|Iranian Journal of Pharmaceutical Research (2006)
Received: January 2005
Accepted: October 2005
Copyright ? 2005 by School of Pharmacy
The Pharmacological Effects of Odonthobuthus doriae Scorpion Venom and Its Extracted Fractions on Neuro-Muscular Transmission
Amir Jalalia, Zahra Hossininasabb, Zynab Ehsanib,
Amir-Abbas Zarec, Afshin Zarghid and Hossein Vatanpourb*
aDept. of Toxicology and Pharmacology, Faculty of Pharmacy, Jundishapour University of Medical Sciences, Ahwaz, Iran. bDept. of Toxicology and Pharmacology, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. cRazi Vaccine and Serum Research Institute, Karaj, Iran. dDept. of Pharmaceutical chemistry, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran.
The effect of Odonthobuthos doriae (O.d) scorpion venom at 0/3, 1 and 3, 10 ?g/ml concentrations were investigated on nerve-muscle transmission, using the Twitch tension technique. A concentration of 0.3 ?g/ml caused a small change in the twitch height in response to indirect muscle stimulation, but higher concentrations (1, 3, 10 ?g/ml) caused a transient augmentation in twitch response followed by a large contracture in the chick biventer cervices (CBC) preparation. This effect could be defined as a complex action of the venom, predominately presynaptic, in which its? effects on postjunctional synapses is also maintained.
?In order to find out which bioactive fraction could explain the venom effects, the soluble crude venom was partially separated by the gel filtration method, using a Sephadex G50 column, and four fractions were separated. Two of the four purified fractions (O.d F3, O.d F4) were characterized as toxic and their LD50 values were lower than the crude venom. Unlike the O.d F1 and O.d F2 fractions, O.d F3 and O.d F4 fractions caused a significant block in the twitch and contracture, in comparison to the control sample . in conclusion, fractions O.d F3 and O.d F4 are supposed to be as the biological active components of the O.d. venom.
Scorpion toxins have had instrumental effects in defining both structural and mechanistic properties of ion channels and various conductances normally present in cells involved in its blocking activity (1).
Up to now, more than 1500 genus of scorpions are known word wide, and the most hazardous scorpions to human are those belonging to the Buthidae family. Odonthobuthus belongs to the Buthidae family and Buthus genus which has two species, doriae and odonthurus. This scorpion has an abundant geographical distribution in Iran. Its? sting can cause various effects ranging from pain, inflammation, and necrosis at the site of sting, to muscle paralysis, hematuria and occasionally death in children.
Pharmacological characterizations of this venom, as well as its? sub-fractions has not yet been reported. In this study, the in vitro pharmacological effects of the venom and its? toxic fractions extracted by gel filtration have been investigated on the neuromuscular transmission, using the Chick Biventer Crevices (CBC) preparation.
Crude scorpion venom was gifted by Dr. Zare and Dr. Akbary, Department of Poisonous animal, Vaccine and Serum Research Institute of Razi, Karaj, Iran. Crude venom was obtained by electrical stimulating telson of scorpion and stored after freeze-drying at ?50?C, until use.
Krebs physiological solution salts, CO2/O2 cylinder, HEPES physiological buffer, O2 cylinder and other materials were also used.
Twitch tension recording
Twitch tension recording experiments were performed on the isolated CBC nerve-muscle preparation. This model is suitable for studying the nerve-muscle transmission effects of drugs or venom. It is small enough to be mounted within a 2-5 ml tissue organ bath and retain its? normal function in vitro, over a few hours.
Chick biventer cervices preparations
Biventer cervices muscles and associated nerves were dissected from 7-10 days old chicks, sacrificed by exposure to ether as described by Ginsborg and Warriner (2). Two muscles were mounted, with a resting tension of approximately 1 g; within a 5 ml tissue bath containing modified physiological solution (mM) as follows: NaCl, 118.4; KH2PO4, 1.2; Glucose, 11.1; NaHCO3, 25; CaCl2, 2.5; MgSO4, 1.4 and KCl, 4.7. Twitches were evoked by stimulating the motor nerve at 0.1 Hz with pulses of 0.2 ms duration and a voltage greater than that which produced a maximal response, using ring electrodes and a Grass S88 B stimulator. To detect any changes in postsynaptic sensitivity, responses to sub-maximal concentrations of exogenously applied acetylcholine (1-2 mM), carbachol (30-40 μM) and KCl (20-40 mM) were recorded in the absence of nerve stimulation, prior to the addition of venom and at the end of the experiment. The preparations were exposed to acetylcholine and KCl for 30 sec, and carbachol for 1 min (3). After the wash-out stage of these drugs, the preparations were allowed to stabilize for 15-20 min before the addition of the venom.
Twitches and contractures were recorded isometrically, using the Washington Grass model 79B and Grass model 79 polygraphs and SRI or Grass FT03 force transducers.
Fractioning of the soluble venom was accomplished using a Sephadex G50 column (3.5?230 cm, 1 ml/min flow) equilibrated and eluted with a pH 8.3 0.1 M ammonium acetate buffer, as previously described (4). Protein content was estimated spectrophotometrically at 215, 260 and 280 nm. All the procedures were carried out at room temperature.
