Toxteon, 1973, Vol. 13, pp . 277-283. Peraamoa Pray . Prlated In Great Hrltain.
EFFECTS OF MELLITIN ON THE CENTRAL NERVOUS SYSTEM J. ISHAY,
D. BBN-SxACxAR, Z. ELAZAR and E. 11A1'LINSKY
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel (Accepted for pubffeation 26 January 1975)
J. Iaxev, D. HaN~c1~1e, Z. EL~zr.1t and E. K~PLU1scY. F8'ects of mellitin on the central nervous system. Toxfcon l3, 277-283, 1975 .-Mellitin, the main toxic component of bce venom was injected in cats intravenously and through the carotid or vertebral arteries . The main effect on the eloctrocortiwgram was depression . This may be due to changes in blood properties (hemolysis, hemoconcentration, hyperkalemia) and to hypotension . INTRODUCTION
effect of bee venom is usually attributed to apamin which represents approximately 2 ~ of dry venom and produces in experimental animals long-lasting hypermotility and convulsions (HABERMANN, 1965). Mellitin, the main constituent of bee venom (40-50 per cent of dry venom) is thought to have mainly hemolytic effects (HABBRMANN, 1972). Recently, melittin was also shown to have neurotoxic central effects (VYATCIIANIVIKOV and SINKA, 1973). In this article we describe results of our studies on the effxt of mellitin on the electrical activity of the cat brain. TY~ NBIntoTOxic
MA~?ulA~c AND METHODS These experiments were performed on 36 cats of both sexes weighing between 2~5 and 3~5 kg . In some of our preparations anesthesia was first obtained by ether and subsequently maintained throughout the experiment at a light level by intravenous injections of sodium pentobarbital (total dose 25-30 mg per k~. In other animals anesthesia was started with ether and maintained during the surgical procedures with sodium thiopental (approximate dosage 20 mg per kg). At the conclusion of surgery in these animals, the surgical areas were infiltrated with 1 ~ procaine ; the animals were then paralysed with gallamine triethiodide (4 mg pet kg each ~} hr), and artificially ventilated . No further general anesthesia was given and experiments wero started 2 hr after stopping sodium thiopental administration . Surgery included cannulations of the femoral vein and artery and cannulation of the trachea. The vertebral artery was cannulated through the subclavian artery without opening the thorax thus permitting spontaneousr~pirations in the non-paralysed preparations. El halographic recordings were taken from screw electrodes placed on the cerebral cortex through the varium . Respiration was recorded with a thermocouple inserted into the trachea and blood pltssutt with a Statham transducer . All data were recorded on a Grass polygraph. Quantitative determinations of frce hemoglobin in plasma (~osaY, 195 were performed as an indication of intravascular hemolysis. Sodium and potassium levels west determined with a Bearel atomic Same photometer. RESULTS
Effects ofmellitin on the electrocorticogram
Small 5 mm' cotton pledgets were imbibed with 0~1 ~ mellitin and applied on the pial surface of the cerebral cortex (suprasylvian gyros) . Recordings were taken from the vicinity ToxrcoN r9~s voG rs
277
278
J. ISHAY, D. BEN-SHACHAR, Z. ELAZAR and E. KAPLINSKY
of the cotton pledget for periods exceeding 1 hr. No obvious changes in the electrocorticogram were seen. In animals anesthetized with pentobarbital and spontaneously respiring mellitin was injected through the femoral vein or the vertebral artery . When mellitin was injected into the femoral vein great variations in the effective dose were seen (1~ mg per kg). The effect consisted in a slowing of the electrical activity and with repeated doses or with high doses death occurred shortly after an abrupt flattening of the recording simultaneously in all cortical leads. Injections of mellitin into the vertebral artery were much more effective with less variation between different animals; the effective dose being between 0"2 and 0 "4 mg per kg. The following sequence of effects was observed: immediately after the injection, a marked drop in amplitude of the electrocorticogram occurred, then the amplitudes rose again. Spindles disappeared and the record showed groups of fast spiky waves arising from a background of low, irregular fast activity. This pattern could last for a few minutes and then the former spindling pattern returned . With higher doses or in more sensitive preparations the first spiky pattern was replaced gradually by slow low amplitude waves interspersed with sharp waves. The amplitudes gradually diminished until the record was almost flat. This development progressed over 10-30 min, after which the animal died. In some preparations, after