OPTOGENETIC STIMULATION OF INTESTINAL ENTEROENDOCRINE CELLS CAUSES EPITHELIAL CHLORIDE SECRETION THROUGH NEUROGENIC AND PARACRINE MECHANISMS

BACKGROUND: Enterochromaffin cells (ECs) are specialized gastrointestinal (GI) epithelial cells that transduce luminal forces and chemicals into release of signal molecules including serotonin (5-HT) to alter GI function including mucosal ion transport. To determine the role of ECs in ion transport, direct activation of ECs is required to localize the downstream effects to ECs versus other cell types.
AIM: Determine whether optogenetic activation of ECs modulates intestinal ion transport.
METHODS: Full-thickness ileum preparations of EC^ReaChR (Tph1^Cre-ERT/+::Rosa26^{LSL-ReaChR-mCitrine/+}) mice treated with tamoxifen (50mg/kg p.o.) four consecutive days prior to tissue harvest were either cryosectioned and immunostained for 5-HT or mounted in 4mL Ussing chambers with Kreb’s solution (NKS). The mucosal side was intermittently stimulated using a fiber optic cable (625-nm LED, Prizmatix) at 2Hz, 40% duty cycle for 20 cycles. Veratridine (30µM), a sodium channel opener, and Substance P (0.3 µM), a neurokinin receptor agonist, were also used to stimulate I_sc. TTX (1µM) was used to block neurotransmission and ondansetron (1µM) and GR113808 (30nM) were used to block serotonin receptors. Responses were measured as change in short-circuit current (ΔI_sc) = I_sc_peak − I_sc_baseline (µA/cm²) with differences detected with Wilcoxson ranked sum tests.
RESULTS: 57% of 5-HT-immunoreactive (ir) cells expressed mCitrine and 96% of mCitrine+ cells were 5-HT-ir illustrating the recombination efficiency and specificity of EC^ReaChR expression. Light stimulation increased I_sc in the ileum from EC^ReaChR mice but did not change I_sc from negative control mice that lacked either Tph1^Cre-ERT or Rosa26^{LSL-ReaChR-mCitrine} transgenes suggesting that light selectively stimulated ReaChR expressed in ECs. Removal of Cl^- from NKS, blocked light-induced I_sc (79 ± 20 µA/cm², NKS, 3.2 ± 0.6 µA/cm², Cl^- free, n=17, P<0.001). While TTX blocked the neurogenic stimuli of veratridine (37 ± 6 µA/cm², veratridine, 3 ± 1 µA/cm², veratridine+TTX, n = 11-23, P<0.001) and Substance P (42 ± 13 µA/cm², Substance P, 9 ± 3 µA/cm² Substance P+TTX, n = 6, P<0.05), TTX reduced but did not fully block light-induced I_sc (73 ± 20 µA/cm² control, 21 ± 5 TTX µA/cm², n=18, P<0.0001). 5-HT receptor antagonists (5HTRa) had no effect on light-induced I_sc (23 ± 6 µA/cm², control, 17 ± 3, 5HTRa, n = 8, P>0.05). However, when TTX and 5HTRa were delivered together, the light induced I_sc was completely blocked (19 ± 5 µA/cm², control, 1.3 ± 0.4 µA/cm², TTX+5HTRa, n = 9, P<0.01).
CONCLUSIONS: Optogenetic stimulation of ECs represents a novel approach to understand mechanisms of EC-initiated ion transport. Optogenetic stimulation of ECs induce mucosal chloride secretion via a combination of neurogenic and paracrine signaling from ECs to secretory epithelial cells.
Supported by NIH AT010875, DK123549, DK129315 and NS118790