Differential sensitivity of gastric and small intestinal muscles to inducible knockdown of anoctamin 1 and the effects on gastrointestinal motility.

Abstract

Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium-activated chloride conductance anoctamin-1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity. Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca2+ -activated Cl- conductance (CaCC), and constitutive knockout of the channel protein anoctamin-1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin-1 channels. Furthermore, the significance of anoctamin-1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell-specific knockdowns of anoctamin-1 in ICC (KitCreERT2/+ ;Ano1tm2jrr/+ ) in GI muscles. The recombination efficiency of KitCreERT was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin-1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin-1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin-1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin-1 knockdown and weak efficacy of anoctamin-1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin-1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity.