Gen. Pharmac , 1975, Vol 6, pp 325 to 331 Pergamon Press Printed m Great Britain

ASPECTS OF THE PHARMACOLOGY OF LATRODECTUS MACTANS VENOM

MAUREEN F. PALMER

Department of Physiology, Queen Elizabeth College University of London, Campden Hill Road, London W8, England

(Received 19 December 1974)

Abstract--1. The actmn of Latrodectus venom on the neuromuscular junctaon Is entirely pre-synaptlc. 2 The end-plate may be protected by reductmn m acetylcholine synthesis or by reduction in calcaum

concentratmn. 3. The actmn of the venom may be a disruptmn of the normal career mechanisms in the pre-

synapUc ternunal

INTRODUCTION

BITES of the black widow spider, Latrodectus mactans are frequently reported throughout the American continent, Australia and in the Mediter- ranean area. Manifestations of the bite in mammals are pain at the sRe of the bite followed by muscle contracture and sometimes paralysis of skeletal muscle. The venom also affects the autonomic nervous system since envenomatlon in both man and experimental animals causes increased motility, secretion of the gastro-mtestinal tract and very large swings in blood pressure. Sampayo (1944) reported changes m cardiac activity, particularly reversal of the T-wave of the ECG following bites in man but in preliminary experiments in rats there does not appear to be any disturbance of cardiac activity suggesting that the blood pressure changes observed are due to changes in the peripheral circulation.

Early investigations of the effect of the venom on the frog nerve-muscle preparation led D 'Amour et al. (1936) to beheve that the muscular paralysis seen after a sp~der bite was due to a central effect since they were unable to demonstrate any change in the response of the muscle to electrical stimulation foUowmg addiUon of venom to the nerve, to the muscle or to the whole nerve-muscle preparation. Russell et al. (1961) demonstrated that the actmn of the venom on the mammalian nerve-muscle prepara- tion was varmble, sometimes causing paralysis but at other times without effect. Electrophysmlogical recording techmques employed by Longenecker et a/. (1970) showed that injection of venom in the frog, caused increased transmitter release from the nerve t~rt~iinal which proceeded until the total pre- existant store of transmitter was exhausted. A similar release of transmitter has been demonstrated by Cull-Candy (1973) at the glutaminergic neuro- muscular junction of the locust. Earlier work by Grasso & Paggi (1967) and Vicari et al. (1965) suggested that m the invertebrates the venom acted directly on the nerve cell membrane causing trans- mitter release.

The present study was undertaken to examine further the effect of the venom on a mammalian preparation and its mode of action.

MATERIALS AND METHODS

Latrodectus mactans venom was obtained as whole dried glands Extracts were prepared by crushing 25 dried glands m 0 01 M phosphate buffer pH 7-4 followed by centrffugation or filtraUon to remove the debris

LD60 of six batches of dried glands was determined by intrapentoneal injection into 20-25 g male mtce of a two-fold series ddutmn of the venom m 0 9% saline using 7 dose levels with 6 mice/dose The number of survwors at 24 hr was observed and from a sample dose response plot LDs0 was calculated. In each case the toxicity was about 10 LDso'S per gland.

Rat phremc nerve-diaphragm preparattons These were obtained from female albino rats welghmg

from 170-250 g as originally described by Buibring (1946). They were suspended m Krebs bicarbonate saline con- talmng 0-2% glucose and gassed continuously wtth 95 % oxygen]5% carbon dioxide. Two organ baths, each containing 50 ml of this sahne, were mounted in a common water bath maintained at 32-35°C unless otherwise stated, and fight and left hemidiaphragms used alternately as test and control. An electromc square wave stimulator (C.F. Palmer Ltd) was used for both direct (maxtmum 5 msec 50-100 V) and indirect stimula- tion (50 Bsec, 2-5 V) The contractions of the muscle were recorded on a rotating drum using an isntomc ink-writing lever. SUmulation rate was 12/rain throughout the experiments unless otherwise stated

Following an equlhbration period of 30 nun the saline was replaced and when the response of the muscle to indirect stimulation at 12/rmn was constant 100 Bg 8-tubocuradne was added to each bath and the prepara- tions left until complete paralysis had occurred The re- sponse to direct sttmulatmn was recorded and this used as a reference value for all subsequent responses. The 8-tubocurarme was washed out and when the preparation had recovered, venom was added to one and an equal volume of phosphate buffer or boiled venom in phosphate buffer to the other

