NEUROMUSCULAR ACTIVITY OF Bothrops neuwiedi pauloensis SNAKE VENOM IN MOUSE NERVE-MUSCLE PREPARATIONS

The pharmacological effects of Bothrops neuwiedi pauloensis venom on mouse phrenic nerve-diaphragm (PND) preparations were studied. Venom (20 μg/ml) irreversibly inhibited indirectly evoked twitches in PND preparations (60 ± 10% inhibition, mean ± SEM; p<0.05; n=6). At 50 μg/ml, the venom blocked indirectly and directly (curarized preparations) evoked twitches in mouse hemidiaphragms. In the absence of Ca, venom (50 μg/ml), produced partial blockade only after an 80 min incubation, which reached 40.3 ± 7.8% (p<0.05; n=3) after 120 min. Venom (20 μg/ml) increased (25 ± 2%, p<0.05) the frequency of giant miniature end-plate potentials in 9 of 10 end-plates after 30 min and the number of miniature end-plate potentials which was maximum (562 ± 3%, p<0.05) after 120 min. During the same period, the resting membrane potential decreased from – 81 ± 1.4 mV to – 41.3 ± 3.6 mV 24 fibers; p<0.01; n=4) in the end-plate region and from – 77.4 ± 1.4 to –44.6 ± 3.9 mV (24 fibers; p<0.01; n=4) in regions distant from the end-plate. These results indicate that B. n. pauloensis venom acts primarily at presynaptic sites. They also suggest that enzymatic activity may be involved in this pharmacological action.


INTRODUCTION
According to the Brazilian Ministry of Health, from 1990 to 1993, bites by Bothrops spp.accounted for 90.5% of the accidents involving snakes in which the genus was known or suspected and for about 70% venomous snakebites in Brazil (14).The main complications in lethal cases are acute renal failure, shock, acute respiratory failure, and sepsis (2,5,6,15).In some cases, respiratory failure has been associated with pulmonary edema (15).Although Bothrops venoms do not produce signs of neurotoxicity after snakebite, in recent years venoms of several Bothrops species, including B. jararacussu (17), B. insularis (8, 9), B. neuwiedi (3,4,21,22), and B. pirajai (10) have been found to affect neuromuscular transmission in avian and mammalian isolated nerve-muscle preparations and in chick in vivo.
B. neuwiedi venom caused head-drop, loss of balance and respiratory failure in chicks in vivo, and inhibited twitch-tension response in biventer cervicis preparations, without decreasing responses to acetylcholine or KCl, all of which suggested a presynaptic action (21).More recently, Borja-Oliveira et al. (3) investigated the neurotoxicity of several lots of B. neuwiedi venoms in biventer cervicis and observed that not all lots exhibited neurotoxicity in vitro.B. n. pauloensis venom neurotoxicity in these preparations is temperature-dependent (4).In this work, the initial observations on neuromuscular action of B. n. pauloensis venom in chick preparations were extended using myographic and electrophysiological techniques in mouse nervemuscle preparations.

Venom and reagents
B. n. pauloensis venom, collected from snakes caught in São Paulo State, was provided by the Instituto Butantan (São Paulo, SP, Brazil).D-tubocurarine chloride was from Abbot Laboratórios do Brasil Ltda and neostigmine methylsulfate from Roche (Basel, Switzerland).

Mouse phrenic nerve-diaphragm preparation
Adult male Swiss white mice (28-35 g) were supplied by the University Animal House.Phrenic nerve and diaphragm were obtained from mice anesthetized with chloral hydrate (300 mg/kg, IP) and sacrificed by exsanguination.Nerve-muscle preparation was mounted as described by Bülbring (7).Hemidiaphragms and phrenic nerves were mounted in 5 ml tissue baths containing Tyrode solution (pH 7.4, 37°C) of the following composition (mM): NaCl 137, KCl 2.7, CaCl 2 1.8, MgCl 2 0.49, NaH 2 PO 4 0.42, NaHCO 3 11.9 and glucose 11.1.The preparations were mounted on an electrode that enabled indirect (supramaximal voltage, 0.1 Hz, 0.2 ms) and direct (50 V, 0.1 Hz, 2 ms) muscle stimulation with impulses delivered from a Grass S4 stimulator.The resulting muscle tension was recorded using a force displacement transducer (BG 25 GM Kulite) coupled to a Gould RS 3400 recorder.The preparation was allowed to stabilize for at least 15 min before venom addition (10, 20, 50, or 100 µg/ml).In some cases, neostigmine (7.4 µM) was used to reverse venom effects.

Electrophysiology
Membrane resting potentials and miniature end-plate potentials (MEPPs) were recorded in mouse hemidiaphragm muscle using conventional microelectrode techniques.The dissected muscle was mounted in a lucite chamber containing aerated (95% O 2 , 5% CO 2 ) Tyrode solution (pH 7.4) at 30°C.The resting potential and MEPPs were recorded using glass microelectrodes filled with 3 M KCl (resistance 10-20 MΩ) inserted into the muscle fiber and the recordings were displayed on a Tektronix oscilloscope.Measurements were obtained 15, 30, 60, 90, and 120 min after the addition of venom (20 µg/ml) and were recorded on a Gould RS 3400.The resting potential was measured at or distant from the end-plate regions.

