Effects of H2O2 at rat myenteric neurones in culture

Oxidants, produced e.g. during inflammation, alter gastrointestinal functions finally leading to diarrhoea and/or tissue damage. There is only scarce information about the action of oxidants on enteric neurones, which play a central role in the regulation of many gastrointestinal processes. Therefore, the effect of an oxidant, H2O2, on cultured rat myenteric neurones was studied with the whole-cell patch-clamp and imaging (fura-2) techniques. H2O2 (5 mmol/l) induced an increase in the cytosolic Ca2+ concentration. Both an intracellular release via IP3 and ryanodine receptors as well as a Gd3+-sensitive Ca2+ influx contributed to this response. Measurement of the membrane potential revealed that the neuronal membrane hyperpolarized by 11.3 ± 0.8 mV in the presence of H2O2. Inhibition of Ca2+-dependent K+ channels prevented this hyperpolarization. Voltage-clamp experiments revealed a second action of the oxidant, i.e. a strong inhibition of the fast Na+ current responsible for the generation of action potentials. This effect seemed to be mediated by the hydroxyl radical (·OH), as Fe2+ (100 µmol/l), which leads to the generation of this radical from H2O2 via the Fenton reaction, strongly potentiated the action of an ineffective concentration (100 µmol/l) of the oxidant. Protein phosphorylation/dephosphorylation seems to be involved in the mechanism of action of H2O2, as the protein phosphatase inhibitor calyculin A (100 nmol/l) strongly reduced the inhibition of Na+ current by H2O2. This effect was mimicked by the protein phosphatase 2A specific inhibitor endothall (100 nmol/l), whereas the PP1 blocker tautomycin (3 nmol/l) was less effective. These results suggest that H2O2 reduces the excitability of rat myenteric neurones by a change of basal membrane potential and an inhibition of Na+ currents.