The Effect Of Botulinum Toxin Type A On The Rat Duodenal Smooth Muscle In Vitro

Objective: The present study is to investigate whether botulinum toxin type A (BTX-A) inhibits spontaneous contractility of isolated duodenal smooth muscle and acetylcholine (ACh)-induced contractility, whether the inhibitory characteristic of BTX-A is same manner with antagonist of muscafinic receptor. Pretreatment of duodenal smooth muscle with BTX-A, ACh administration still enhance the contractile response or not. The aim of this study is to demonstrate the efficiency of BTX-A to treat the disorder of intestinal function such as irritable bowel syndrome.Methods: Sprague-Dawley rats weighing 250 to 350 g were used in this study and fasted for 24 hours before operation. Duodenal smooth muscle strip of 1.5 cm was quickly removed from distal pylori after the rat unconsciousness by hitting head. The smooth muscle strip rinsed with Krebs bicarbonate buffer (in mmol/1: NaCL 120.6, KCL 5.9, NaH₂PO₄ 1.2, MgCL₂ 1.2, NaHCO₃ 15.4, CaCL₂ 2.5, C₆H₁₂O₆ 11.5), pH 7.4 and oxygenated with 95% O₂ and 5% CO₂. Each isolated muscle strip was put in an organ bath containing 5 ml Krebs solution, one end of the strip was fixed to a hook on the bottom of the bath, the other end was connected to an isometric force transducer. Muscle strips were prepared to 1 g loading tension, and subdivided randomly into BTX-A group (n=12), Atropine group (n=12), ACh + BTX-A group (n=12), ACh + Atropine group (n=12), BTX-A + ACh group (n=12). The contractile graph in motility of the muscle strips were simultaneously recorded with physiological experimental system.Results: (1) BTX-A (10 U/ml) reduced the tension, frequency and amplitude of spontaneous contractility of duodenal smooth muscle compared to incubation in Kreb's solution as control (P＜0.01). The inhibitory effect of BTX-A persisted more than 1 h. (2) The administration of atropine (1μmol/L) might completely suppress the tension, frequency and amplitude of spontaneous contractility of duodenal smooth muscle compared to control (P＜0.01), but the inhibitory effect of atropine persisted only 5 min, and then the duodenal spontaneous contractility was gradually recovered (compared with control P＞0.05). (3) The administration of ACh (100 μmol/L) enhanced the tension, frequency and amplitude of spontaneous contractility of duodenal smooth muscle compared to control (P＜0.01). Subsequently, the respective addition of BTX-A (10 U/ml) or atropine (1μmol/L) inhibited the contractile response to ACh (P＜0.01). (4) The administration of ACh (100μmol/L) did not induce the duodenal smooth muscle contractility after the inhibition with BTX-A (10 U/ml).Conclusion: The inhibitory effect of BTX-A on spontaneous contractility of duodenal smooth muscle in vitro suggests BTX-A prevents neurotransmitter vesicle containing ACh from fusion with pre-synaptic membrane and subsequent release. Furthermore, the inhibitory efficient time of BTX-A was longer than that of atropine. BTX-A also reduced duodenal muscle contractile response to external addition of ACh. After BTX-A treatment, ACh did no longer agitate duodenal muscle contractile response. These data demonstrate that BTX-A inhibits duodenal smooth muscle contractility suggesting not only inhibition of ACh release from cholinergic nerves but also inhibition of cholinergic muscarinic muscular transmission.