Peripheral versus central action of a toxin from indian cobra (Naja naja naja) venom

Toxk~on, Vol . 19, No. 2 pp . 305 317, 1981 Printed in Great Britain

0041 0101/81~D20305-I3 902 .00/0 ® 19R I Pergamon Preis Ltd

PERIPHERAL VERSUS CENTRAL ACTION OF A TOXIN FROM INDIAN COBRA (NAJA NAJA NAJA) VENOM A. K. CHARLES*

and S. S.

DESHPANDEt

C.S.L R. Centre for Biochemicals and Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India (Acceptedfor publication 29 September 1980) A. K . CHARLES 8nd S. S. DESHPANDE . Peripheral versus central action of a toxin from Indian cobra

(Naja raja raja) venom. Toxicon l9, 30$-317, 1981 .-A homogeneous toxin (NT fraction) purified from Indian cobra venom produced (at 025-1-0 mg/kg) predominant respiratory paralysis prior to anyeffect on thecontractions of tibialis anterior muscle (cats) andgastrocnemius muscle (rabbits). NT fraction produced no direct effects on cardiovascular system or nerve action potential . The preferential action of the toxin on the respiratory process, over the tibialis anterior muscle twitches, was well differentiated at lower doses of NT fraction . Recovery from the respiratory and peripheral muscle paralytic effects was observed in cats assisted with artificial ventilation, suggesting that the toxin could be removed from the neuromuscular receptor sites, possibly by metabolic degradation. Phrenic nerve discharges and diaphragm contractions were unaffected at a time when animals stopped spontaneous breathing. In contrast, the electrical activity of the intercostal muscles disappeared at a faster rate, concurrent with therespiratory impairment . It is concluded that the effect of NT fraction on respiration is of peripheral origin and is possibly related to paralysis of the muscles ofrespiration among which the intercostal muscles appear to be the most susceptible. The neurotoxin does not seem to exert any direct effect on central respiratory mechanisms .

INTRODUCTION

feature of cobra venom intoxication is flaccid muscular paralysis ensuing from the action ofneurotoxic components ofthe venom. There are different postulations regarding the primary cause of death in cases of cobra bite. Increased force of heart contraction, arrhythmia, systolic failure and cardiovascular collapse are the major symptoms caused by Naja nigricollis venom (RADOMSKI and DEICHMANN, 19$7) and by the venoms of other Naja subspecies in aIlln7813 (See reVlewS by ,TIMENFZ-PORRAS, 1968 ; LEE, 1971). BHANGANANDA ând PERKY (1963), using heart-lung preparations in dogs, have shown that the loss of vascular resistance leading to pooling ofblood in the peripheral vascular bed and causing progressive blood pressure drop is the cause of death in animals treated with Naja raja naja venom . On the other hand, experiments in dogs (VICK et al., 1967) and data on human studies (REID, 1964 ; CAMPBELL, 1964) have indicated that the changes caused by the venom on cardiovascular system were only secondary to respiratory failure. In support ofthese reports, it has been suggested that paralysis of the respiratory system is the most common cause of death In âII1Ina1S (MELDRUM, 196$ ; ,TIMENEZ-PORRAS, 1968 ; LEE, 1971) and lII humans (DE VRIES and CONDREA, 1971). THE COMMON

Present address: Department of Biochemistry, School of Dentistry of Maryland, Baltimore, MD 21201, U .S .A. t Present address : Department of Pharmacology and Experimental Therapeutics, School of Medicine, University of Maryland, Baltimore, MD 21201, U .S .A .

