The muscarinic receptor agonist carbachol evokes Cl^– secretion in rat colonic epithelial cells by increasing intracellular Ca²⁺ (Greger et al. 1997). Previous studies from our laboratory have shown that carbachol-induced anion secretion in rat distal colon is predominantly through cystic fibrosis conductance regulator (CFTR) channels (Bellingham et al. 2002a), regulated by protein kinase phosphorylation and intracellular cAMP (Jia et al. 1997; Dahan et al. 2001). Here we have examined the involvement of protein kinases A and C (PKA and PKC) in carbachol-induced I_SC in rat distal colon.

The terminal colon (5 cm) was removed from male Wistar rats humanely killed by cervical dislocation. The mucosal portion was mounted in an Ussing chamber (area 0.5 cm²) containing Krebs solution (mM: NaCl 119, KCl 4.7, NaHCO₃ 24.8, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 2.5 and glucose 11.1) at 37°C gassed with 95 % O₂-5 % CO₂. Short circuit current (I_SC) was recorded and measurements taken from baseline to peak. All values are means ± S.E.M. and Student’s paired t test was used to test significance (P < 0.05).

Basal I_SC was 5.2 ± 3.2 µA cm^-2 (n = 20). Carbachol (100 µM basolateral) stimulated a triphasic response consisting of an increase in I_SC (phase 1, 8.7 ± 3.2 µA cm^-2) followed by a decrease towards baseline (phase 2, 4.9 ± 4.8 µA cm^-2) then a large increase (phase 3, 92.0 ± 14.3 µA cm^-2, n = 5) that decayed to a stable plateau. We have previously shown that phases 1 and 3 of the carbachol-induced I_SC are caused by Cl^– and HCO₃^– secretion (Bellingham et al. 2002b). In the presence of the PKA inhibitor H-89 (Sigma, 100 µM apical) phase 3 of the carbachol-induced I_SC was significantly reduced but phases 1 and 2 were unaltered (phase 1, 6.8 ± 2.0 µA cm^-2; phase 2, 1.1 ± 1.7 µA cm^-2; phase 3, 42.3 ± 6.5 µA cm^-2, P < 0.05, n = 5). In the presence of the adenylate cyclase inhibitor MDL 12330A (Sigma, 100 µM basolateral and apical), phase 3 of the carbachol response was also significantly reduced compared to control (phase 1, 7.0 ± 2.1 µA cm^-2; phase 2, -8.4 ± 0.8 µA cm^-2; phase 3, 31.2 ± 2.4 µA cm^-2, P < 0.05, n = 5). However, in the presence of the PKC inhibitor chelerythrine chloride (Sigma, 100 µM apical) the triphasic carbachol response was unaffected (n = 5).

The present results would suggest that carbachol-induced anion secretion in the rat distal colon occurs partly through activation of cAMP-dependent PKA, which is a potent activator of CFTR (Dahan et al. 2001) since both adenylate cyclase and PKA inhibition reduced the carbachol-induced I_SC. PKC inhibition had no significant effect on the carbachol-induced I_SC suggesting that PKC plays a negligible role compared to PKA in carbachol-induced ion secretion in rat distal colon.

MB is supported by a GCU studentship