Toad Toxin from the Skin of Bufo regularis has Bimodal Effects on Neuronal Electrical Properties of Cray Fish Stretch Receptor and Rat Subfornical Organ (SFO)
Background: The skin of various amphibians contains potentially diverse bioactive principles. Some of these are speculated to have pharmaceutical, medicinal, toxicological or other chemical importance. Moreover, such active principles have the potential to be used as chemical tool in biomedical research in quest for other discoveries. Objectives: The present study aims at isolating and purifying bioactive principles from the skin of Bufo regularis, and studying their effect on the neurons of Cray fish stretch receptors and rat subfornical organ (SFO). High performance liquid chromatography (HPLC) was used to isolate the toad toxin. The HPLC fractions were initially tested on guinea pig ileum for bioactivity and potency using organ bath method. The effects of potent semi-purified and purified extracts were then tested on neurons of Cray fish stretch receptors and hypothalamic SFO using two-electrode voltage clamp and patch clamp methods. Results: The HPLC purification resulted in potent bioactive components with a λmax UV absorbance pattern at around 295 nm. From the methanol preparative run, the 79 min elute had potent bioactivity. This was further purified using acetonitrile run, where the Fraction 40 min showed the maximum bioactivity of 57% of the weighed response on guinea pig ileum. When this was tested on cray fish stretch receptor neurons it had persistent inhibitory effect on action potentials generation to current steps under voltage clamp. Action potential generation was completely abolished (100% inhibitory response) in this neuron in response to the toxin. When the SFO neurons were studied under patch clamp, the neurons exhibited sustained depolarizing inward current (5.2 ± 3.4 pA) with complete inhibition of action potential generation. Conclusion: The HPLC eluate at 79th min in methanol preparative run corresponding to the eluate at 40th min in the acetonitrile run has complete inhibitory effect on action potential generation and a depolarizing effect on the resting membrane potential of neurons, which is deduced as a bimodal response.
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