The response to indirect stimulatmn was calculated

325

326 MAUREEN F PALMER

Control p r e p a r a t i o n

Phosphate buffer D S added

Test Preparation

DS

I rrl'

V e n o m e x t r a c t 5' 10 ~ 15 / 20' 25 / 45" DS added D$

Fig 1. Record of the normal course of paralys~s of a rat phrenic nerve dmphragm preparaUon following addition of Latrodectus venom The upper trace ]s the control preparation to winch phosphate buffer was

added. IS is redirect stimulation at 50 lasec and 2 V. DS is direct sUmulatlon at 2 msec and 50 V.

as a percentage of the fully curansed direct response for both the test preparatlon and the control The per- centage change in twitch stze of the control was used throughout to correct the test response for the small temperature changes winch mewtably occur during the course of the experiment. The changes were usually 5-7 ~o over the three hour course of the experiment

RESULTS

Normal course o f paralysis Following addition of Latrodectus venom to the

bath there was a rapld fadure of the response to indirect stimulation, 50~o paralysis was achieved m 14 5-t-2.2 min whde paralysis was complete after 35 min (range of 15--40 min) One minute following venom addmon there was a very marked merease m twitch size before the rapid progresswe paralys~s (Fig. 1). There was no recovery of the response to in&reet stimulation m spite of numerous washes with fresh sahne; however direct stimulation of the diaphragm muscle was stdl at all times stdl fully effective in producing a response.

The speed of entry and fixation of the toxin was determined by measuring the speed and degree of muscular paralysis after varying the length of time for wluch the venom was allowed to remain m con- tact with the preparatlon; these periods of contact varied from 2-5--60rain. Complete paralysm oc- curred after contact t~mes of 10 min or longer the time course of the paralysis vaned very little. By contrast, contact times of less than 10 rain were insufficient for complete paralysis to occur (Table 1 and Fig. 2).

There appeared to be a certain stolchlometry about the venom-receptor combination. Thus bath fluid from a preparaUon completely paralysed by the venom, when added to a second preparation always caused some degree of paralysis, whilst that from one preparation showing incomplete paralysis had no effect upon a second dmphragm.

100"

.

i 80- 60" 40"

~ 20" g

- - - - - - - - - - - - - ' - - - 4

t~ 2~ ~o 4~ 5'0 e'o T i m e minutes

Fig 2 To show the relatlonsinp between duration of contact of Latrodectus mactans venom and % paralys~s

after 60 mm

The effect o f varying temperature on the acttvity o f the venom

Experiments were earned out at temperatures, between 22-45°C to determine the effect of bath temperature on the speed of entry, fixation and actwity of the venom.

From the results gwen m Table 2 st ,s apparent that the venom is extremely heat sensmve. At 45 0°C there appears to be approximately 70 ~o denaturation of the venom since the degree of paralysis is only 24 34-t-5-56 compared wah 87 02+5 64 at 32-0°C. Ttus is even smaller than the value of paralysm obtained with a contact time of 2 5 mm and probably means that the molecules of venom which were effective were those which attached to end plates before temperature mactwatlon of venom had occurred

The effect o f end plate acttvity on the speed of entry and fixation o f the venom

The effect of end plate activity on the speed of entry and fixation of the venom was examined m two ways

Table 1.

Aspects of the pharmacology of Latrodectus mactans venom

Effect of time of contact on the course of paralysm of the rat phrenic nerve-diaphragm preparation with Latrodectus venom

327

Contact tlme (mins)

6O

15

lO

5

2.5

% Paralysis after 60 mins +S.E.

87.o~±5.64

79.01L5.37

81.92~5 98

67.03~4.17

30.07L5.25

% Paralysis after 120 mlns +S.E.

87 06L5.71

76.81+4.60

91.08±6.Ol

66.90Z4 22

52.22d5.11

Time for 50% Paralysis mins.

14.45Z2.21

21.79!1.84

15.66di.66

24.70~4.88

Venom extract was added to all preparations and replaced wRh fresh sahne after the contact time state Stimulation was at 12/ram and there were 10 expenments m each group.

(1) The activity of the end plate was changed by varying the frequency of stimulatmn from almost zero (by stimulating preparatmus for only 30 see in each 5 rain period) to 30/min or 60/rmn.

The results given m Table 3A, show that variations m end plate activity influenced neither the degree nor time course of the paralysis.

(2) End plate activity was increased by the addition of physostigmlne.

