Melatonin exerts dual, concentration‐dependent effects on intestinal pacemaker networks via MT2 receptor activation and redox pathways

BACKGROUND AND PURPOSE: Melatonin, primarily known for regulating circadian rhythms, also modulates gastrointestinal motility. However, its direct impact on network-forming interstitial cells of Cajal (ICCs), whose pacemaker activity enables flexible and coordinated gut movement, remains unclear. This study investigated the effect of melatonin on ICC electrical activity and micro-coordination in the murine ileum. EXPERIMENTAL APPROACH: Electrical activity was recorded from ileal musculature using a dialysis membrane-reinforced 8 × 8 microelectrode array (MEA), allowing visualisation of spatio-temporal coordination. Smooth muscle contraction and neuronal activity were suppressed using nifedipine and tetrodotoxin (TTX), respectively. RT-qPCR was used to assess melatonin receptor and antioxidant enzyme expression. KEY RESULTS: Functionally relevant concentrations of melatonin (10-20 μM) increased the frequency of electrical slow waves without affecting amplitude or propagation patterns. This excitatory effect was blocked by the melatonin receptor antagonists, luzindole and 4P-PDOT, and was associated with MT2 receptor expression. In contrast, supraphysiological concentrations (500 μM-1 mM) suppressed both frequency and amplitude and increased the incidence of expanding activity patterns. This inhibitory effect was luzindole-insensitive but was mimicked by 3-indolepropionic acid (IPA), a gut microbiota-derived indole with antioxidant properties. High-dose melatonin also upregulated Nrf2 and its downstream antioxidant enzymes. CONCLUSION AND IMPLICATIONS: Melatonin modulates ICC networks in a concentration-dependent manner: stimulation via MT2 signalling at functionally relevant levels and inhibition via antioxidant pathways at supraphysiological levels. These findings indicate that ICC networks contribute to melatonin responses and reveal distinct receptor-dependent and redox-dependent mechanisms regulating gastrointestinal pacemaker networks.