306

A. K. CHARLES and S. S. DESHPANDE

The persistence of central respiratory control activity in dogs injected with venom led Vtcx et al. (1965) to conclude that the respiratory failure observed in animals was of peripheral origin . Clinical studies (REID, 190)4 ; CHATTERJEE, 1965 ; CAMPBELL, 19Ô4) suggested that the neurotoxicity of the venom was effected through respiratory and peripheral muscular paralysis both occurring simultaneously . However, reports by BICHER et al. (1965) ând KRUPNICx et al. (1968) have indicated a central action for cobra venom neurotoxins. The toxins that these workers isolated from cobra venom caused an early depression ofcerebral cortical activity and a diphasic circulatory shock in cats. Some of the early experiments (quoted in review by MELDRUM, 1965a) revealed that direct application of venom to the respiratory centers or the fourth ventricle in brain produced respiratory arrest without affecting neuromuscular transmission in diaphragm muscle . Recently, BHARGAVA et al. (1970) noticed long-lasting convulsions in rats when a neurotoxic fraction was applied to cerebral cortex . On the basis of these observations and from his own studies CHAUDHURI et al. (1971) concluded that neurotoxins from Indian cobra venom have a definite action on higher respiratory centers. Whether these multiple and controversial effects of toxins from cobra venoms are primarily central in origin or secondary to the peripheral action remains to be clarified. It should be mentioned that the above described cobra venom toxins might be biochemically or pharmacologically identical . Yet no systematic studies have been carried out to examine the similarities or differences of various neurotoxins isolated from the venom of the same species of cobra by different workers using different purification techniques. Therefore, it is diû~cult to attribute a specific mode of action to a unique fraction of the venom. In this paper we have studied the actions ofthe major homogeneous neurotoxin (NT fraction) obtained from Indian cobra (Naja naja naja) venom. An analysis has been made on the effects of the toxin on peripheral and central respiratory centers. MATERIALS AND METHODS The Naja naja naja venom neurotoxin (NT fraction) used in this study was purified and biochemically characterized according to the procedures described by CHARLES et a/. (1981).The dose of the toxin was calculated in terms of protein content as determined by the method of LOWRY et aI . (1951). x-Chloralose (E . Merck, Germany), atropine andheparin (British Drug House, U.K .), neostigmine (Prostigmine, Roche, NJ) and urethane (Riedel) were used for i.v . injections. Anesthetic grade ether was obtained from Alembic Chemical Works, Baroda . All other chemicalswere of analytical grade purchased from E. Merck or British Drug House. Cats (2-4 kg)of either sex were obtained from Vallabhbhai Patel Chest Institute animal house. Healthy rabbits (average weight, 1 kg) were purchased from a local supplier . Animals were anesthetized with ether. In cats a-chloralose (1","é solution) in a dose of 75 mg/kg was injected i.v . through a polyethylene catheter inserted into the antecubital vein . The catheter allowed additional anesthetic to be injected when required andi.v. injections of NT fraction and other drugsto be made .The tail end of a'Y' shaped glass cannula was introduced into the trachea to facilitate breathing and to keep the respiratory passage clear of secretions . When the cat required artificial ventilation, the respiratory pump was connected to the open ends of the tracheal cannula. The animals were given dextrose saline (5% dextrose in 085% saline by i.v . drip) whenever necessary. The body temperatures of all cats were recorded by a thermometer inserted under the scapula through a small incision. The temperature was maintained at 37 f 1°C . The effects of the toxin on the cardiovascular system, respiration, nerve conduction velocity, tibialis anterior muscle contraction, phrenic nerve discharges, electromyogram,otc . were studied in separate sets of experiments, although in some cases more than one parameter were recorded from the same cat. Measurement of expiratory volumes was conducted in two groups of cats, five in each group, which received0~5 and 1 mg of NT fraction per kg body wt, respectively. Expiratory volumes were recorded on a Spirometer (Palmer, U.K.) . One side of the tracheal cannula was connected to the spirometer with rubber tubings through an unidirectional low resistance Douglas valve which regulated the expiratory and inspiratory air. The other side of the cannula was clamped during expiratory volume measurements in order to prevent exposure to outside air. The speed of the drum was kept constant at 32 cm/min and the spirometer was calibrated at this speed by injecting known volumes of air. Volume of expired air (Ve), expressed as crna/min, was calculated from the slope of the spirometer tracings . Blood pressure was monitored routinely in cats receiving NT fraction (or in some experiments, cobra venom 0~5 mg/kg) either from a mercury manometer or using a blood pressure transducer (Type P23-Gb), the output of

