Evidence for the involvement of chloride channels in carbachol-induced contractions of longitudinal smooth muscle in mouse isolated distal colon M.R.Chaluvadi, A.D. Corbett & D.L. Bovell, Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, G4 0BA.	Print Abstract Search PubMed for: Chaluvadi MR Corbett AD Bowell DL

Activation of chloride channels in smooth muscle cells is one of the mechanisms proposed to underlie the depolarization which leads to increased entry of calcium through voltage-dependent calcium channels and results in smooth muscle contraction (Bolton, 1979; Van Helden, 1988). We have shown previously that chloride channels are involved in carbachol-induced contractions of rat colonic smooth muscle (Chaluvadi et al., 2003). In this study, we have examined the possible involvement of chloride channels in carbachol-induced mechanical responses of longitudinal smooth muscle from the mouse distal colon.

A segment of distal colon was excised from DBA/2 mice (27 - 32 g) and the contents were flushed gently with Krebs solution (composition, mM: NaCl, 118; KCl, 4.74; CaCl₂, 2.54; KH₂PO₄, 1.19; MgSO₄.7H₂O, 1.20; NaHCO₃, 25; glucose, 11). Colonic rings of about 1.0 cm length were mounted vertically under 1 g tension in 3 ml siliconised organ baths containing Krebs solution maintained at 37°C and bubbled with 95% O₂ and 5% CO₂. Preparations were allowed to equilibrate for approximately 60 min and non-cumulative concentration-response curves (CRCs) to carbachol were carried out in all tissues. Tissues were then incubated either with Cl^--depleted Krebs solution (NaCl was replaced in an equimolar fashion with Na gluconate) for 60 min 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, 30 µM) glibenclamide (200 µM) and diphenylamine 2-carboxylate (DPC, 300 µM); 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 and
p < 0.05 is considered significant.

Replacement of normal Krebs solution with Cl^--depleted Krebs solution (n=18) reduced the pEC₅₀ for carbachol from 6.31 ± 0.08 to 5.70 ± 0.04 (p < 0.001) and the maximum contractile response from 1.90 ± 0.14 to 0.76 ± 0.08 g (p < 0.001). Incubation with NPPB (n=8) also resulted in reduction of carbachol pEC₅₀ from 6.42 ± 0.08 to 5.80 ± 0.05 (p < 0.001) and maximum response from 1.54 ± 0.24 to 0.8 ± 0.12 (p < 0.05). Glibenclamide reduced both pEC₅₀ and maximum response for carbachol whereas DPC reduced the pEC₅₀ but not the maximum response. The contractile responses to KCl (n=5) were unaltered by Cl^- depletion, NPPB and DPC.

The reduction of contractile responses to carbachol but not to KCl with Cl^- depletion and with chloride channel blockers would support the hypothesis that Cl^- channels are involved, at least to some extent, in carbachol-induced contractions of mouse colonic longitudinal smooth muscle. Further investigations are required to determine which subclass of Cl^- channels is involved in these contractions in rodents.

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