Figure 4. Enterochromaffin cell activation mediates Ca_V-dependent 5-HT release.

A. Representative 5-HT “biosensor” experiment. 5HT₃R-expressing HEK293 (mCherry, red) adjacent to an EC cell (GFP, green) for simultaneous Ca²⁺ measurements from EC cells and whole-cell current measurements from biosensor cells.

B. Epinephrine (EP, 1µM) or high extracellular K⁺ induced a Ca²⁺ response in EC cells that correlated with a large 5HT₃R current in biosensor cells. EP responses were inhibited by yohimbine (yoh, 5µM). The 5HT₃R agonist mCPBG (10µM) elicited a large biosensor current, but no EC cell Ca²⁺ response. When biosensor cells were moved away from EC cells, neither epinephrine nor K⁺ induced Ca²⁺ responses in GFP⁻ epithelial cells or biosensor currents, but mCPBG elicited a large 5HT₃R current. Scale bars: 0.6 Fura-2 ratio, 50s, 500pA.

C. EP-evoked Ca²⁺ responses and 5HT₃R currents were not affected by vehicle but were blocked by the TRPC4 inhibitor ML204 (10µM). The Ca_V inhibitor ω-agatoxin IVA (300nM) slightly reduced Ca²⁺ responses and abolished 5HT₃R currents. Scale bars: 0.3 Fura-2 ratio, 50s, 500pA.

D. AITC (150µM)-evoked Ca²⁺ responses and 5HT₃R currents were blocked by the TRPA1 antagonist A967079 (A96, 10µM). ω-agatoxin IVA (300nM) did not significantly affect Ca²⁺ responses, but abolished 5HT₃R currents. Scale bars: 0.1 Fura-2 ratio, 25s, 500pA.

E. Isovalerate (IVL, 200µM)-evoked Ca²⁺ responses and 5HT₃R currents. ω-agatoxin IVA (300nM) inhibited Ca²⁺ responses and abolished 5HT₃R currents. Scale bars: 0.1 Fura-2 ratio, 25s, 500pA.

F. Average agonist-evoked biosensor currents normalized to mCPBG-induced current (I_mCPBG). n=4 – 5 per condition. Data represented as mean ± sem. Responses to epinephrine (EP, blue), AITC (red), and isovalerate (IVL, green) in the presence of indicated antagonists. p<0.001 for control versus treatments. One-way ANOVA with post-hoc Tukey’s test.