Objective Our previous studies has demonstrated that botulinum toxin type A (BTX-A) intrasphincteric injection through guide cannula in free move rats dose-dependently caused an inhibition of slow wave in amplitude but not in frequency, a diminishment of spike activity in amplitude and spike burst of pyloric myoelectrical activity, and a reduction of substance P (SP) content and cell number of SP-immunoreactivity in pylorus.These data suggest that botulinum toxin may inhibit not only the acetylcholine (ACh) release, but also SP release from the autonomic and enteric nervous terminals in pylorus.SP, a major excitatory non-cholinergic neurotransmitter, depolarizes the membrane potential and thus induces contraction in gastrointestinal smooth muscle.It is an undecapeptide belonging to the tachykinin family and can induce strong contractions in pylorus via neurokinin 1 receptor (NK1R), the preferred receptor for SP.However, no experimental evidence that BTX-A inhibits the pyloric contractile response induced by SP release from nervous terminals or SP dosed has been documented until now.The aim of this study was to determine the inhibitory effect of BTX-A on pyloric smooth muscle contractile response to substance P in rats in vitro.Methods The effect of BTX-A on contractile tension and frequency of pyloric muscle strips incubated with Krebs bicarbonate buffer with electric field stimulation (EFS, 100 V, 4 Hz, 0.5 ms pulse width duration) were detected after atropine (1 μM) abolishing cholinergic action and compared with the effect of [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-SP (APTL-SP), a NK1R antagonist.The pyloric smooth muscle contractile response to SP (1 μM) added at intervals of 30 min under a range dose of BTX-A (from 4 to 10 U/mL) incubation for 4 h was measured, and the distributions of SP and NK1R cells in pylorus were examined by immunohistochemistry.Results BTX-A (10 U/mL) as APTL SP (1 μM) further decreased EFS-induced pyloric contractile tension (from 1.524±0.029 to 1.362±0.027, P < 0.01) and frequency (from 3.798±0.042 to 2.250±0.067, P < 0.001) after atropine and then repeat APTL-SP administration does not act on contractility.Pyloric smooth muscle strips incubated with different concentrations of BTX-A gradually and dose-dependently suppressed SP-induced contractility.After 4 hour, incubation with BTX-A at 4 U/mL and 10 U/mL respectively decreased SP (1 μM)-induced contractions by (26.64±5.12)% and by (74.92±3.62)%.SP immunoreactive (IR) nerve fibers were found in myenteric nerve plexus of circular and longitudinal muscles and densely near to the enteroendocrine cell (ECI) of pyloric gland in mucosa and submucosa.NK1R-IR expressions were observed on cytomembrane of circular and longitudinal muscle cells, and both on cytomembrane and in cytoplasma in pyloric gland cell of mucosa and submucosa, Conclusion BTX-A, similar to NK1R antagonist further inhibits EFS-induced contractile responses after atropine blocking muscarinic cholinergic receptors and then NK1R antagonist administration does not act on suggesting inhibition of SP release from pyloric nervous terminals, in which SP through NK1R innervates pyloric muscle for regulation of gastric emptying.BTX-A dose-and time-dependently directly abolishes SP-induced pyloric smooth muscle contractility suggesting an additional site for BTX-A in regulating muscle contractility, besides the classical effect of acting on presynaptic neurons to reduce neurotransmitters release, though the exact cellular mechanism of BTX-A inhibiting SP-induced contractions was not detailed in the current study.