Role of chloride channels in garbachol-induced contractions of longitudinal smooth muscle in rat isolated distal colon M.R.Chaluvadi, A.D. Corbett & D.L. Bovell, Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, G4 0BA.

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Activation of chloride channels has been proposed as one of various possible mechanisms underlying the agonist-induced depolarisation required to activate voltage-gated calcium channels in mammalian smooth muscle (Bolton, 1979; Van Helden, 1988). Evidence for the involvement of chloride channels in colonic contractions came from the work on muscarinic and tachykinin receptor-mediated contraction of rabbit longitudinal smooth muscle (Kolbel et al., 1998). The aim of the present study was to investigate the role of chloride channels in carbachol-induced contraction of longitudinal smooth muscle from rat distal colon.

Male Wistar rats (275 - 300 g) were stunned and killed by cervical dislocation and exsanguination. A segment of distal colon was excised, everted and the mucosa and submucosa removed from the muscularis. Muscle rings of about 1.0 cm length were mounted vertically under 1 g tension in 3 ml siliconised organ baths containing Krebs solution (composition, mM: NaCl, 118; KCl, 4.74; CaCl₂, 2.54; KH₂PO₄, 1.19; MgSO₄·7H₂O, 1.20; NaHCO₃, 25; glucose, 11) maintained at 37°C and bubbled with 95% O₂ and 5% CO₂. Preparations were allowed to equilibrate for at least 60 min prior to drug addition. Non-cumulative concentration-response curves (CRCs) to carbachol were carried out in all tissues. Tissues were then incubated either with C1^--depleted Krebs solution (NaCl was replaced in an equimolar fashion with Na gluconate) for 1 hr or with a chloride channel blocker for 30 min and CRCs to carbachol were repeated. The chloride channel blockers used were 5-nitro-2-(3-phenylpropylamino)- benzoate(NPPB, 50 µM), glibenclamide (200 mµM) and diphenylamine 2-carboxylate, (DPC, 300 µM) (Wangemann et al., 1986) ; all were dissolved in dimethylsulphoxide (DMSO). The contractile responses to 120 mM KCl were also tested before and after each treatment. Values are mean±s.e.m. Statistical analysis was carried out using Student's t-test, p<0.05 is significant.

Replacement of normal Krebs solution with C1^--depleted Krebs solution (n=7) reduced pEC₅₀ for carbachol from 6.22 ± 0.03 to 5.62 ± 0.05 (p < 0.001) and maximum response from 5.79 ± 0.04 to 2.90 ± 0.33 g (p < 0.001). Incubation with NPPB (n=9) also resulted in reduction of carbachol pEC₅₀ from 6.16 ± 0.03 to 5.58 ± 0.04 (p < 0.001) and maximum response from 6.41 ± 0.42 to 1.93 ± 0.25 (p < 0.001). Glibenclamide reduced both the carbachol pEC₅₀ and maximum response whereas DPC reduced the pEC₅₀ but not the maximum response. The contractile responses to KCl (n=4) were unaltered by C1^--depletion, NPPB and DPC. DMSO had no effect.

Thus, C1^- depletion and treatment with chloride channel blockers resulted in a reduction of contractile responses to carbachol but KC1-induced contractions were unaltered. These results provide evidence for the contribution of C1^- channels in agonist-induced excitation-contraction coupling in the longitudinal muscle layer of the rat colon.

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Van Helden D.F. (1988) J. Physiol., 401, 489-501.