The blood pressure decreased (with recovery) after a l-rain administration of 0.02 mg phospholipase or 0.06 mg PC-complex (1:1, w/w)/kg (3 experiments per each dose). Tachyphylaxis was observed, too. In Langendorff preparations of rat hearts the heart rate, contractility (dp/dt) and coronary flow were not altered during and 90 min after a 10-min exposure to 6 • 10 -6 M phospholipase, 10 -5 M crotapotin or a combination of both (2 experiments for each concentration).

DISCUSSION

Toxicity (in vivo)

Fig. 5. Twitchresponses of isolated chick biventer cervicisnervemuscle preparations to 3 • 10.6 and 6 x 10.6 M crotalus phospholipaseA and to 10-6M PC-complex (10 6M phospholipase together with 10-6 M crotapotin). The lowest graph demonstrates the impaired contractility of a chick biventer muscle prepared 30 rain after i.v. injection oi'0.08 mg of PC-complexper kg (0.05 mg phospholipase and 0.03 mg crotapotin/kg). Vertical bars: Tension (g) of twitch response to indirect stimulation, which was applied once every 10 s. PhA Crotalus phospholipaseA. ACh Acetylcholine. 20 rain Interruption of recording

ministration of 0.01 to 0.1 mg phospholipase/kg, the arterial blood pressure decreased within 4 rain with spontaneous recovery within 15 rain (3 experiments). No recovery occurred after a l-rain administration of 0.13 mg phospholipase/kg (n = 3). No hypotensive reaction was observed after a 1-min administra-

The toxicity tests with chicks, mice, rabbits and rats showed the same order of susceptibility to the basic crotalus phospholipase A as to the highly toxic complex of the basic phospholipase A and the acidic peptide crotapotin. In all species tested PC-complex was more toxic than crotalus phospholipase. For mice the PC-complex (1:1.7, mol/mol) was found to be 3.9 time more toxic (LDs0) than crotalus phospholipase alone. Therefore, on a molar base the toxicity of I mole phospholipase was increased 7.7 times by the addition of 1.7 mole crotapotin. Though crotapotin potentiated the toxicity of crotalus phospholipase A, crotapotin was non-toxic in the range of doses tested. After a lag period of 20 to 100 min, crotoxin as well as crotalus phospholipase A elicited a progressive flaccid paralysis. The signs of toxicity were similar to those of the Crotalus durissus terrifieus venom and the crotoxin as described by Brazil et al. (1966). These authors emphasized the extreme differences in the sensitivity of pigeons (LDs0 i.v. 0.002 mg crotoxin/kg) and rats (LDso i.p. 0.75 mg crotoxin/kg). Clonic convulsions which shortly interrupted the flaccid paralysis occurred spontaneously or were elicited by noise or touching. The convulsant action of the toxins may be due to hypoxia or to a direct

H. Breithaupt: Toxicity of Crotalus PhospholipaseA and Its Complex with Crotapotin

277

Fig. 6. Responses of arterial blood pressure of rabbits to phospholipaseA alone or together with crotapotin. Upper pannel: Response of arterial pressure (Pa~) to a 1-min i.v. injection of 0.05 mg phospholipase (PhA)/kgrabbit. No hypotensiveresponse of the same rabbit to repeated injectionsof phospholipase. Lower pannel: Hypotensiveresponse of a second rabbit to a 1-min i.v. injectionof 0.05 mg of phospholipase together with 0.05 mg of crotapotin/kg. No response to a second injection of a ten-fold amount of this PC-complex. The same time scale for both experiments

central nervous action produced by small amounts of toxin that may have passed the blood-brain barrier. The latter hypothesis is supported by reports of convulsant actions of crotoxin (Brazil et al., 1966) and of basic crotalus phospholipaseA (Habermann and Cheng-Raude, 1975) when these toxins were injected by intracerebroventricular route. Crotalus phospholipase A and PC-complex decreased the blood pressure only after rapid administration. The acute hypotensive reaction faded quickly and was followed by tachyphylaxis. These effects may be due to histamine liberation with a consecutive exhaustion of the mast cells. Bee venom phospholipase A elicited a similar hypotension with tachyphylaxis (Habermann, 1957). The long-time survival experiments in rabbits as well as the in vitro studies with rat hearts demonstrated the lack of cardiotoxic activity for crotalus phospholipase A and its complex with crotapotin. The haemolytic activity of phospholipase A was low when this enzyme was combined with crotapotin. Since crotapotin potentiates' the toxicity we conclude that intravasal haemolysis is not an important factor for toxicity. The paralyzing effect on the respiratory system represents the principal mechanism of toxicity. The survival time was increased by artificial ventilation up to 10 h without signs o f cardiovascular and renal

failure. Recovery of spontaneous respiration occurred very slowly. As judged by the phrenic nerve discharges, the respiratory center activity as well as the conduction of action potentials in the nerve remained unaffected by the toxins. This indicates that central nervous effects are irrelevant for the paralysis.