This drug was added to the bath (final concentration of 1 lag/ml) and the preparations left for 5-10 rain until a new steady state had been reached. Venom was then added to one preparation of a pair and allowed to remain in contact for either 60 min or 5 rain.

The results given in Table 3B, show that for venom and physostlgrnine in contact for 60mln, no significant difference in the time course of the paralysis occurs although the time taken for 50~0 paralysm was somewhat larger. In the presence of physostigmme venom addition caused an enormous increase in twitch size, in many cases to an extent as great as the maximal cnrarmed dzrect response; in

addition the preparations showed spontaneous contractions. The increase in the control, due solely to the physostigmme was very much smaller.

With the venom and physostigmine in contact for only 5 mm there was as before enormous enhance- ment of the response at first but the final degree of paralysis was significantly less.

The effects o f pre-synaptie blocking drugs on the speed o f entry and fixation on the venom

Since the venom causes increased transmitter release (Longenecker et aL, 1970) which is a pre- synaptic effect, hemichohnium No. 3 (HC-3) was used to determine whether it could in any way protect the end plate from the venom.

Hemicholinium (final concentrations 0.5, 1.0 or 2.0 ~tg/ml) was added to both test and control preparations at zero time, which were then stimula- ted for 20 see at 50/sec once every 2 rain returning to 12/min between tetanL This caused them to become completely unresponsive to indirect stimula- tion in 15-20 rain. Venom was then added to the test preparation, left in contact and washed out

Table 2. Effect of temperature on the time course and degree of paralysm

Temperature °C

22.5-24.0 (IO)

32.0-35.0 (io)

40.0 (6)

45.0 (6)

Contact time for each group was md,cate number of experiments

% Paralysis after 60 mlns +S.E.

76.07~6.60

87.02!5.64

76.83±5.31

24.34!5.56

% Paralysis after 120 mins +S.E.

93.0O±5.35

87.06±5.71

68.00+6.74

20.64+4.50

Time for 50% Paralysis mins.

43.2~5.89

14.45~2.21

9.50±O.96

60 mm and rate of stimulation 12/rmn. Figures in parentheses

328 MAUREEN F PALMER

Table 3 Effect of end plate acUwty on speed of entry and fixation of the venom

A. Varlatlon In rate of stlmulatlon of the preparatlon. Contact tlme 60 mlnutes

Preparatlon Stlmulat~on Stimulation St~mulatlon rate 12/mln rate 30/mln rate 60/mln

stlmulated

% Paralysls at 60 mlnutes

Time taken for 50% Paralysis (minutes)

No. of experiments

not

93.91d2.80

17 35Z4.66

i0

87.02L5.64

14.45Z2.21

I0

89.24+6.11

15.14~3.24

lO

90 12Z3.60

16.28Z2.89

i0

B. Effect of Ph~sost16snlne 1 ~5/ml. Stlmulation rate 12/min

% Paralysls at 60 minutes

Time taken for 50% Paralysis

No. of experiments

Venom alone

Contact time 60 m_ins.

87.02Z5.64

14.45Z2.21

i0

Venom ÷ Fnysostigmine Contact time

60 mins.

99.30~2.45 NS.

26.75+1.94 S~g. a'% o.1%

]srel

13

Venom alone

Contact time 5 mins.

67.03Z4.17

24.90~4.88

I0

Venom + Fnysostigmin~ Contact time

5 mins.

38.73+7.17 S~g. ~t 0.1%

level

10

after 5 min. Table 4A gives the results o f these experiments.

With the smallest dose o f h e m i c h o h n i u m there was slgmficant protection o f the end plate against the venom but as the dose o f h e m l c h o h m u m was m- creased this was lost.

The effect o f ca l cmm concentration on venom fixation and activity has also been demonstrated. Krebs solutions were prepared in which the CaC1, was either orrutted (Ca' +-free) or where the concen- tration was doubled to 5 m M Ca '+ (normal Krebs

contains 2 5 m M Ca'+). Diaphragms were bathed m these solutions until either paralysm occurred (Ca t +-free) or until the twitch size became constant (Ca'+-nch) after which venom was added. Fol low- mg a 5 rain contact, the venom was washed out and the bath re-filled with normal saline.

The results o f changes in calcium concentration can be seen in Table 4B. It is evident that reduction in calcium concentration causes protection o f the end plate whereas increase in calcium causes increase in the degree o f paralysis.