Respiratory Effects of Cobra Neurotoxin

30 7

which was fed into a two~hannel Beckmann Type RS Dynograph through a Type 9872 strain gauge coupler. The right carotid artery was cannulated with a polyethylene catheter filled with heparin (50 units/ml) to prevent clotting One end of the catheter was connected to a stainless steel 3-way stopcock which in tuna was wnnected to the Dynograph and recorded at a paper speed of 1 mm/sec . The mean blood pressure (mmHg) before and after NT injection was calculated from the tracings. For recording the electrocardiogram (ECG), electrodes were connected to the left forelimb and to the left hindlimb of the animal ; giving a type IIi ECG. The impulses were fed into an oscilloscope (Tektronix, Type 422) througha preamplifier (Tektronix, Type 122) having again of 1000.The ECG wasphotographed by a camera (Grass Instruments, U.SA., Model C4N) at a film speed of 50 mm/sec . when heart rate was measured, the film speed was kept at 2~5 mm/sec . In some animals the heart rate and blood pressure were recorded simultaneously on a Dynograph ; the former was then recorded at a paper speed of 5 mm/sec in order to see the individual heart beats. The tibialis anterior muscle-sciatic nerve preparation was made from the left hindlimb of the cat as described by Beowty (1938). The attachment of the tendon to the transverse ligament of the ankle was cut and the muscle was separated from the overlying extensor digitorum longus. A hook was tied to the tendon . After fixing thecat to a rigid steel frame in a horizontal position, the skin flaps were stretched and tied to theframeso as to form apool for liquid paraffin . The hipwas fixed with steel pins and the leg was immobilized by pointed pins drilled through the lower end of thetibiaat theanklejoint and thecondyles of thefemurin order to minimize the movements of the limbswhen the muscle contractions were recorded . In experiments using rabbits, instead of tibialis anterior muscle, gastrocnemius muscle was dissected along with sciatic nerve as described above. The tendon was connected to a transducer (Statham GI-80-350 oz) giving a proper initial tension . The isometric contraction of the muscle was recorded following the stimulation of sciatic nerve placed on a pair of silver electrodes . Stimulation was kept constant at a frequency of 15 shocks/min for 0~1 msec duration, given by square wave pulses delivered from an electrophysiological stimulator. The contractile tension was amplified through a bridge-circuit (Tektronù Typc 132 plug-in unit) and fed into an oscilloscope for display on a stationary spot . Contractions were photographed at required intervals with the help of a camera . The phrenic nerve was dissected carefully from cats after an incision was made along the ventral surface of the neck. A few filaments from the nerve were cut, separated for sufficient length, and the cut ends were placed on chlorided silver electrodes which were wnnected to a preamplifier (Tektronù, Type 122), the output of which was fed simultaneously into an oscilloscope and sudioamplifier .The discharges of the nerve were recorded by acamera from the oscilloscope display in a stationary spot . Soon after the discharges from the filaments were seen, all cats were allowed to breathe O=-CO, (95 :5) from a Douglas bag connected to the inlet of the respiratory pump. Diaphragm muscle contractions were recorded (from cats used for recording phrenic nerve discharges) after performing thoracotomy . Diaphragm was stimulated directly and indirectly and the twitches were recorded as described under tibialis anterior muscle contractions . Electromyograms were recorded in cats and rabbits from intercostal muscles using electrodes made of hypodermic needles (23 G) through which two insulated copper wires were pulled out to a length of approximately 3 mm . The insulation was stripped oß the tip of the wires and the electrodes were carefully inserted into the intercostal muscles through an incision made on the overlying skin . The output was recorded as described under phrenic nerve discharges . Nerve action potential was studied by placing oneportion of thesciatic nerve close to thepopliteal space on a pair of stimulating electrodes and the other portion of the nerve high up in the hip region on the recording silver electrodes. The parameters for stimulation and recording were as described under tibialis anterior muscle except that the display at a sweep speed of 0~5 msec per division was photographed at a film speed of 50 mm/sec. RESULTS

Action of NTfraction on respiration One group of5 cats received i.v. 0~5 mg and the other group 1 ~0 mg of NT fraction per kg. The expiratory volumes (Ve) were recorded until the animals were given artificial ventilation. Figure 1 shows the Ve measured in 5 cats injected with the toxin (0~5 mg/kg) . In two cats of this group a tendency of slight elevation of Ve was seen within 3-5 min ofNT injection and Ve eventually showed a gradual decline. Results of one experiment from this group are presented in Fig. 2 together with blood pressure measurements and muscle twitches . Such an initial rise in expiratory volume was not noticed in any cat given a higher dose ofNT fraction (1 mg/kg). In both groups, Ve showed a progressive reduction within 15 min after the injection. The extent of reduction in this particular cat (Fig. 2) was 43~ of the control at a time (17 min) when the cat failed to breathe spontaneously and needed to be put on artificial respiration. It was noticed that there was a gradual decrease in Ve in all five cats and most of the points fell in the area between the two dotted lines (Fig. 1). In animals injected with 025 and 1 ~0 mg/kg of the toxin the times required for assistance in breathing were 21 f 4 and

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FIG. l. EFFECT OF NT FRACTION ON RESPIRATION IN CATS. Expiratory volumes (Ve) of five cats (each represented by symbols, p ~ ~ ~ ~) were recorded on a spirometer as described in the text. Animals were injected i.v . with NT fraction (03 mg=g). Ve (cm3/min) before and after injection was measured and converted into % of control. Note the tendency of gradual decline of Ve (shown in the area marked by dotted lines) with the time . All cats were artificially ventilated at 130 min.

