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PHARMACOLOGICAL CHARACTERIZATION OF MURINE LOWER ESOPHAGEAL SPHINCTER (LES) RELAXATION
S Carmichael, J Sauvé, DV Miller, Y Zhang, WG Paterson
GI Diseases Research Unit, Queen’s University, Kingston, Ontario
AIM: Because of the availability of genetic knockouts, the mouse has been increasingly utilized as a model for studying GI motility. To date there is limited data available on the physiology of the LES motor function in normal or mutant mice.
METHODS: We characterized pharmacologically the putative cholinergic, nitrergic and purinergic innervation of the LES circular smooth muscle from CD-1 mice using isometric tension recording from isolated LES rings. The magnitude of LES relaxation (LESR) was expressed as a percent of maximal LESR induced by 1 µM isoproterenol. Preliminary experiments were also conducted in W/Wv mutant mice, which lack intramuscular interstitial cells of Cajal (ICCs).
RESULTS: Rings of LES muscle from CD-1 mice developed spontaneous tone shortly after suspension in the tissue bath. Electrical field stimulation of intrinsic nerves (100V, 10 Hz, 0.5 ms pulse duration, 5 sec) evoked a reproducible LESR (45.4 ± 6.8 %; n=30) followed by a post-relaxation contraction. This relaxation was abolished by application of the nitric oxide synthase antagonist N(G)-nitro-L-arginine methyl ester (L-NAME; 100 µM). In the presence of L-NAME, cumulative application of atropine (3 µM) restored LESR to a level not significantly different than control (55.8 ± 8.4%). Furthermore, atropine virtually abolished the post-relaxation contraction. Subsequent application of apamin (300 nM), a small conductance Ca2+-activated K+ channel blocker, significantly attenuated the LESR to 20.4 ± 4.2% (p<0.05) suggesting a purinergic component to LESR. Application of the neurokin-2 receptor antagonist MEN-10376 (3 µM) or Substance P tachyphylaxis had no consistent effects on the post-relaxation contraction. LES rings from W/Wv mutant mice developed significantly less spontaneous tone and weaker KCl-induced contraction than strips from control mice. When tone was restored by pre-application of carbachol in LES rings from W/Wv mutant mice, electrical field stimulation induced relaxation that was abolished by L-NAME.
CONCLUSIONS: These experiments indicate that in the mouse LES both nitrergic and purinergic inhibitory neurotransmission is present. Acetylcholine appears to be the major excitatory neurotransmitter. In ICC deficient mice, LES muscle contractility is impaired, but nitrergic relaxation is intact, suggesting that ICCs are not required for nitrergic neurotransmission.
Supported by CIHR