the first spiky pattern the normal spindling pattern returned but at a certain point the record became abruptly and simultaneously flat in all leads. Figure l illustrates the immediate effect ofmellitin (A), the later fattening of the record and recovery of activity after adrenaline injection (B), and the final abrupt and generalized flattening which preceded death by 20 min. The animals which had received gallamine triethiodide, without general anesthesia and kept under artificial respiration were very sensitive to the effects of mellitin ; usually a single dose of 0"3 mg per kg was sufficient to produce electrocorticographic effects and death. Figure 2 shows the effect of injecting 1 mg per kg mellitin into the femoral vein of a cat which had received gallamine triethiodide . The first effect (B) is a weak increase in synchronization consisting in more prolonged rhythmic trains of waves of 9-10 Hz and short (1-2 sec) spindles of 7-8 Hz ; 300 sec after the injection this effect decreased and the pre-injection pattern reappeared . Shortly after a second injection of 1 mg per kg the rhythmic activity disappeared and the record became almost flat 60 sec later (C and D). Figure 3 shows the effect of intracarotid injection of mellitin . A diminution of amplitude and a general slowing of the electrocorticogram progressing to a completely flat record prior to death was usually seen. Figure 4 shows the effects of mellitin injection into the vertebral artery. Only a few seconds after the start of the injection (B) the amplitude of the electrocorticogram markedly decreased. Later, (C) spindling activity of low amplitude was observed in the anterior leads followed by the complete loss of electrical activity prior to death. Cardiovascular effects ofmellitin and correlation: with the electrocorticographic effects Effects or: bloodpressure . When injected through the vertebral artery in the anesthetized
(pentobarbital) spontaneously respiring animal, mellitin produced a rise in systemic blood pressure, often preceded by a very briefdecline in pressure. The respiration became irregular and spastic and eventually stopped. At the same time generalized uncoordinated movements were seen. The electrocorticogram showed, as described above, first flattening and then an TOXICON 1973 Yor. 13
Effects of mellitin on the central nervous system
FIG. I. EFFECT OF MPr i rrnv ON ELECI'ROCORIICOGRAM, BLOOD PRE93IJRE AND RESPIRATION.
Cat anesthetized with pentobarbital. Mellitin injected through the vertebral artery. Electrocorticographic leads: FR = right frontal ; FL = left frontal ; PR = right parietal ; PL = left parietal . FBP = Femoral artery blood pressure. T = Time, small divisions 1 sec, heavy linedrug injection. RESP = Respiration . In A two injections of mellitin (0~5 mg total dose). Note changes in electrocorticogram and the two-phase blood pressure change. In B blood pressure at shock level, electrocorticogram depressed . An injection of 2~5 llg total dose of epinephrine is followed by increase in blood pressure and recovery of electrocorticogram . In C an addi tional injection of 0~5 mg total dose of mellitin is followed by abrupt depression of electrocorticogram while blood pressure remains unchanged .
EKG
FIG. 2. EFFECT OF ME~-~T~ ON EIECTROCORTICOORAM AND ELECTROCARDIOGRAM. Cat paralysed with gallamine triethiodide, without general anesthesia and under artificial respiration. Mellitin injected through the femoral vein. RA = Right anterior electrocorticographic lead . A = Control. B = 4 sec after 1 mg per kg mellitin . C = An additional 1 mg per kg mellitin products disappearance of spindles and profound depression 10 min later, in D. Calibration = vertical, 200 ~; horizontal, 1 sec. TOXICON 1975 Yol. I3
279
280
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EFFECT OF MELLITIN ON
11
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ELECTROCARDIOGRAM AND HLOOD
Cat paralysed with gallamine triethiodide, without general anesthesia and under artificial respiration. Mellitin injected through the carotid artery. RA = Right anterior electrocorticographic lead . BP = Blood pressure ; vertical line, 100 mm Hg. ECG = Electrocardiogram . A = Control. B = 2 min after 1 mg per kg mellitin ; slight slowing of the electrocorticogram, no change in blood pressure. C = 5 min aftermellitin injection ; note depression of the electrowrticogram, and decrease in blood pressure . D = 15 min aftermellitin injection ; depression of the electrocorticogram, temporary Increase in blood pressure and disturbances in the electrocardiogram . LP A
B
RA
C
FIG. 4. EFFECT OF AiELL1'ITN ON ELECTROCORTICOGRAM .
