On-line version ISSN 1678-4936
J. Venom. Anim. Toxins vol. 2 n. 1 Botucatu 1996
IN VITRO ACTIVITY OF DUVERNOY'S GLAND SECRETIONS FROM THE AFRICAN BOOMSLANG, Dispholidus typus, ON NERVE-MUSCLE PREPARATIONS.
1 Oak Ridge National Laboratory, Health Sciences Research Division, Oak Ridge, TN, USA.
ABSTRACT: In vitro toxicity assays were performed using Duvernoy's gland secretions from the African boomslang, Dispholidus typus, and isolated rat duodenum and frog sciatic nerve-gastrocnemius muscle preparations. The Duvernoy's gland secretions of D. typus had no apparent effect on the frog sciatic nerve-gastrocnemius muscle preparation. The secretions produced an increase in baseline tonus and an increase in the rhythmic contractile force of the rat duodenum, but had no significant effect on the frequency of contractions. Atropine sulfate (10 M) effectively antagonized the excitatory effects of the Duvernoy's gland secretions at concentrations 200 µg/ml. Although the coagulopathic effects of D. typus are well documented, this report provides preliminary data indicating effects of this venom on neuromuscular preparations.
KEY WORDS: Duvernoy's gland, African boomslang, Dispholidus typus.
The African boomslang, Dispholidus typus, is an opisthoglyphous colubrid responsible for serious envenomations of humans (2,4,8) Relative to other colubrids, D. typus has a well-developed Duvernoy's gland and venom delivery apparatus (11). Clinical reports have shown that serious envenomations by D. typus are characterized by consumptive coagulopathy (5,6,10). Grasset et al. (3) isolated a glycoprotein that affected blood clotting mechanisms, and Robertson & Delpierre (9) reported proteolytic and amino acid esterase activity in the venom of D. typus. This report presents data indicating cholinomimetic activity in D. typus Duvernoy's gland secretions.
Duvernoy's gland secretions were extracted from five specimens of D. typus (1 to 1.5 m in total length) by placing 20 µl capillary pipets over the enlarged rear teeth in a manner similar to that reported by Vest (13). The secretions were lyophilized and stored over CaSO4 in a desiccator at 0°C. The volume of the Duvernoy's gland secretions extracted from individual snakes ranged from 2.0-20.0 µl (5.0 ± 1.5 µl; ± S.D.) and the dry weight ranged from 0.1-1.5 mg (0.5 ± 0.25 mg; ± S.D.). The extraction yields were similar to those reported by Robertson & Delpierre (9) for D. typus.
To confirm the activity of the extracted material, three groups of five male Swiss-Webster mice were administered Duvernoy's gland secretions reconstituted in saline at doses of 0.1, 1.0, or 10.0 mg/kg i.p. (injected volumes not exceeding 0.1 ml). A control group received 0.1 ml of saline. All mice receiving the Duvernoy's secretions were sacrificed in extremis at 48 hours, as would be expected based upon the reported i.v. LD50 values of 0.071 µg/g (12) and 0.10 mg/kg (7), and an s.c. LD50 value of 12.5 mg/kg (7) for this venom. Although an i.p. LD50 was not estimated, the activity of the Duvernoy's gland secretions used in this study was affirmed by necropsy findings showing a dose-dependent severity of hematoperitoneum, thoracic hemorrhage, and subcutaneous hemorrhage at the injection site. In a separate experiment, sciatic nerve-gastrocnemius muscle (SN-GM) preparations were obtained from adult Rana pipiens and suspended in a Lexan chamber with stimulating and recording electrodes contacting the sciatic nerve. The muscle was attached to a Bionix F-200 isotonic transducer connected to a Tektronix 502A oscilloscope and Beckman Type K Dynograph. A Grass S-88 stimulator was used to apply both direct stimulation (muscle) and indirect stimulation (via the sciatic nerve) to the preparation. Duvernoy's gland secretions at concentrations up to 100 µg/ml had no observable effect on the frog SN-GM preparation. Threshold voltages for both direct and indirect stimulation remained unchanged for 4 hours after application of the secretions (Figure 1). The Duvernoy's gland secretions had no effect on the generation or propagation of the nerve action potential.
FIGURE 1. In vitro effects of Dispholidus typus Duvernoy's gland secretions on the muscle twitch amplitude (indirect stimulation) of the frog sciatic nerve-gastrocnemius muscle preparation. Over a period of 270 min, Duvernoy's gland secretions from D. typus applied at a concentration of 100 µg/ml ( ) failed to significantly inhibit muscle twitch amplitude compared with the control preparation ( ). Neither the nerve action potential nor the muscle response to direct stimulation were affected by the Duvernoy's gland secretion.
The effects of the Duvernoy's gland secretions on the rat duodenum in vitro are depicted in Figure 2 A and B. The rat duodenum preparations were prepared as described by Barlow et al. (1). The preparations were suspended in a 10-ml organ bath with physiological saline at 37°C, and were connected to a Bionix F-100 isometric transducer and chart recorder. The Duvernoy's gland secretions produced a concentration-related increase in the tonus and an increase in the amplitude of the rhythmic contractions of the smooth muscle preparation (Figure 2A). The excitatory effect was barely discernible at a concentration of 50 µg/ml, but a concentration of 100 µ/ml produced a two to three-fold increase in the tonus level and a 26-fold increase in the amplitude of the rhythmic contractions compared with controls. The frequency of contraction was unaffected. The excitatory action of the Duvernoy's gland secretions were effectively inhibited by prior addition of atropine sulfate (10M) to the bathing solution (Figure 2B). However, atropine sulfate (10M) was ineffective in preventing the excitatory effects of the Duvernoy's gland secretions at higher concentrations (>200 µg/ml).
FIGURE 2. Contractile response (force in g [ordinate], time in seconds [abscissa]) of isolated rat duodenum following application of D. typus Duvernoy's gland secretions. a) Concentration response effect of venom (arrows) showing a slight increase in tonus at venom concentrations of 50 and 75 µg/ml and a greater increase in tonus accompanied by an increased amplitude of rhythmic contractions at a concentration of 100 µg/ml. b) antagonistic effect of 10M atropine (first arrow) applied prior to the addition of 100 µg/ml of D. typus venom (second arrow) to the bathing solution.
The results of the in vitro bioassays conducted in this investigation showed that D. typus venom had no observable effect on the neuromuscular function of anuran skeletal muscle preparation. This is consistent with the absence of observable neuromuscular effects (e.g. flaccid paralysis) following envenomation by D. typus. The results of experiments using the isolated rat duodenum showed that the venom exerted an excitatory effect on mammalian smooth muscle which could be prevented by the antimuscarinic agent, atropine, thereby implying the presence of a muscarine-like activity in the whole venom. These data are preliminary, but suggest the existence of a muscarine-like agonist in the venom of D. typus and, therefore, an activity seemingly unrelated to the coagulopathic effects of this venom. Additional studies are required to affirm this contention and to characterize the active component(s).
Portions of this work have been extracted from a dissertation submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Physiology and Pharmacology, Southern Illinois University. This work was supported in part by a Dissertation Research Award Fellowship from the Southern Illinois University Graduate School and a Grant-in-Aid of Research from Sigma Xi Scientific Research Society. Final preparation of this work was supported, in part, by Oak Ridge National Laboratory, Oak Ridge, TN.
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