The Effect And Mechanism Of Botulinum Toxin Type A On Electrical Field Stimulation- And Substance Pinduced Contractility In Pyloric And Antral Smooth Muscle In The Rat In Vitro

Objective: The effect of botulinum toxin type A (BTX-A) on spontaneous contractility, electrical field stimulation (EFS)- and substance P (SP)-induced contractility in pyloric and antral smooth muscles was investigated.Methods: Sprague-Dawley rats weighing 200 to 250g were used in this study and fasted for 24 hours before operation. Stomach was quickly removed along greater curvature after the rat losed consciousness by hitting head. The stomach was dissected along the longitudinal axis and rinsed with Krebs bicarbonate buffer (in Mm: 118 NaCL, 2.5 CaCl₂, 1.2 MgSO₄, 24.9 NaHCO₃, 1.2 NaH₂PO₄, 4.7 KCL and 12.2 glucose), pH7.4 and oxygenated with 95% O₂ and 5% CO₂. The mucosa was removed from the pylorus and antrum. A pyloric and an antral smooth muscle strips were taken from per stomach. 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 suspended between argentine electrodes placed adjacent and parallel to the long axis of the muscle strip. Electrodes were connected to an electric stimulator. Muscle strips were prepared to 1 g loading tension, and subdivided randomly into control group (n=12), EFS treated group (n=12), BTX-A treated group (n=12), EFS+BTX-A treated group (n=12), SP treated group (n=12), SP+BTX-A treated group (n=12), SP+antagonist of NK₁ receptor of SP ([d-arg¹, d-phe⁵, d-trp^7.9,leu¹¹]-substance P) treated group (n=12). the contractile graph in motility of the muscle strips were simultaneously recorded with physiological experimental system.Results: (1) EFS enhanced the tension (P<0.01) and amplitude (P<0.05) in pyloric contractility and similarly did in antral contractile tension (P<0.01) and amplitude (P<0.01). (2) BTX-A inhibited EFS-induced tension (P<0.01) and amplitude (P<0.01) in pylorus and antrum. (3) BTX-A decreased tension (P<0.01) and amplitude (P<0.01) in pyloric and antral smooth muscle spontaneous contractility. (4) SP enhanced the amplitude (P<0.05) in pyloric contractility and in antral contractile tension (P<0.01) and amplitude (P<0.01), but the tension was undifferentiated(P>0.05) in pyloric contractile. (5) BTX-A inhibited SP-induced amplitude (P<0.01) in pyloric contractility and antral contractile tension (P<0.01) and amplitude (P<0.01), but BTX-A did not inhibit SP-induced the tension (P>0.05) in pyloric contractility. (6) Antagonist of NK₁ receptor of SP inhibited SP-induced tension (P<0.01) in pyloric contractility and antral contractile tension (P<0.01) and amplitude (P<0.01), but antagonist of NK₁ receptor did not inhibit SP-induced the amplitude (P>0.05) in pyloric contractility. Conclusion: (1) EFS enhances smooth muscle spontaneous contractility in pylorus and antrum. (2) BTX-A inhibits EFS-induced contractile response in pylorus and antrum. (3) BTX-A inhibits pyloric and antral smooth muscle spontaneous contractility. These results suggest BTX-A inhibits smooth muscle contractility in pylorus and antrum in vitro via inhibition of the fusion of synaptic vesicles containing neurotransmitters with the plasma membrane to undergo exocytosis and release, or preveals directly EFS effect. (4) SP enhances smooth muscle contractility in pylorus and antrum. (5) BTX-A inhibits SP-induced smooth muscle contractile response in pylorus and antrum. (6)Antagonist of NK₁ receptor of SP inhibits SP-induced smooth muscle contractile response in pylorus and antrum. These results suggest SP enhances smooth muscle contractility via binding NK₁ receptor on postsynaptic membrane, antagonist of NK₁ receptor of SP inhibits SP-induced smooth muscle contractility via blocking the binding of SP with NK₁ receptor on postsynaptic membrane. BTX-A inhibits smooth muscle contractility in pylorus and antrum in vitro via inhibiting the fusion of synaptic vesicles containing neurotransmitter with the plasma membrane to undergo exocytosis and release, or preventing SP from binding NK₁ receptor.