13 t 3 min, respectively. However, irrespective of the doses the Ve remained close to 45% of the control when the cats were connected to the respiratory pump. It is evident that

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Records shown are taken from one cat (4~3 kg) anesthetized with chloralose. Upper tracing shows expiratory volume . Middle tracing shows the blood pressure monitored by a mercury manometer before and after NT injection. Note that blood pressure returns to control level afterthe cat is put on artificial respiration (17 min) . Lower tracing shows the twitches of tibialis anterior muscle recorded as described under Materials and Methods. After neuromuscular block had occurred (40 min) the cat was atropinized (1 mg=g) and the first dose of neostigmiae was injected. This cat showed reversal of neuromuscular block and respiration after a dose of 1 ~9 mg of neostigmine . NT, NT fraction ; AR, artificial respiration; NEO, neostigmine.

Respiratory Effects of Cobra Neurotoxin TABLE 1 . EFFECT OF NT FRACTION ON TiBIALIS ANTERIOR MUSCLE CONTRACTION AND RESPIRATION IN CATS

NT fraction (mB/kg)

tAV (~)

Max. TA block (~)

Ve at tAV (%)

TA block at tAV (~)

0~25 (2) 050(5) 1 ~00 (5)

21 t 4 18f2 13 t 3

114 f 36 51f17 27 t 10

46 t 8 44t8 46 t 6

15 t 0 13f3 34 t 16

Control records of expiratory volume (Ve) and tibialis anterior (TA) muscle contractions were obtained for 10 min prior to NT fraction injection . Measurements of Ve were continued until the animals required artificial ventilation (AV). Magnitude of TA block was calculated as ~ of control from the heights of contractions measured at various periods. The values given are mean f S .E .M . of the number of experiments indicated in parentheses for each dose of NT fraction . tAV denotes the time at which the animals were connected to the respiratory pump.

respiratory failure was a predominant effect observed in cats and this effect showed a tendency of dependence on the dose of the toxin injected (Table 1). E,~ect on cardiovascular system

In Fig. 2 and Table 2 are given the data showing the effects ofthe toxin on blood pressure in cats. As seen in Fig. 2, the mean blood pressure of the cat before toxin injection was 145 mmHg . Two minutes after injection, the blood pressure rose gradually to 200 mmHg at which point the respiration was also severely affected . A similar increase was observed in most cats treated with 025-1-0mg/kg of the toxin (Table 2). The change was often associated with the course of respiratory impairment while in some cases the blood pressure changes were insignificant. These changes in blood pressure are most likely secondary to disturbances in respiration since the blood pressure in all cats showed a tendency to regain the control level soon after the animals were put on artificial respiration (Table 2). Heart rate was measured in 6 cats after NT fraction, 075 and 1-0mg/kg, was injected (Table 2). There was no appreciable changes observed in heart rate in any cat over a period of 3-6 hr after injection, suggesting that the fraction did not produce any cardiotoxic effect . Furthermore, no apparent arrhythmia or any other obvious change in electrocardiographic pattern was observed either immediately following injection or up to 4-5 hr after injection. Only in one cat in this group a slight enhancement of heart rate was noticed from 4 hr onwards. This cat, however, recovered completely from the paralytic effects (results to be discussed in next section). TABLE 2. EFFECT OF NT FRACTION ON BLOOD PRESSURE AND HEART RATE IN CATS

NT fraction (mg/kg) Blood pressure (mmHg)

0~25 0~50 0~75 1 -00

(2) (5) (2) (9)

Heart rate (per min)

075 (2) 1 ~00 (4)

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f f f t

7 14 36 11 260 1 5 185 t 23

at tAV 125 193 200 189 260 193

f 45 f 26 f 25 t 17 15 f 24

30 min after tAV 100 102 142 139 261 189

t 00 ±9 f 18 t 14 f4 f 23

All cats required artificial ventilation (AV) between 13 and 25 min after the NT injection. After ventilation the blood pressure and heart rate were well maintained at the level shown throughout the experiment. The values represent mean t S.E .M . of the number ofexperiments indicated in parentheses for each dose of NT fraction. tAV denotes time at which the animals were connected to the respiratory pump.

