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EVIDENCE FOR TRPC CHANNEL INVOLVEMENT IN THE ACTIONS OF CHOLECYSTOKININ (CCK) ON GASTRIC AND MESENTERIC VAGAL AFFERENTS
MJ Beyak, C Williams
Gastrointestinal Diseases Research Unit (GIDRU), Queen’s University, Kingston, Ontario
The gut peptide CCK is known to excite gastric and mesenteric vagal afferents, and this action is important in the satiety effects of CCK. In isolated vagal afferent cell bodies, CCK results in an inward current with pharmacological and biophysical properties similar to TRPC channels. It is unknown whether these effects are relevant at the peripheral site of action of CCK, namely the afferent terminals. The following experiments were undertaken to examine the effects of TRPC channel blockade on GI afferent responses to CCK at the level of the nerve terminal.
METHODS: Segments of proximal stomach and jejunum were removed from CD1 mice and placed in an organ bath continuously perfused with carbogenated Kreb’s solution. Multiunit in vitro extracellular afferent nerve recordings were performed on vagal afferent fibres innervating the mouse stomach and on mesenteric nerve bundles innervating the mid jejunum. CCK (1-300nM) was applied and the effects of the TRP channel blockers ruthenium red (10µM), 2-APB (100µM) and SKF96365 (10µM) were examined. Immunohistochemistry for TRPC4 and TRPC5 subunits using specific rat anti human antibodies (Alomone) was performed on nodose ganglion sections. Immunoreactivity was visualized using goat anti-rat secondary conjugated to Cy3.
RESULTS: CCK induced a dose dependent increase in afferent firing rate in both gastric and mesenteric afferents. The EC50 was approximately 30nM. All TRP channel blockers used reduced the response to CCK as follows. For gastric vagal afferents (n=5) Ruthenium red 82±9.4%; 2-APB 78±9.7%; SKF96365 75±8.7%. For mesenteric afferents (n=6), ruthenium red 78±7.6%, 2-APB 82±7.3%, SKF96365 74±10.4%. The effects of these blockers were partially reversible on washout. Immunohistochemistry on nodose ganglion sections revealed the presence of widespread TRPC4 and TRPC5 immunoreactivity.
CONCLUSIONS: Blockade of TRPC channels at the level of the nerve terminal nearly abolishes the afferent nerve response to CCK. This is consistent with previous reports demonstrating the presence of a non selective cation current in nodose ganglion neurons. TRPC4 and TRPC5 are widespread in vagal afferent neurons. These results suggest that TRPC channels may be critical in the actions of CCK on gastrointestinal vagal afferents.