Cat paralysed with pallamine triethiodide, without general anesthesia and under artificial respiration. Mellitin m~ected through the vertebral artery . LP = Left posterior, RA = right anterior electrocorticognphic leads. Calibrations as in Fig. 2. A = Control. B = Immediately after 1 mg total dose mellitin. C = 5 min later . TOXIC0IV 1973 Yol. I3
Effects of mellitin on the central nervous systean
281
imitative, spiky pattern. When the animals were put on artificial respiration the motor reaction gradually ceased and the electrocorticographic activity became more regular but the blood pressure level remained elevated. Figure 5 shows graphically the changes in the A
mm Hg zso
zoo
tso
too zo
so C 0
I
M
to
PIG. S . BLOOD PRES4IJRE C1iANGFS II~UCED BY }NIECITON OF ARTERY.
I~LLI'riN 77EIROUGH T1iE
VBitTapR~3
Cats anesthetized with pentobarbital . A = Effects on arterial systolic blood pressure. B = Effects on pulse plnesure. C = Control before in~'xtton . I = Changes daring the first 3-S sec after injection . M = The vasopressive phase of the main effect, measurod during the first minute after injection. Later, the blood pressure decreased. In cat 1 the extent of change is pulse pressure could not be evaluated. On cat S we included the initial change in pulse pressure only.
systolic pressure (A) and in pulse pressure (B) in several animals in the first minute after mellitin injection. At a later stage the blood pressure gradually declined to shock level. These changes in blood pressure, respiration and electtocorticogram can be seen in Fig. 1. In Fig. 1B it can be seen that when blood pressure decreased to shock level an injection of 2"50 lIg epinephrine had a powerful and long-lasting (see also Fig. 1G~ effect. When a second and third dose of mellitin was injected on a background of low blood pressure produced by the preceding dose, an increase in blood pressure occurred but this vasopressive effect was not seen with further repeated doses. When mellitin was injected in the anesthetized animals through the femoral vein the effects on blood pressure were less dramatic and more variable . An increase or decrease was first noted but with repeated doses or with high doses a decrease in blood pressure was the rule . Tn%ICON 1975 YoL 13
282
J. LSHAY, D . HEN-SHACHAR, Z . ELAZAR and E. KAPLINSKY
In the animals paralysed with gallamine triethiodide the same sequence of decrease, increase and then prolonged and profound decrease in blood pressure was seen but the changes were less abrupt. An example of these effects can be seen in Fig. 3 after injection of mellitin into the carotid artery . Note also the disturbances in the electrocardiogram in Fig. 3D. The final decrease in blood pressure to shock levels was correlated with depression of electrocorticographic activity as described above. Elevation of blood pressure after epinephrine administration could in some instances induce a rapid recovery of brain electrical activity (Fig. 1 B). However, it can be seen in the same figure (1C) that an additional injection of 0~5 mg mellitin produced a rapid depression of the electrocardcogram while the systemic blood pressure was still at the same level as in the former part of this figure (1B). Effects of mellitin on blood. in six animals mellitin was injected through the femoral vein and blood was taken for an analysis of electrolyte content. Significant changes were found in potassium levels which increased after 2 and 10 min. The plasma potassium concentration (mequiv. per liter) in six cats prior to mellitin administration was 3~03~0~3 (mean ~ s.n.). Corresponding values 2 and 10 min after 1 mg per kg mellitin were 3'6 ~ 0~3 and 5~5 ~ 0~5. Ten minutes after injection of mellitin the blood was hemolytic and no further samples were taken. Sodium levels did not change significantly after mellitin . It was also observed that mellitin caused hemoconcentration; in fact, 15 or 20 min after mellitin injection the blood became so concentrated that sampling was difficult. The animals died soon thereafter. DISCUSSION
