Botulinum Toxin Type A Still Inhibits ACh-induced Pyloric Muscle Contractile Response Following Cleavage Of SNAP-25 Antagonized By Toosendanin In Vitro

Background and Aim: Botulinum toxin type A (BTX-A) selectively cleaves synaptosomal-associated protein of 25 kDa (SNAP-25) and results in inhibition of the fusion of synaptic vesicles containing neurotransmitters with the presynaptic membrane to undergo fusion with presynaptic membrane, exocytosis and release. Toosendanin (TSN) was recently demonstrated a potential antibotulismic agent by antagonizing BTX-A-mediated cleavage of SNAP-25. The study was to decide whether BTX-A effected on the pyloric smooth muscle contractility induced by acetylcholine (ACh) after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin and investigate in which way BTX-A did.Methods: Three groups of Sprague-Dawley rat pyloric muscle strips were studied in vitro. All strips were allowed to equilibrate for 52 min under a basal loading tension of 1 g in Kerbs solution and its spontaneous contractile waves regularly emerging as its own control before adding respectively ACh, BTX-A, TSN and atropine. The first group (n=10), after initial response to ACh 100μM for 10 min, atropine 1μM (n=5) or BTX-A 10 U/ml (n=5) was respectively added for recording 4 h. The second group(n=10), after initial response to BTX-A 10 U/ml (n=5) or atropine 1μM (n=5) for 30 min. ACh 100μM was added for recording 4 h. The third group, after initial incubation of toosendanin (TSN) 29.6μM for 20 min, subsequent BTX-A 10 U/ml for 30 min and then ACh 100μM were added for recording 4 h.Results: In the first group the addition of ACh 100μM into Krebs solution enhanced significantly pyloric muscle contractile tension (P<0.001) and frequency (P<0.001), but not contractile amplitude (P=0.769) in preparations. The further addition of BTX-A 10 U/ml suppressed ACh-induced contractile responses including tension (P<0.001), frequency (P<0.001) and amplitude (P=0.014). Atropine 1μM suppressed comparably ACh-induce contractile responses. Nevertheless, the inhibition of atropine was almost complete. The results showed that BTX-A 10 U/ml directly inhibits ACh-induced pyloric contractile response.In the second group, BTX-A 10 U/ml inhibited directly pyloric muscle spontaneous contractions, such as contractile tension (P=0,006), frequency (P<0.001) and amplitude (P<0.001). Atropine 1μM similarly inhibited pyloric muscle contraction (unshown). Sequential addition of ACh 100μM did not agitate any more pyloric muscle contraction.In the third group, the addition of TSN 29.6μM in initial period of 20 min did not influence pyloric muscle spontaneous contractility, subsequently BTX-A 10 U/ml addition still decreased pyloric muscle contractile (tension, P<0.001; amplitude, P<0.001). Though this inhibition was not similar to the one BTX-A did without TSN. BTX-A 10 U/ml still inhibited pyloric smooth muscle contractility so that ACh 100μM did no longer agitate it.Conclusion: These data demonstrate that BTX-A inhibits not only pyloric smooth muscle spontaneous contraction but also ACh-induced contraction. Even after BTX-A-mediated cleavage of SNAP-25 antagonized by toosendanin, BTX-A inhibits ACh-induced pyloric smooth muscle contraction. Such suggest that BTX-A inhibits not only acetylcholine release from cholinergic nerves but also cholinergic muscarinic muscular transmission.