Statistical analysis
Each experiment was repeated at least three times.The results were expressed as mean ± S.E.M. Student's t-test was used for statistical analysis of data.Values of p < 0.05 were considered significant.
The twitch inhibition was not reverted by neostigmine (not shown) or by several washes with Tyrode solution (Figure 1B-D).No significant neuromuscular blockade was observed at concentrations < 20 µg/ml (not shown).
Changes in MEPPs amplitude and frequency were observed in preparations incubated with B. n. pauloensis venom when compared to preparations incubated with Tyrode solution (control).There was also an increase (25 ± 2%, p<0.05) in the number of giant miniature end-plate potentials observed in 9 of 10 end-plates after a 30 min incubation (Figure 5).The intracellular recordings showed an increase in MEPPs frequency, which was maximum (562 ± 3%, p<0.05) after 120 min incubation with venom (20 µg/ml).There was also a visible increase in the frequency of 0.6-0.7 mV potentials after 30 min and 0.4-0.5 mV after 120 min, and the generation of MEPPs with large amplitude (>1.2 mV) during 120 min incubation, which was not observed in control experiments (Figure 6).B. neuwiedi venom is not the first of the Bothrops genus to show presynaptic activity.
B. insularis venom also has a presynaptic action (8) which probably involves the Ca 2+ -and temperature-dependent presynaptic phospholipase A 2 present in this venom (9).The observation that B. n. pauloensis venom neuromuscular effect was Ca 2+ -dependent indicated that enzymatic activity was necessary for neuromuscular action.Several divalent ions, including Sr 2+ , can bind to the same site, allowing neuromuscular transmission, but cannot substitute for Ca 2+ in catalysis.For this reason, they are useful phospholipase A 2 activity inhibitors (20).Similar to B. insularis venom (8), B. n. pauloensis venom caused the early appearance of giant MEPPs that may be considered as the initial manifestation of a presynaptic action.This early agrees with the action of this venom on membrane resting potential seen at the same time (30 min).Crotoxin, the major neurotoxin from Crotalus durrissus terrificus venom, also causes an initial fall in MEPPs frequency in frog neuromuscular junction, followed by a secondary rise which was characterized by the appearance of large spontaneous potentials, i.e., giant MEPPs (12,18).At frog neuromuscular junction, giant MEPPs are described as spontaneous potentials with amplitude of more than twice the modal MEPPs average and a slower rising phase (1).A recent comparative study about the pharmacological activities of B. insularis, B. neuwiedi, and C. d. terrificus in chick neuromuscular preparations (19) indicated that bothropic venoms may contain components which act presynaptically at the skeletal neuromuscular junction in a manner similar to C. d. terrificus venom and its main toxin, crotoxin.
The neuromuscular blocking action of some bothropic venoms reported in recent years appears to involve Ca 2+ -independent (B.jararacussu) and Ca 2+ -dependent (B.insularis and B. n. pauloensis) pathways.The first pathway is intriguing because high Ca 2+ inhibits the neuromuscular blockade seen with bothropstoxin-I from B. jararacussu venom (13), the second pathway, which requires Ca 2+ for its toxic action, may involve PLA 2 activity of some as yet unidentified venom constituents.Thus, the classic concept of neurotoxicity involving mainly elapid and some crotalid venoms may need reexamination in view of the presynaptic actions of some bothropic venoms.

Figure 1 .
Figure 1.B. n. pauloensis venom effect on indirectly stimulated mouse phrenic nervediaphragm preparations.A shows average responses to venom compared to Tyrode control (*p<0.05).Each point represents mean ± S.E.M. of 3-6 experiments.B-D shows responses to venom (added at arrow) in indirectly stimulated preparations.Recordings are representative of 3-6 experiments.

Figure 2 .
Figure 2. The effect of replacing 1.8 mM Ca 2+ for 4.0 mM Sr 2+ in the blocking activity of B. n. pauloensis venom (50 µg/ml) as measured by the development of twitchtension in indirectly stimulated mouse diaphragm.Preparations incubated with normal Tyrode solution plus venom ( ) and Ca 2+ -free Tyrode solution plus 4.0 mM Sr 2+ and venom (▲).Each point represents mean ± S.E.M. of 3-6 experiments.All points indicated by an asterisk differed significantly from control preparations (p< 0.05).

Figure 4 .
Figure 4. Resting potential (RP) of mouse diaphragm preparations obtained at or distant from the end-plate regions.There was no significant difference in effects between these two regions.Preparations incubated with Tyrode solution showed no change in RP.The depolarizing effect of B. n. pauloensis venom (20 µg/ml) was gradual.The points are mean ± S.E.M. of four experiments.All points indicated by an asterisk differed significantly from the control preparations (p < 0.05).

Figure 5 .
Figure 5. B. n. pauloensis venom (20 µg/ml) effect on MEPPs amplitude in mouse diaphragm.In (a), preparations exposed to Tyrode solution and, in (b), venom effect.Note in (a) that most MEPPs have amplitudes ≤0.9 mV and; in (b), the large potentials (>1.8 mV) appear 30 min after venom addition.These phenomena are indicative of presynaptic events.Recordings are representative of four experiments.

Figure 6 .
Figure 6.B. n. pauloensis venom (20 µg/ml) effect on MEPPs frequency and amplitude in mouse diaphragm after 30, 60, and 120 min incubation compared to Tyrode control (white columns).The columns indicated by an asterisk are significantly different from control responses.Symbol (▲) on the X-axis indicates non-existent reading of bioelectric potentials.Note that 60 min after venom addition to the bath, an evident increase in MEPPs frequency is observed (p<0.05).Each point represents mean ± S.E.M. of four experiments.

Table 1 :
Time to 50% blockade by B. n. pauloensis venom of twitches elicited by indirect stimulation in phrenic nerve-diaphragm preparations.All data in the table were significantly (p<0.05)different from each other.Values are mean ± SEM of the number of experiments (n) shown.