The effect of mellitin on the electrical activity of the brain was studied recently by VYATCHANHIKOV and SINKA (1973) . These authors found in non-anesthetized cats paralysed with gallamine that low intravenous doses (1 mg per kg) of mellitin produced slowing of electrocortical activity and enhancement of spindling, while higher doses (3 mg per kg) produced desynchronization and epileptic seizures followed by post-ictal depression. Still higher doses (5 mg per kg) produced depression . Intraventricular introduction of mellitin induced spiking and seizure discharges. Similarly, in our cats paralysed with gallamine the intravenous or arterial injection of mellitin into the carotid or vertebral arteries produced depression of the electrocarticogram . In the anesthetized non-paralysed preparations injection of mellitin into the vertebral artery elicited a strong reaction consisting in irritative changes in the electrocardcogram, general contractions of the skeletal musculature, spastic respiration and large changes in blood pressure . Since this reaction was not seen in the paralysed preparations we propose that it was a result of a peripheral effect of mellitin . Mellitin produces depolarization and shortening of skeletal muscle and also pain (HABQtMANN, 1972). The main difference between our results and those of VYATCHAIdNIKOV and Snaxn (1973) is that we did not see seizures or other epileptiform phenomena in the electrocardcogram or convulsions. Noteworthy is also the fact that topical application of mellitin on the pial surface did not cause obvious changes in electrical activity. These findings would suggest that the depression observed with high doses of mellitin might be the result of hypoxia due to vascular reactions or changes in blood characteristics . The depression in the electroencephalogram was usually correlated with decreases in systemic blood pressure and serious disturbances in the electrocardiogram . Blood appeared to become concentrated, hemolytic and a rapid hyperkalemia developed. Anoxia and T1U%ICON 1975 Yol. 13
Effects of mellitin on the central nervous system
283
hyperkalemia may produce depolarization of brain cells (VnN HARREVELD, 1966) and this may be the mechanism of the electrical depression. In artificially respired animals the heart continues to function 10-30 min after depression of the electrocorticogram but death was clearly the result of cardiovascular failure. The reduction of blood pressure to shock levels was the final effect of mellitin . The first effect was vasopressor and in many preparations the vasopressor effect was preceded by a brief but sharp decline in blood pressure . Similar phenomena were reported for other venoms . CORRADO et al. (1974) mention a similar short-lived hypotension preceding the hypertensive effect of scorpion venom. Snake venoms were found to produce a similar three-phase effect on blood pressure (Vicx et al., 1965 ; FELnsERG and ICELLAWAY, 1937). This effect was shown by FELDBERG and 11nLLAWAY (1937) to be similar to that produced by the injection of histamine in dogs. It is known that mellitin releases histamine from rat mast cells (HABERMANN, 1972). Epinephrine injection during the stage of circulatory shock produced by mellitin, caused a powerful and long lasting vasopressive effect and may indicate that the heart and blood vessels were still able to respond to sympathetic stimuli. Changes in the electro corticographic activity were not always correlated with the change in systemic blood pressure following epinephrine administration . This is probably due to a greatly decreased blood flow as a consequence of hemoconcentration or due to anoxia which follows hemolysis. It was indeed shown that the effect of epinephrine on cerebral circulation is greatly attenuated in conditions of blood $ow reduction or anoxia (CARPI et al., 1962). This observation supports our conclusion that depression of electrocorticographic activity after mellitin administration is due to blood or circulatory factors . REFERENCES Cwxrr, A., ÜRSILLO, R. C. and Bovsr, D. (1962) Local circulatory and metabolic factors influencing the cerebral vascular response to adrenaline in the dog. Archs int. Pharmacodyn. Thér. 89, 355. CoxRwno, A. P., Rtcciorro Nsro, F. and Arrrorno, A. (1974) The mechanism of the hypertensive effect of Brazilian scorpion venom (Tttyus serrulatus Lutz e Mello) . Toxicon 12, 145. CROSSV, W. H. (19517 A modification of the benzidine method for the measurement of haemoglobin in plasma and urine. Blood 11, 380. FsiasExß, W. and KELLwWwv, C. H. (1937) Circulatory effects of the venom of the Indian Cobra (NaJa naJa) in cats . Aust. l. exp. Biol . med. Scf. 15, 159. HzHexMwxx, E. (1965) Recent studies on Hymenoptera venom. In : Recent Advances in the Phmmacology of Toxins, Proc. 2nd Int. Pharmac. Meeting, p. 53 . New York : Pergamon Press. HwHeRMwxx, E. (1972) Bee and wasp venoms. Science 177, 314. Vwx HwRnav$Ln, A. (1966) Brain Tissue Eleetrolites . Waslv'ngton : Butterworths. Vicx, J. A., Cnrcnzw, H. P. and Pota ev, E. H. (1965) The et~ect of cobra venom on the respiratory mechanism of the dog. Archs int. Pharmacodyn . 77iér. 153, 424 . VYwTC{iAPINI80V, N. K. and Snvxw, A. Yw . (1973) T'he effect of mellitin-the major constituent of the bce venom on the central nervous system. Farntak. Toksik . 36, 526 .