Table 4 Effect of hemlchohmum and Ca '+ on the degree of paralysm obtained after 5 mm contact time of the venom

A. HEMICHOLINIUM

% Paralysis at 60 mlnates

No. of experiments

Venom alone

67.03Z4.17

lO

Venom + 25 ~g/ml

HC3

24.22+4.04

S1g. at 0.1%

i0

Venom + 50 ~g/ml

HC3

39.35d7.37 Sig. at 0.1%

lO

Venom lO0 ~g/ml

HC3

58.74Z7.08

NS

8

B. CALCIUM

% Paralysis at 60 minutes

No. of experlments

Ca ++ Free Sallne

3O.71Z6.38

2.5 mM Ca ++ I.e. normal Krebs

67.03±4.17

5.0 mM Ca ++

82.90!4.95

~g. at 0.1% level

i0 lO

Sig. at 0.1% level

i0

Aspects of the pharmacology of Latrodectus mactans venom 329

The effects o f post-synaptic blocking drugs on the speed of entry and fixation of the venom

8-Tubocurarme at a concentration of 2 lag/mi bath fired was added to both test and control preparatmns and the preparation left until paralysm to indirect stlmulat~on had occurred. Venom was added to the bath fired of the test preparation and left m contact for 5 nun. The sahne was then replaced without 8- tubocurarme and recovery from the drug occurred and the diaphragms subsequently became paralysed due to the venom

Atropine, although not commonly regarded as a blockmg agent m skeletal muscle can nevertheless cause paralys~s if the dose is sufficiently large. Atropine m both paralysmg and non-paralysing doses were used in conjunction w~th the venom in a manner similar to that described for &tubocurarine above.

There was no slgmficant difference in the percent- age paralys~s between the 5 nun contact t~me and the 5 nun contact t~me m conjunctmn w~th atropine or 8-tubocurarme Table 5. However particularly with atropine the recovery after atropine plus venom was extremely slow, commencement of recovery some- times being delayed for 30 rain. Control prepara- tions which had atropine alone showed full recovery after about I0 min.

DISCUSSION

Using the isolated rat phrenic nerve-diaphragm preparations the venom of Latrodectus mactans dear ly possesses a neuromuscular blocking action. The varmbihty of the response reported by Russell & Long (1961) may perhaps he in the stoictuometric properties of the venom. The venom of Latrodectus tredectmguttatus has been fractionated by Frontali & Grasso (1964) and Vican et al. (1965) and three fractions have been collected, the third fraction of which (LVs) will produce characteristic symptoms, including muscular paralysis, in n'ace and guinea pigs irrespective of the route by which it is given. Fraction LVx wdl produce characteristic symptoms in mice only if given in larger doses and by the intravenous route, while fractmn LV~ is ineffective in mammals. Both groups of workers

state that the fraction LV1 is extremely heat labile, losing 90~o of its actiwty when kept for 21 hr at 26°C. The fractmn LV8 is heat s table--at least for 21 hr at 26°C. The results of the present experiment (Table 2) suggest that the neurotox~c agent in the crude venom of Latrodectus mactans is extremely heat labile above 40°C when approxmmtely 70yo of the aeUvity ~s lost; above 45°C there is complete denaturation.

In the sequence of events at the neuromuscular junction; namely the depolarisation of the nerve ternunal by passage of a nerve action potentml, release of transmitter and subsequent depolarisaUon of the post-synapUc membrane, ~t would appear from the experimental results presented here that Latro- dectus venom is nnplicated at two specific s~tes.

The synthesis of acetylchohne is a continuing process and the nerve ending itself has only enough chohne stored to synthesme 8 - 9 ~ of the total acetylchohne store (Potter, 1970) the rest must be derived from outside sources. Normally, following the breakdown of acetylchohne by chohnesterase, the choline ~s reabsorbed rote the nerve terminal using a career mechanism which ~s sensmve to hermchohnlum-3. Thus, acetylchohne synthesis is blocked. Longenecker et aL (1970) observed that there was increased release of acetylchohne at the neuromuscular junction of the frog following addi- Uon of venom and that the acetylchohne is released until the pre-stored transmRter is exhausted. Mmmture end plate potentials fall to zero about 80 ram after addmon of the venom and paralysis of the muscle is complete at about the same time. From Table 4 ~t appears that the venom is less effective when acetylcholine release cannot occur. As the concentration of HC-3 increases the protection is progressively lost. Although HC-3 blockade can clearly affect the fixation and activity of Latrodectus venom this may not be a &rect effect upon the acetylcholine-synthesls system since varmtion m acetylchohne usage, caused by changes m the stnnulatmn frequency, do not appear to affect the actiwty of the venom.