31 0

A . K . CHARLES and S. S. DESHPANDE

Autopsy examination of the thoracic cavity of NT injected cats revealed no hemolytic patches, hemorrhage or edematic fluid accumulation in lungs. This observation is consistent with the earlier observation by CHARLES et al. (1981) that NT fraction did not produce hemolytis using various erythrocyte suspensions. Effect on tibialis anterior muscle contractions The data obtained from 12 cats showing the effect ofNT fraction on the twitch responses of tibialis anterior muscles are presented in Table 1 . The reduction in the height ofcontractions of the muscle was taken as the criterion to assess the percentage block produced by the neurotoxin . Records of twitches from the same cat studied for the reduction in expiratory volume are shown in Fig. 2. Muscle contractions (neuromuscular transmission) were affected (17%) at 20 min following NT injection. Within 40 min the neuromuscular block attained a maximum level. It was seen that at a time when the cat required a pump (17 min), less than 17~ of tibialis anterior muscle twitch was affected (Fig. 2). This preferential effect on respiration was more marked at lower doses ofneurotoxin while these two effects occurred concurrently at higher doses of the toxin (Table 1). Interestingly, tibialis anterior muscle was not affected at all even after 106 min in another cat (0~5 mg NT per kg) while the animal required a pump within 17 min following injection. Thus an important observation is that the neurotoxic effect on tibialis anterior muscle took place much later than respiratory cessation and that the muscle was little affected at a time when animals were artificially ventilated . A comparison of the ~ of Ve and tibialis anterior muscle block at this point of artificial ventilation makes the primary effect of NT on respiration more obvious. When the Ve was reduced to approximately 50~ of the control (Table 1), tibialis anterior muscle twitches were decreased only to 15-34% regardless of the dose. Since the onset of respiratory failure occurred first using different doses, the primary effect ofthe toxin on the respiratory process did not appear to be dose-dependent . Effect on nerve action potential Action potentials were recorded from sciatic nerve trunk in 5 cats simultaneously with tibialis anterior muscle twitches to see if the toxin affected the nerve conduction velocity . Portions of records obtained from one such experiment are shown in Fig. 3(d) . The nerve action potential in this cat was not affected even after 30 min while all the cats showed stoppage of respiration when NT fraction (0~5 mg/kg) was injected (Fig. 1). Similarly, in all cats studied nerve action potentials were unaltered at a stage when there was complete neuromuscular block of the tibialis anterior muscles. Recovery from paralysis Seven cats (NT dose, 0~5-1 ~0 mg/kg) were observed for recovery from respiratory and peripheral paralysis. Animals were artificially ventilated during the time when respiration was not voluntary. All cats spontaneously breathed after a few hours. The time for recovery depended on the quantity of toxin injected. The cats injected with the lower dose recovered within 3-6 hr and those with the higher dose recovered within 8-11 hr. In two cats ofthe first group (0~5 mg/kg) tibialis anterior muscle twitches were recorded at intervals throughout the time over which the animals were observed for recovery from both respiratory and neuromuscular paralysis. The tibialis anterior muscle twitches recovered 60-90 min earlier than the respiration. The data clearly indicate that regardless of the doses administered, the effects ofneurotoxin are completely reversible provided that the animals were assisted with artificial ventilation. Also it must be noted that the peripheral muscles ofthe limbs, which are

Respiratory Effects of Cobra Neurotoxin

affected much later than respiration, recovered from paralysis earlier. It may be assumed that the toxin might be metabolized in the body or detoxified over a course of time. EQ`ect of neostigmine

It was of interest to know whether neostigmine could reverse the paralysis of peripheral and respiratory muscles . Figure 2 shows that after complete neuromuscular block had A

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FIG 3. EFFECT OF NT FRACTION ON PHRENIC NERVE DISCHARGES, DIAPHRAGM CONTRACTION, ELECTROMYOGRAM AND NERVE ACTION POTENTIAL IN THE CAT. (A) Cat (2~75kg) was artificially ventilated with S~ CO z as described in the text . NT fraction

(0~5 mg/kg) was injected i.v . and the phrenic nerve discharges were recorded from the cut central end of the nerve forup to 72 min. Control records (top tracing) shows some movement artifacts, prior to a buustof discharges, wlvchstabilize as theneurotoxic effects progress. (B)Dynograph trocords showing thecontractions ofdiaphragm muscle in cat(3 3 kg)after injection of NT fraction (0 ~5 mg/kg).Thecat required artificial ventilation 26 min after the injection. At 45 min thoracotomy was performed and the diaphragm was stimulated . The figure shows : initial twitches (indirect stimulation, 15 shacks/min) ; tl and t2, tetanic indirect stimulation (100 and 50 shocks/sec, respectively, for5 sec) ; t3, tetsmic dit~ect stimulation (100 ahocke/sec for S sec) ; final twitches (direct stimulation, 30 shocks/min). In this particular cat neuromuscular block (diaphragm level) was absent while the respiration was severely affected. (C) Electromyogram records obtained from acat {3 ~2 kg) receiving NT fraction (0~5 mg/kg) . The burstof rhythmic electrical discharges seen in the control was affected in thefirst 5 min and disappeared completely at 15-20 min. This catwas put on the pump at 26 min. (D) Sciatic nerve action potential records taken from a cat (2~6 kg) before and 30 min after NT injection (0~5 mg/kg). In this cat complete neuromuscular block oarorrect at 26 min.