TOXICON 1975 Yol. 13
EFFECTS OF MELLITIN ON THE CENTRAL NERVOUS SYSTEM J. ISHAY,
D. BBN-SxACxAR, Z. ELAZAR and E. 11A1'LINSKY
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel (Accepted for pubffeation 26 January 1975)
J. Iaxev, D. HaN~c1~1e, Z. EL~zr.1t and E. K~PLU1scY. F8'ects of mellitin on the central nervous system. Toxfcon l3, 277-283, 1975 .-Mellitin, the main toxic component of bce venom was injected in cats intravenously and through the carotid or vertebral arteries . The main effect on the eloctrocortiwgram was depression . This may be due to changes in blood properties (hemolysis, hemoconcentration, hyperkalemia) and to hypotension . INTRODUCTION
effect of bee venom is usually attributed to apamin which represents approximately 2 ~ of dry venom and produces in experimental animals long-lasting hypermotility and convulsions (HABERMANN, 1965). Mellitin, the main constituent of bee venom (40-50 per cent of dry venom) is thought to have mainly hemolytic effects (HABBRMANN, 1972). Recently, melittin was also shown to have neurotoxic central effects (VYATCIIANIVIKOV and SINKA, 1973). In this article we describe results of our studies on the effxt of mellitin on the electrical activity of the cat brain. TY~ NBIntoTOxic
MA~?ulA~c AND METHODS These experiments were performed on 36 cats of both sexes weighing between 2~5 and 3~5 kg . In some of our preparations anesthesia was first obtained by ether and subsequently maintained throughout the experiment at a light level by intravenous injections of sodium pentobarbital (total dose 25-30 mg per k~. In other animals anesthesia was started with ether and maintained during the surgical procedures with sodium thiopental (approximate dosage 20 mg per kg). At the conclusion of surgery in these animals, the surgical areas were infiltrated with 1 ~ procaine ; the animals were then paralysed with gallamine triethiodide (4 mg pet kg each ~} hr), and artificially ventilated . No further general anesthesia was given and experiments wero started 2 hr after stopping sodium thiopental administration . Surgery included cannulations of the femoral vein and artery and cannulation of the trachea. The vertebral artery was cannulated through the subclavian artery without opening the thorax thus permitting spontaneousr~pirations in the non-paralysed preparations. El halographic recordings were taken from screw electrodes placed on the cerebral cortex through the varium . Respiration was recorded with a thermocouple inserted into the trachea and blood pltssutt with a Statham transducer . All data were recorded on a Grass polygraph. Quantitative determinations of frce hemoglobin in plasma (~osaY, 195 were performed as an indication of intravascular hemolysis. Sodium and potassium levels west determined with a Bearel atomic Same photometer. RESULTS
Effects ofmellitin on the electrocorticogram
Small 5 mm' cotton pledgets were imbibed with 0~1 ~ mellitin and applied on the pial surface of the cerebral cortex (suprasylvian gyros) . Recordings were taken from the vicinity ToxrcoN r9~s voG rs
277
278
J. ISHAY, D. BEN-SHACHAR, Z. ELAZAR and E. KAPLINSKY
of the cotton pledget for periods exceeding 1 hr. No obvious changes in the electrocorticogram were seen. In animals anesthetized with pentobarbital and spontaneously respiring mellitin was injected through the femoral vein or the vertebral artery . When mellitin was injected into the femoral vein great variations in the effective dose were seen (1~ mg per kg). The effect consisted in a slowing of the electrical activity and with repeated doses or with high doses death occurred shortly after an abrupt flattening of the recording simultaneously in all cortical leads. Injections of mellitin into the vertebral artery were much more effective with less variation between different animals; the effective dose being between 0"2 and 0 "4 mg per kg. The following sequence of effects was observed: immediately after the injection, a marked drop in amplitude of the electrocorticogram occurred, then the amplitudes rose again. Spindles disappeared and the record showed groups of fast spiky waves arising from a background of low, irregular fast activity. This pattern could last for a few minutes and then the former spindling pattern returned . With higher doses or in more sensitive preparations the first spiky pattern was replaced gradually by slow low amplitude waves interspersed with sharp waves. The amplitudes gradually diminished until the record was almost flat. This development progressed over 10-30 min, after which the animal died. In some preparations, after the first spiky pattern the normal spindling pattern returned but at a