Lethality tests and LD50 determination in mice
The mice (white, of both sexes, weighing 20 g) used in this research were treated in compliance with the US Public Health Service policy on human care and the use of animals. The mouse lethality of various protein fractions was observed after i.v. injections to mice (5). The Reed and Muench method, which provides a moderately accurate estimate of the LD50 based upon data smoothened by counting cumulative number of survivals, was used (6). Two designations of ?toxic? and ?nontoxic?? were used to express venom fractions. A fraction or peptide was considered non-toxic, if during the first 24 h of its injection the mouse lived or showed no more toxicity signs (heperexcitability, lachrymation, apnea, partial paralysis or respiratory failure). Toxic fraction means a fraction that immediately caused death of the test animal. The control group received an injection of normal saline at pH 7.4.
Data analysis was performed using the one-way (repeated) analysis of variance (ANOVA) for multiple comparison and student?s t-ttest for comparing two set of data as Mean ? SEM (p<0.05).
Results and Discussion
Effects of the venom on chick biventer cervices preparations
Figure 1 shows that the twitch responses of CBC evoked by nerve stimulation were reduced in the presence of Odonthobuthus scorpion venom (0.3-10 μg/ml), in a time-dependent manner. In contrast to other concentrations, the effect of 10 μg/ml venom was extremly strong and without any transient increase in the twitch height. It blocked muscle twitching within 13 min. As could be seen in figure 1, there was a direct relation ship between concentration and the maximum contracture effect.
With 3 and 10 μg/ml concentrations, the effect of venom was much faster and stronger in the indirectly stimulated preparation than the direct preparation (Figure 2).
Effects of the venom on acetylcholine, carbachol and potassium chloride responses, without electrical stimulation
Figure 3 shows a significant (P<0.05) decrease of Acetylcholine (1-2 mM) for 30 sec, Carbachol (30-40 ?M) for 1 min and KCl (30-40 mM) for 30 sec CBC contracture responses, after exposure to 1, 3, and 10 ?g/ml concentrations of the venom. Meanwhile, the responses to a concentration of 0.3 ?g/ml of venom in acetylcholine and carbachol was not significant. As shown in Figure 3, the decreasing pattern of acetylcholine and potassium chloride is similar at different concentrations of venom.
The effect of O.d F3 fraction on twitch height in indirect electrical stimulation
Figure 4 shows a gradually significant reduction in the twitch height responses of CBC preparations stimulated indirectly at different concentrations of 1, 3, and 10 ?g/ml of O.d F3, which was followed by complete blockage.
The effect of O.d F4 fraction on twitch height in indirect electrical stimulation
Figure 5 shows a gradually significant reduction in twitch height responses of CBC preparations stimulated indirectly at different concentrations of 1, 3, and 10 ?g/ml of O.d F4, in comparison to the control, leading to muscle paralysis at 1 and 10 ?g/ml concentrations. A complete blockage at 1 and 10 ?g/ml concentrations was observed after 21 and 18 min, respectively.
Skeletal muscle paralysis could occur due to either a presynaptic action via blockade/acceleration of acetylcholine release, or to a postsynaptic effect through blockage of acetylcholine receptors or direct muscle damage. In this study, experiments were performed by the indirect, direct and without electrical stimulation of CBC preparation, in order to investigate the pre- or post-synaptic effects of the O.d venom and its? toxic fractions.
According to the data obtained, it seems that the effects of the venbom are mostly mediated through presynaptic effects on nerve terminals. Our results are in agreement with the Pandinus imperator scorpion effects on CBC preparation (7). Furthermore, the same effects were noted earlier with Pandinus exitialis and Tityus serrulatus scorpion venoms on mouse hemi-diaphragm preparation (8, 9).
At higher concentrations, the inhibitory responses of CBC to Ach and Carb were boosted. This effect may be due to the blockage of neuro-muscular transmission, arising from the effects of the venom on nicotinic receptors of the postsynaptic membrane (10). The inhibitory effects of venom in response to Ach and Carb, suggest the curar?mimetic effects of venom. However, the contracture response observed as a result of direct stimulation, after the addition of d-Tubocurarine, can rule out this suggestion (11). Further more, the significant reduction in response to KCl at a venom concentration of 0.3 ?g/ml, contributes to the direct effect of venom on muscle (12).
The influence of 1, 3 and 10 ?g/ml concentrations of two non-toxic fractions, O.d F1 and O.d F2, were also studied on CBC preparations (table 1). There were no significant pharmacological changes in response to indirect stimulation. In contrast, O.d F3 and O.d F4 fractions result in significant changes in twitch height and yield in a complete blockage. Comparison of different concentrations of O.d F3 and Od F4, show a similar potency in paralysis of twitch tension responses (64% and 63%, respectively).
Finally, according to our obtained data, O.d venom acts mostly prejunctionally to inhibit the neuromuscular transmission. Further studies to find out which bioactive molecule is responsible for the venom action, showed that O.d F3 and O.d F4 fractions, which have a similar potency, are the active components of this venom. These findings may warrant research into the development of purification and extraction of a specific pharmacological agent against channels, from the Odonthobuthus venom.
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