31 2

A . K . CHARLES and S . S. DESHPANDE

occurred (40 min), the first dose of neostigmine (0 ~ 1 mg) was given (80 min). It was seen that the muscle twitches in response to nerve stimulation slowly started recovering. The reversal, however, was generally transient and in this particular experiment a cumulative dose of neostigmine up to 2~1 mg was necessary to maintain the muscle twitches . Moreover, the complete recovery of respiration and reversal of neuromuscular block with repeated administration of neoatigmine was not a consistent feature. Out of four cats receiving NT &action (0"5 mg/kg), one recovered from both respiratory and tibialis anterior muscle block while only partial recovery was seen in muscle twitches in one cat and in respiration in another cat. Likewise, among four cats in the second group (1 mg/kg), complete reversal to spontaneous breathing and partial recovery in tibialis anterior muscle contractions were seen only in one cat. The results suggest that neostigmine is not a very useful antagonist to NT fraction although some beneficial effects are produced in some cases. Effect on phrenic nerve discharges The action of NT fraction on the phrenic nerve discharges was studied in three cats artificially ventilated with OZ-COZ (95 : 5). Control records were taken 10 min after the animals were stabilized with 5% COZ inhalation. The nerve discharges were recorded periodically following NT injection (0~5 mg/kg) . This procedurewas adopted to eliminate the acute state of asphyxia and anoxia which would develop during the course of respiratory depression . Asphyxia would affect the phrenic nerve discharges due to its effect on higher centers and hence any depression or enhancement seen in the phrenic nerve activity under this condition would be meaningless. Phrenic nerve discharges recorded from one cat under the influence of toxin are shown in Fig. 3(a). Discharges prior to injection of toxin were synchronous and occurred just before the inspiratory phase ofrespiration. The background disturbances in the control records were due to the respiratory excursions and were abolishedcompletely with the progress ofrespiratory failure. Records taken at 42 and 72 min after NT injection showed a slight increase in the rate of discharges. It is clear that higher centers responsible for the respiratory control continue to function for the entire period, even after complete cessation of respiration occurred. Complete respiratory arrest might have occurred in this cat within 30 min, according to the observations shown in Fig. 1. The fact that the discharges were not affected as long as the cats were put on the respiratory pump suggests that NT fraction causes respiratory depression independent of any central control mechanisms . Effect on diaphragm muscle From four cats, including three studied for phrenic nerve activity, diaphragm contractions were recorded. Sixty minutes following NT injection (0"5 mg/kg) the left diaphragm was stimulated both directly and indirectly (through phrenic nerve) . In three cats diaphragm showed mild response to indirect stimulation thereby indicating a partial neuromuscular block at the end plates of the muscle. Twitch response to direct stimulation was unaffected, implying that the toxin did not affect the contractile mechanism of the muscle. Interestingly, the records reproduced from the fourth cat (Fig. 3b) show that the muscle responded to both types of stimulation at a period when total arrest of voluntary breathing had occurred (26 min). Tetanic tension (tl, t2 and t3) was well maintained during the stimulation period . The results obtained from this limited number of experiments point out that the neuromuscular block at diaphragm level need not necessarily be associated with the onset of respiratory arrest. Diaphragm activated respiration seems to be comparatively less affected by the NT fraction.

Respiratory Effects of Cobra Neurotoain

Effect on intercostal electromyogram

31 3

Electromyogram recorded from the intercostal muscles offour cats, studied for diaphragm contractions, indicated that NT fraction possessed a profound inhibitory activity on the electrical activity of these muscles. Records obtained from one cat (0~5 mg/kg) are presented in Fig. 3(c). Control records prior to NT injection showed bursts of electrical discharges (5 bursts/15 sec) at intervals synchronizing with the inspiratory phase, in addition to the intermittent single spikes seen in between the bursts . Immediately after the injection of NT (5 min), the intercostal muscle activity (bursts of discharges) diminished considerably in frequency with a very slight increase in pulse height . However, the activity was completely abolished both in pulse height and frequency within 15-20 min following the NT injection (Fig. 3c). This cat needed artificial ventilation within 20 min. It is clear that complete disappearance of electromyogram occurred at a stage when the cats could no longer breathe on their own. Datagiven in Fig. l support this assumption since all cats treated with this dose of neurotoxin failed to breathe voluntarily. Early disappearance of intercostal electrical discharges suggests that the motor end plates of these muscles are preferentially affected by NT fraction . In three rabbits the electromyogram from the intercostal muscles was recorded simultaneously with the contractions of gastrocnemius muscle twitches evoked by sciatic nerve stimulation. The records (Fig. 4) showed that the electromyogram waves decreased in amplitude within 13 min after the injection of the toxin (0.05 mg/kg) and were abolished almost completely at 17 min. It is evident from the records that throughout the period the contractions of gastrocnemius muscle remained unaffected, thereby suggesting that in rabbits also the intercostal neuromuscular junction could be vulnerable to the deleterious action of NT fraction .