Calcium is heavily lmphcated m the release of acetylchohne from the nerve terminal and both Katz & Mlledi (1968) and Hubbard (1968) have argued

Table 5 Effect of non-depolarmng post synapt~c blocking drugs on degree of paralysm obtained after a contact Ume of 5 nun wRh the venom st~mulaUon rate 12/nun

Venom alone

167.o5_+4.17

lO

Vanom + 3TC 2 pg/ml

56.72~4.98

I0

Venom ** Atropine i mg/ml

64.51±7.53

I0

$ Paralysis at 60 minutes

No. of experiments

Venom @ Atropine 2 ~g lml

62.13±6.23

6

*Recovery after atropine I mg/ml was so slow that the figure given is paralysis after 120 rain when the steady state condition had been reached

330 MAUREEN F. PALMER

for a earner mechamsm on the membrane surface such that depolarlsatlon of the nerve terminal permits entry of calcmm on a carrier which in turn causes enhanced transmitter release. When the calcium concentration is very low the carrier is prevented from moving and transnutter release ~s impaired; conversely htgh calcmm concentrations give enhanced transmatter release. To some extent the results w~th HC-3 at low concentrations and wath calcium-free saline are s~mllar if It is the release of transmitter which enhances or determines venom action. In both cases paralysis is delayed suggestmg that transmitter synthesis or release is necessary for the venom to be fully effectave. Against th~s view is the observation that alterations m frequency of stimulation is lneffectwe m modifying the paralysis time as well as the demonstrataon by Longenecker et al. (1970) that the mcrease In MEPP's and increased transmitter release could take place even at low calcmm suggests that the venom ~s acting on the calcium carrier system in some way.

Electron microscopy stu&es of the frog neuro- muscular junction by Clark et al. (1972) have shown disappearance of the synaptlc vesicles and infolding of the pre-synaptac membrane following envenoma- tlon and samdar studies by Okamoto et al. (1971) on the cat neuromuscular junction have shown loss of synaptlc vesicles 14 mln following addition of venom and after 58 man, complete disruption of the pre- synaptlc membrane and organelles in the nerve terminal.

Physost~gmme, which prevents breakdown of acetylchollne appears to slow the fixation or actwaty of the venom, since the degree of paralysis after 5 man contact was s~gmficantly less although ff the venom is allowed to remain m contact it does not prevent full paralysis. This might suggest that a delay in repolansatlon after the passage of an im- pulse can slow either the attachment of the venom to a career or membrane damage Alternatively if the acetylchohne is not being hydrolysed, the choline carrier will not be operative and the protection found after physostigmine could andicate that the venom does act on the carrier or synthesis mechan- Ism

Fmally neither of the non-depolarlsmg post- synaptlc blocking drugs affected the degree of paralys~s nor the speed of fixatton of the venom. This agrees with the studies of Okamoto et al. (1971) who found in electron micrographs of the cat neuromuscular junctaon that although there was gross d~sorgamsatlon of the pre-synaptic membrane the contractde apparatus and the post-synaptac membrane were totally unaffected.

It may be concluded that the actaon of the venom of Latrodectus mactans is entirely pre-synaptic and that in the early stages of envenomat~on it perhaps acts on the career systems within the membrane but w~th longer contact t~mes ~t causes complete mem- brane disruption.

SUMMARY

1. The speed of fixation of the venom is fast and it is stolchlometrlc i e. ~t will attach to receptor sites according to the number of molecules present.

2. The venom is very heat labile and ~s completely inactivated at temperatures above 45°C.

3 Increased frequency of electrical stimulation of the end plate does not change the degree of paralysm nor the speed of fixation of the venom

4. Increased activity of the end plate caused by physost~gmlne causes slowing of the speed of entry of the venom

5 Small doses of hemlchohnlum i e block of acetylchohne synthes~s can protect the end plate from the effects of the venom. With larger doses of hemlchohnlum this protection is lost.

6. Low Ca ~ + IS able to protect the end plate from the effect of the venom whereas high Ca ~ + enhances the speed of entry of the venom.

7 Post-synaptic blocking agents do not appear to affect the speed of entry or the degree of paralysis caused by the venom. The action of the venom is entirely pre-synaptlc.

REFERENCES

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Aspects of the pharmacology of Latrodectus mactans venom 331

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Key Word Index--Latrodectus mactans, rat phrenie nerve--diaphragm preparatton, temperature, end-plate act~vlty, pre- and post synaptic blocking drugs on venom activity