AB310. SPR-37 Cholinergic excitatory motor responses in the colon are mediated through the calcium-activated chloride conductance Ano1

OBJECTIVE: There is a growing body of evidence that gastrointestinal smooth muscle excitability is regulated by several different classes of interstitial cells [interstitial cells of Cajal (ICC) and PDGFRα(+) cells] that are electrically coupled to SMC. Thus, ‘myogenic’ activity results from the integrated behavior of the SMC/ICC/PDGFRα(+) cell (SIP) syncytium. Inputs from excitatory and inhibitory motor neurons are required to produce the complex motor patterns of the gut and coordinate GI motility. Motor neurons innervate these three cell types in the SIP syncytium, and receptors, second messenger pathways, and ion channels in these cells mediate postjunctional responses. Cholinergic neurotransmission in GI muscles from several species has long been thought to be dependent upon activation of a non-selective cation conductance in smooth muscle cells and the molecular candidates for mediating cholinergic excitation have been reported to be the transient receptor protein channels Trpc4 and Trpc6. However, we have shown that cholinergic responses in the GI tract involve ICC and in their absence these motor responses are greatly diminished or absent. We sought to determine the conductance(s) responsible for cholinergic motor responses in the colon. METHODS: Cre-LoxP recombinase technology was utilized to determine the role of the calcium-activated chloride conductance, Ano1 in post-junctional motor responses in the mouse colon in a cell-specific manner (Kit(+) ICC). c-Kit(CreERT2/+) (Kit-Cre) mice and Ano f/f mice were crossed to generate c-Kit(CreERT2)(/+); Ano1(f/f) (mutants) and c-Kit(CreERT2/+); Ano1(f/+) (controls) animals that were subsequently treated with tamoxifen to induce Cre recombinase expression in ICC. Confocal microscopy was used to determine the cell type Cre expression was switched on. Intracellular microelectrode recordings were performed to determine changes in post-junctional neural responses to nerve stimulation in c-Kit(CreERT2/+) (Kit-Cre) mice and Ano(f/f) mice to generate c-Kit(CreERT2/+); Ano1(f/f) and c-Kit(CreERT2/+); Ano1(f/+) animals mice treated with tamoxifen using electrical field stimulation (EFS). RESULTS: Knock down of Ano1 in Kit(+) ICC using the Cre/Lox P technology caused a marked reduction or loss of excitatory junction potentials (EJPs) in colonic muscles of c-Kit(CreERT2/+); Ano1(f/f) in response to EFS compared to c-Kit(CreERT2/+); Ano1(f/+) controls. These EJPs were atropine sensitive providing evidence that cholinergic muscarinic receptors were responsible for these excitatory responses. CONCLUSIONS: These data provide evidence that Ano1 expressed in ICC and not smooth muscle cells is critically important for cholinergic excitatory post-junctional neural responses in colonic muscles. FUNDING SOURCES: NIH PO1 41315 and RO1 DK57236