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FIG 4. EFFECT OF NT FRACTION ON ELECTROMYOGRAM (upper trace) AND GASTROCNEMIUS MUSCLE CONTRACTIONS (IOWCr trace) IN RABBTT.

The rabbit (1 kg) was anesthetized with i .v . urethane (1~5-2~Omg,/kg) . Records at 13 and 17 min following the i.v. injection of NT fraction (0 05 mghg) are shown. Even at this time (17 min) the gastrocncmius muscle contraction (lower tracing) induced by sciatic nerve stimulation (15 shocks/min) was unaffected. This animal required a supplementary dose of NT for gastrocnemius muscle block . Note the early disappearance of electromyogram (upper tracing) .

314

A. K . CHARLES and S . S. DESHPANDE DISCUSSION

Since the cat is very susceptible to the cardiotoxic effects of the venom (LEE and TSENG, 1969), it is a useful model to test the cardiotoxicity ofvenomconstituents . Our results indicate that the major neurotoxin (NT fraction) of Naja raja naja venom has no direct effects on blood pressure and heart rate in cats. Any increase in blood pressure prior to artificial ventilation could be totally attributed to hypoxia and/or anoxia caused by respiratory impairment. When respiration ofthese cats was supported by a respiratory pump, the blood pressure was maintained more or less at a steady level close to that shown before injection of the toxin (Table 2). Electrocardiographic records from cats given a highdose of the toxin also showed no obvious abnormalities. These results suggest that NT fraction is devoid of any cardiotoxic activities and is, therefore, quite different from the Naja raja neurotoxin of SARKAR et al. (1942) which affected heart rate and blood pressure due to its contamination with some factors, most likely cardiotoxins (LEE et al., 1968). The NT fraction is biochemically homogeneous (CHARLES et al., 1981). Crude venom, on the other hand, was shown to produce drastic effects on the above-described cardiovascular parameters (CHARLES and DESHPANDE, unpublished results ; LEE and PENG, 1961 ; BHANGANANDA and PERKY, 1963 ; VicK et al., 1965, 1966, 1967). Studies conducted in dogs (Vlcx et al., 1967) revealed that cobra venom produced a significant drop in expiratory volume and respiratory rate. In our experiments, we have observed that all cats injected with venom (0~5 mg/kg) were able to breathe spontaneously until death occurred due to circulatory failure (CHARLES and DESHPANDE, unpublished observations). This observation seems justifiable since the quantity ofNT fraction present in 0~5 mg venom was approximately 001 mg (calculated on the basis of NT fraction as 2% of crude venom, CHARLES et al., 1981) and this quantity was probably inadequate to cause marked changes in respiration. It is worth recalling that the skeletal muscles ofcats are highly resistant to cobra venom neurotoxins (LEE and TSEIVG, 1969). Unlike mice (LDS p, 0~2 mg/kg) the rabbits are more susceptible to the action of NT fraction (0025 mg/kg) (CHARLES et al., 1981). These findings indicate that the lethality of NT in cats and rabbits is due to its primary paralysing effect on respiration. Any respiratory distress seen at terminal stages in cobra venom-injected animals could be secondary to the effects of venom components on the cardiovascular system . Measurement of expiratory volume provides an objective assessment of the functional derangement of the respiratory muscles. In the present study it was observed that, in contrast to the minimal effects on the cardiovascular system, the respiration was severely affected in all cats treated with various doses oftoxin. Only in two out of ten cats was there an initial brief increase in the expiratory volumes (Fig. 2). Whether this stimulation is related to the excitation of chemoreceptors or is perhaps due to direct action on the higher respiratory centers cannot be ascertained. The latter possibility seems unlikely in view of the fact that in all cats studied, the phrenic nerve activity remained unaffected. However, it should be noted that a Crotalus durissus terrificus venom component was shown to stimulate the respiratory activity by an action on chemoreceptors of aortic and carotid sinus bodies along with its direct action on respiratory centers (cl: Tu, 1971). Phrenic nerve discharges continued to persist for a long time (72 min, Fig. 3a) while all cats at this dose of the toxin (0~5 mg/kg) showed marked difficulty in spontaneous breathing within 10-30 min. As long as the animals were maintained on artificial ventilation, the phrenic nerve activity and the withdrawal reflexes on pinching on the foot were not abolished. These findings indicate that the neurotoxin-induced respiratory arrest is independent of any control from the higher respiratory centers and are in good agreement