certain point the record became abruptly and simultaneously flat in all leads. Figure l illustrates the immediate effect ofmellitin (A), the later fattening of the record and recovery of activity after adrenaline injection (B), and the final abrupt and generalized flattening which preceded death by 20 min. The animals which had received gallamine triethiodide, without general anesthesia and kept under artificial respiration were very sensitive to the effects of mellitin ; usually a single dose of 0"3 mg per kg was sufficient to produce electrocorticographic effects and death. Figure 2 shows the effect of injecting 1 mg per kg mellitin into the femoral vein of a cat which had received gallamine triethiodide . The first effect (B) is a weak increase in synchronization consisting in more prolonged rhythmic trains of waves of 9-10 Hz and short (1-2 sec) spindles of 7-8 Hz ; 300 sec after the injection this effect decreased and the pre-injection pattern reappeared . Shortly after a second injection of 1 mg per kg the rhythmic activity disappeared and the record became almost flat 60 sec later (C and D). Figure 3 shows the effect of intracarotid injection of mellitin . A diminution of amplitude and a general slowing of the electrocorticogram progressing to a completely flat record prior to death was usually seen. Figure 4 shows the effects of mellitin injection into the vertebral artery. Only a few seconds after the start of the injection (B) the amplitude of the electrocorticogram markedly decreased. Later, (C) spindling activity of low amplitude was observed in the anterior leads followed by the complete loss of electrical activity prior to death. Cardiovascular effects ofmellitin and correlation: with the electrocorticographic effects Effects or: bloodpressure . When injected through the vertebral artery in the anesthetized
(pentobarbital) spontaneously respiring animal, mellitin produced a rise in systemic blood pressure, often preceded by a very briefdecline in pressure. The respiration became irregular and spastic and eventually stopped. At the same time generalized uncoordinated movements were seen. The electrocorticogram showed, as described above, first flattening and then an TOXICON 1973 Yor. 13
Effects of mellitin on the central nervous system
FIG. I. EFFECT OF MPr i rrnv ON ELECI'ROCORIICOGRAM, BLOOD PRE93IJRE AND RESPIRATION.
Cat anesthetized with pentobarbital. Mellitin injected through the vertebral artery. Electrocorticographic leads: FR = right frontal ; FL = left frontal ; PR = right parietal ; PL = left parietal . FBP = Femoral artery blood pressure. T = Time, small divisions 1 sec, heavy linedrug injection. RESP = Respiration . In A two injections of mellitin (0~5 mg total dose). Note changes in electrocorticogram and the two-phase blood pressure change. In B blood pressure at shock level, electrocorticogram depressed . An injection of 2~5 llg total dose of epinephrine is followed by increase in blood pressure and recovery of electrocorticogram . In C an addi tional injection of 0~5 mg total dose of mellitin is followed by abrupt depression of electrocorticogram while blood pressure remains unchanged .
EKG
FIG. 2. EFFECT OF ME~-~T~ ON EIECTROCORTICOORAM AND ELECTROCARDIOGRAM. Cat paralysed with gallamine triethiodide, without general anesthesia and under artificial respiration. Mellitin injected through the femoral vein. RA = Right anterior electrocorticographic lead . A = Control. B = 4 sec after 1 mg per kg mellitin . C = An additional 1 mg per kg mellitin products disappearance of spindles and profound depression 10 min later, in D. Calibration = vertical, 200 ~; horizontal, 1 sec. TOXICON 1975 Yol. I3
279
280
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3.
Yt~i`~
EFFECT OF MELLITIN ON
11
~,`~+~~hy~'1w'~J!~~~ V~4Vir4~~ll'~Vr~y"'~~i
ELECTROCORTICO(iRAM, PRESSURE .
I~~~
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1I
l i l%~~ I I
ELECTROCARDIOGRAM AND HLOOD
Cat paralysed with gallamine triethiodide, without general anesthesia and under artificial respiration. Mellitin injected through the carotid artery. RA = Right anterior electrocorticographic lead . BP = Blood pressure ; vertical line, 100 mm Hg. ECG = Electrocardiogram . A = Control. B = 2 min after 1 mg per kg mellitin ; slight slowing of the electrocorticogram, no change in blood pressure. C = 5 min aftermellitin injection ; note depression of the electrowrticogram, and decrease in blood pressure . D = 15 min aftermellitin injection ; depression of the electrocorticogram, temporary Increase in blood pressure and disturbances in the electrocardiogram . LP A
B
RA
C
FIG. 4. EFFECT OF AiELL1'ITN ON ELECTROCORTICOGRAM .