Neurotoxic and Myotoxic Effects (in vitro) In vitro experiments with nerve-muscle preparations strongly supported the hypothesis that the respiratory failure was caused by peripheral paralysis. Whereas Russell (1967) found only a gradual block of the indirectly stimulated guinea pig phrenic nerve-diaphragm, the present study demonstrates both a neurotoxic and a myotoxic action in rats. The latency before paralysis was similar to that of the effect of p-bungarotoxin (Chang and Lee, 1963), black widow spider venom (Longenecker et al., 1970), notexin (Harris et al., 1973), and botulinum toxin (Burgen et al., 1949). In contrast to/~-bungarotoxin, black widow spider venom and botulinum toxin, crotalus phospholipase A and its complex with crotapotin exerted a direct myotoxic action, which was also reported for notexin (Harris et al., 1973), a basic prejunctional neurotoxin with low phospholipase A activity (Eaker, 1975) and

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Naunyn-Schmiedeberg's Arch. Pharmacol., Vol. 292, No. 3 (1976)

for Naja naja atra phospholipase A (Chang et al., 1972). The mechanism by which crotalus phospholipase A alone or in combination with crotapotin produced the block in neuromuscular transmission is not clear. The studies on the chick biventer cervicis muscle indicate that a depolarizing type of block did not occur. Brazil et al. (1973) demonstrated that crotalus phospholipase acts prejunctionally at the frog neuromuscular junction, and that crotapotin enhances the rate of block produced by phospholipase. Similar to fl-bungarotoxin (Chang et al., 1973) and black widow spider venom (Longenecker et al., 1970) crotalus phospholipase caused an initial increase in m.e.p.p. frequency, before m.e.p.p.s, were finally abolished (Brazil et al., 1973). The latter authors suggested an interference of crotalus phospholipase A with depolarization-secretion coupling possibly involving presynaptic membrane depolarization. Crotalus phospholipase A might be useful as a specific binding agent for a functional macromolecule important in transmitter release.

by crotapotin. In: Animal and plant toxins, E. Kaiser (ed.), pp. 83--88. Miinchen, Goldmann 1973 Btilbring, E. : Observations on the isolated phrenic nerve diaphragm preparation of the rat. Brit. J. Pharmacol. 1, 38-61 (1946) Burgen, A. S. V., Dickens, F., Zatman, L. J. : The action of botulinum toxin on the neuro-muscular junction. J. Physiol. (Loud.) 109, 10- 24 (1949) Chang, C. C., Chen, T. F., Lee, C. Y. : Studies of the presynaptic effect of fl-bungarotoxin on neuromuscular transmission. J. Pharmacol. exp. Ther. 184, 339-345 (1973) Chang, C. C., Chuang, S.-T., Lee, C. Y., Wei, J. W. : Rote ofcardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom. Brit. J. Pharmacol. 44, 752--764 (1972) Chang, C. C., Lee, C. Y. : Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action. Arch. int. Pharmacodyn. 144, 241- 257 (1963) Eaker, D. : Structural nature of pre-synaptic neurotoxins from Australian elapid venoms. Toxicon 13, 90-91 (1975) Ecklebe, G.: Die moderne Cyanhfimiglobinbestimmung. Dtsch. med. Wschr. 84, 1531-1532 (1959) Ginsborg, B. L., Warriner, J. : The isolated chick biventer cervicis nerve-muscle preparation. J. Pharmacol. 15, 410- 411 (1960) Habermann, E. : Beitrfige zur Pharmakologie yon Phospholipase A. Naunyn-Schmiedebergs Arch. exp. Path. Pharmak. 230, 538546 (1957) Habermann, E., Cheng-Raude, D. : Central neuromxicity of apamin, crotamine, phospholipases A and c~-amanitin. Toxicon 13, 465-473 (I975) Harris, J. B., Karlsson, E., Thesleff, S.: Effects of an isolated toxin from Australian Tiger snake (Notechis scutatus scutatus) venom at the mammalian neuromuscular junction. Brit. J. Pharmacol. 47, 141 - 146 (1973) Litchfield, J. T., Wilcoxon, F. : A simplified method of evaluating dose-effect experiments. J. Pharmacol. exp. Ther. 96, 99-113 (1949) Longenecker, H. E., Hurlbut, W. P., Mauro, A., Clark, A. W.: Effects of black widow spider venom on the frog neuromuscular junction. Nature (Lond.) 225, 701- 703 (1970) Riibsamen, K., Breithaupt, H., Habermann, E. : Biochemistry and pharmacology of the crotoxin complex. I. Subfractionation and recombination of the crotoxin complex. Naunyn-Schmiedebergs Arch. Pharmak. 270, 274-288 (1971) Russell, F. E. : Comparative pharmacology of some animal toxins. Fed. Proc. 26, 1206-1224 (1967) Slotta, K. H., Fraenkel-Conrat, H. L.: Schlangengifte. III. Mitteilung: Reinigung und Krystallisation des KlapperschlangenGiftes. Ber. dtsch, chem. Ges. 71, 1076-1081 (1938)

Acknowledgement. The author is indebted to Dr. G. Paar, Innere Medizin der Universit~it Ulm, F.R.G., for help in performing the phrenic-nerve impulse recording.

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Received May i2 / Accepted November 6, 1975