Respiratory Effects of Cobra Neurotoxin

31 5

with the results of Lee and PENC (1961) and VICK et al. (1965) who reported no central effects for Formosan and Indian cobra venoms, respectively. In support of this postulation, the purified Formosan cobra venom neurotoxins labeled with'31 I were shown to accumulate in rat diaphragm (LEE et al ., 1967) and to cross the blood-brain barrier with great difficulty (TSENG et al ., 1968), suggesting that neither crude venom nor purified components can penetrate into the brain in sufficient quantities to account for such central effects. The predominant respiratory depression seen in cats and rabbits in our experiments could possibly be due to paralysis of the respiratory muscles themselves . Studies on cobra venom systemically administered into animals (LEE ând PENG, 1961 ; VtcK et al., 1965, 1967) are in support of the view that respiratory failure is of peripheral origin whereas several other studies suggest that the failure of central respiratory control is the cause of respiratory paralysis (BICHER et al., 1965 ; ICRUPNICK of al., 1968 ; CHALIDHURI et al., 1971) . Whether th15 effect is caused by neurotoxin itself or by a contaminant (like phospholipase A) present in the toxin is not known. The overall changes in the cortical activity, convulsions and other et%cts observed by these investigators could be due to some secondary effects. So far there are no studies available regarding the mode of action ofa purified neurotoxic principle from cobra venom on the respiratory mechanisms except the one by VrcK et al. (1966). These workers showed that certain venom components caused respiratory paralysis in dogs due to their actions on diaphragm by interfering with the actions ofacetylcholine. In our studies it was observed that the contractile response of the diaphragm muscle to direct stimulation was not abolished at a time when cats were breathing with the aid ofa respiratory pump. At this time only partial or no neuromuscular block was seen, as evidenced by the weak or complete twitch response to indirect stimulation of the diaphragm (Fig. 3b). Even the tension ofthe muscle during tetanic stimulation was well maintained and no post-tetanic facilitation was observed. These results lead to the conclusion that the neurotoxin-caused neuromuscular block at diaphragm level is not a requisite condition for the onset of respiratory impairment. Therefore, it appears that at least in cats, amongst the respiratory muscles, the diaphragm might be comparatively less susceptible to the action of NT fraction . This is not in agreement with the reports of VICK et al . (1966) and NAKAr et al. (1971), who showed that the primary cause ofrespiratory failure in animals was due to the paralysis ofthe diaphragm. It is evident that the neurotoxin causes neuromuscular block in the peripheral tibialis anterior muscle (Fig . 2). From the data obtained on the electromyogram, it was found that the frequency and amplitude of the discharges of the intercostal muscles started diminishing at a rapid rate, within 20 min of NT injection in cats and rabbits. In both animals, peripheral muscles, i.e. tibialis anterior (cats) and gastrocnemius (rabbits) muscles, were little of%cted at the time when the animals required artificial ventilation. It should be noted that in spite ofthe fact that the animals had varying susceptibilities to neurotoxic action, both respiratory impairment and loss ofelectromyographic response occurred almost simultaneously in these animals. The two effects are very much interdependent. Respiratory muscles are undoubtedly affected preferentially over the peripheral muscles. The ef~lcacy of neostigmine as an antagonist to the neurotoxin was tested in rabbits and cats. Contrary to expectations, this treatment could neither help the animals to recover from the paralysis nor was their survival time prolonged. Although in some cases neostigmine showed some beneficial effects in counteracting neurotoxic effects, its use as a true antagonist remains doubtful . The poor antagonistic action ofneostigmine seen in our in vivo studies is quite different from what was observed in in vitro nerve-muscle preparations (TA~IEFFDEPIERRE and PIERRE, 1966 ; SU et al ., 1967) . Our studies provide the first evidence for

31 6

A. K. CHARLES and S. S. DESHPANDE

complete recovery from the neurotoxin-induced respiratory paralysis in animals if the respiration is maintained for a sufitcient period of time . During this period, the neurotoxin could be detoxified or metabolically degraded at the receptor binding sites. Lastly, the varying susceptibility of dißerent skeletal muscles to the action of neurotoxin seen in our studies appears to be similar to what was described in the case of d-tubocurarine, a potent neuromuscular blocking agent (UNNA et al., 1950 ; TAYLOR t?t al., 1964). However, the interesting point is that unlike the action of NT fraction, the respiratory muscles are more resistant to the action of curare than other peripheral muscles. Acknowledgement--Part of this study was supported by a fellowship grant awarded to A.K .C . from Council of Scientific and Industrial Research, New Delhi, India. REFERENCES

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