Cat paralysed with pallamine triethiodide, without general anesthesia and under artificial respiration. Mellitin m~ected through the vertebral artery . LP = Left posterior, RA = right anterior electrocorticognphic leads. Calibrations as in Fig. 2. A = Control. B = Immediately after 1 mg total dose mellitin. C = 5 min later . TOXIC0IV 1973 Yol. I3
Effects of mellitin on the central nervous systean
281
imitative, spiky pattern. When the animals were put on artificial respiration the motor reaction gradually ceased and the electrocorticographic activity became more regular but the blood pressure level remained elevated. Figure 5 shows graphically the changes in the A
mm Hg zso
zoo
tso
too zo
so C 0
I
M
to
PIG. S . BLOOD PRES4IJRE C1iANGFS II~UCED BY }NIECITON OF ARTERY.
I~LLI'riN 77EIROUGH T1iE
VBitTapR~3
Cats anesthetized with pentobarbital . A = Effects on arterial systolic blood pressure. B = Effects on pulse plnesure. C = Control before in~'xtton . I = Changes daring the first 3-S sec after injection . M = The vasopressive phase of the main effect, measurod during the first minute after injection. Later, the blood pressure decreased. In cat 1 the extent of change is pulse pressure could not be evaluated. On cat S we included the initial change in pulse pressure only.
systolic pressure (A) and in pulse pressure (B) in several animals in the first minute after mellitin injection. At a later stage the blood pressure gradually declined to shock level. These changes in blood pressure, respiration and electtocorticogram can be seen in Fig. 1. In Fig. 1B it can be seen that when blood pressure decreased to shock level an injection of 2"50 lIg epinephrine had a powerful and long-lasting (see also Fig. 1G~ effect. When a second and third dose of mellitin was injected on a background of low blood pressure produced by the preceding dose, an increase in blood pressure occurred but this vasopressive effect was not seen with further repeated doses. When mellitin was injected in the anesthetized animals through the femoral vein the effects on blood pressure were less dramatic and more variable . An increase or decrease was first noted but with repeated doses or with high doses a decrease in blood pressure was the rule . Tn%ICON 1975 YoL 13
282
J. LSHAY, D . HEN-SHACHAR, Z . ELAZAR and E. KAPLINSKY
In the animals paralysed with gallamine triethiodide the same sequence of decrease, increase and then prolonged and profound decrease in blood pressure was seen but the changes were less abrupt. An example of these effects can be seen in Fig. 3 after injection of mellitin into the carotid artery . Note also the disturbances in the electrocardiogram in Fig. 3D. The final decrease in blood pressure to shock levels was correlated with depression of electrocorticographic activity as described above. Elevation of blood pressure after epinephrine administration could in some instances induce a rapid recovery of brain electrical activity (Fig. 1 B). However, it can be seen in the same figure (1C) that an additional injection of 0~5 mg mellitin produced a rapid depression of the electrocardcogram while the systemic blood pressure was still at the same level as in the former part of this figure (1B). Effects of mellitin on blood. in six animals mellitin was injected through the femoral vein and blood was taken for an analysis of electrolyte content. Significant changes were found in potassium levels which increased after 2 and 10 min. The plasma potassium concentration (mequiv. per liter) in six cats prior to mellitin administration was 3~03~0~3 (mean ~ s.n.). Corresponding values 2 and 10 min after 1 mg per kg mellitin were 3'6 ~ 0~3 and 5~5 ~ 0~5. Ten minutes after injection of mellitin the blood was hemolytic and no further samples were taken. Sodium levels did not change significantly after mellitin . It was also observed that mellitin caused hemoconcentration; in fact, 15 or 20 min after mellitin injection the blood became so concentrated that sampling was difficult. The animals died soon thereafter. DISCUSSION
