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A robust method for evaluation of NANC transmission in human sigmoid colon muscle in vitro
1Academic Department of Surgery, GKT School of Medicine and Dentistry, King's College, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK. ignatius.tavares@kcl.ac.uk
Introduction:
Human tissues are notoriously difficult to work with, giving results that are quantitatively variable within and between studies. Hence, previous investigations of nonadrenergic, noncholinergic (NANC) relaxation in human colon muscle report both partial and complete inhibitions of the NANC response by specific competitive inhibitors of nitric oxide (NO) production. We have established a robust and reproducible model to examine the contribution of NO during NANC relaxation assay in human sigmoid colon muscle strips.
Methods:
Complete control curves to long-train, stepwise, frequency-dependent, continuous electrical field stimulation (EFS) relaxation using vertical platinum electrodes connected to a biphasic pulse train stimulator generated NANC responses in fresh human sigmoid colon circular muscle strips set up in Bennett baths. A second complete curve was generated on the same strip in the presence of specific drugs to determine the contribution of NO to NANC relaxation. Responses to NO were also generated in muscle strips. Results were fitted to the Hill equation.
Results:
The first and second frequency-response curves without test drugs could be fitted to the Hill equation, resulting in similar midpoint locations ([f](50)), maximal asymptotes (alpha), and midpoint slope (n) parameters. L-N(G)-nitro-arginine (L-NOARG), TTX, and haemoglobin produced a tonic contraction in the muscle strips. NANC relaxations to EFS were inhibited by L-NOARG (30-37%), TTX (56-62%), and haemoglobin (48-90%). NO relaxations were concentration dependently inhibited by haemoglobin. Haemoglobin was equipotent in mediating tonic contraction and inhibiting NO relaxation.
Discussion:
We established reproducible assays for human colon muscle strips by the generation of two complete dose-response curves to long-train EFS, thus enabling a "within-preparations" study. The results suggest that NO contributes but is not the sole mediator of relaxations to long-train EFS in human sigmoid colon muscle. Moreover, a basal production of NO may serve to regulate tone of human colonic muscle.