The effect of mellitin on the electrical activity of the brain was studied recently by VYATCHANHIKOV and SINKA (1973) . These authors found in non-anesthetized cats paralysed with gallamine that low intravenous doses (1 mg per kg) of mellitin produced slowing of electrocortical activity and enhancement of spindling, while higher doses (3 mg per kg) produced desynchronization and epileptic seizures followed by post-ictal depression. Still higher doses (5 mg per kg) produced depression . Intraventricular introduction of mellitin induced spiking and seizure discharges. Similarly, in our cats paralysed with gallamine the intravenous or arterial injection of mellitin into the carotid or vertebral arteries produced depression of the electrocarticogram . In the anesthetized non-paralysed preparations injection of mellitin into the vertebral artery elicited a strong reaction consisting in irritative changes in the electrocardcogram, general contractions of the skeletal musculature, spastic respiration and large changes in blood pressure . Since this reaction was not seen in the paralysed preparations we propose that it was a result of a peripheral effect of mellitin . Mellitin produces depolarization and shortening of skeletal muscle and also pain (HABQtMANN, 1972). The main difference between our results and those of VYATCHAIdNIKOV and Snaxn (1973) is that we did not see seizures or other epileptiform phenomena in the electrocardcogram or convulsions. Noteworthy is also the fact that topical application of mellitin on the pial surface did not cause obvious changes in electrical activity. These findings would suggest that the depression observed with high doses of mellitin might be the result of hypoxia due to vascular reactions or changes in blood characteristics . The depression in the electroencephalogram was usually correlated with decreases in systemic blood pressure and serious disturbances in the electrocardiogram . Blood appeared to become concentrated, hemolytic and a rapid hyperkalemia developed. Anoxia and T1U%ICON 1975 Yol. 13
Effects of mellitin on the central nervous system
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hyperkalemia may produce depolarization of brain cells (VnN HARREVELD, 1966) and this may be the mechanism of the electrical depression. In artificially respired animals the heart continues to function 10-30 min after depression of the electrocorticogram but death was clearly the result of cardiovascular failure. The reduction of blood pressure to shock levels was the final effect of mellitin . The first effect was vasopressor and in many preparations the vasopressor effect was preceded by a brief but sharp decline in blood pressure . Similar phenomena were reported for other venoms . CORRADO et al. (1974) mention a similar short-lived hypotension preceding the hypertensive effect of scorpion venom. Snake venoms were found to produce a similar three-phase effect on blood pressure (Vicx et al., 1965 ; FELnsERG and ICELLAWAY, 1937). This effect was shown by FELDBERG and 11nLLAWAY (1937) to be similar to that produced by the injection of histamine in dogs. It is known that mellitin releases histamine from rat mast cells (HABERMANN, 1972). Epinephrine injection during the stage of circulatory shock produced by mellitin, caused a powerful and long lasting vasopressive effect and may indicate that the heart and blood vessels were still able to respond to sympathetic stimuli. Changes in the electro corticographic activity were not always correlated with the change in systemic blood pressure following epinephrine administration . This is probably due to a greatly decreased blood flow as a consequence of hemoconcentration or due to anoxia which follows hemolysis. It was indeed shown that the effect of epinephrine on cerebral circulation is greatly attenuated in conditions of blood $ow reduction or anoxia (CARPI et al., 1962). This observation supports our conclusion that depression of electrocorticographic activity after mellitin administration is due to blood or circulatory factors . REFERENCES Cwxrr, A., ÜRSILLO, R. C. and Bovsr, D. (1962) Local circulatory and metabolic factors influencing the cerebral vascular response to adrenaline in the dog. Archs int. Pharmacodyn. Thér. 89, 355. CoxRwno, A. P., Rtcciorro Nsro, F. and Arrrorno, A. (1974) The mechanism of the hypertensive effect of Brazilian scorpion venom (Tttyus serrulatus Lutz e Mello) . Toxicon 12, 145. CROSSV, W. H. (19517 A modification of the benzidine method for the measurement of haemoglobin in plasma and urine. Blood 11, 380. FsiasExß, W. and KELLwWwv, C. H. (1937) Circulatory effects of the venom of the Indian Cobra (NaJa naJa) in cats . Aust. l. exp. Biol . med. Scf. 15, 159. HzHexMwxx, E. (1965) Recent studies on Hymenoptera venom. In : Recent Advances in the Phmmacology of Toxins, Proc. 2nd Int. Pharmac. Meeting, p. 53 . New York : Pergamon Press. HwHeRMwxx, E. (1972) Bee and wasp venoms. Science 177, 314. Vwx HwRnav$Ln, A. (1966) Brain Tissue Eleetrolites . Waslv'ngton : Butterworths. Vicx, J. A., Cnrcnzw, H. P. and Pota ev, E. H. (1965) The et~ect of cobra venom on the respiratory mechanism of the dog. Archs int. Pharmacodyn . 77iér. 153, 424 . VYwTC{iAPINI80V, N. K. and Snvxw, A. Yw . (1973) T'he effect of mellitin-the major constituent of the bce venom on the central nervous system. Farntak. Toksik . 36, 526 .
TOXICON 1975 Yol. 13
