Oxidants, produced e.g. during inflammation, alter gastrointestinal functions finally leading to diarrhea 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 (n = 30) in the presence of H2O2. Inhibition of Ca2+-dependent K+ channels by the BK-specific inhibitor paxilline (10 µmol/l) or by 100 µmol/l TPA (a broad inhibitor of Ca2+-dependent K+ channels) prevented this hyperpolarization. This indicates that the increase in the cytosolic Ca2+ concentration likely activates BK channels, which carry an outward K+ current to hyperpolarize the neuronal membrane.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. Inhibition of protein phosphorylation by staurosporine (1 µmol/l), a protein kinase inhibitor, prevented the effect of a subsequent administration of the oxidant. Vice versa, the protein phosphatase (PP) inhibitor calyculin A (100 nmol/l) strongly reduced the inhibition of Na+ current by H2O2. This effect was mimicked by the PP2A specific inhibitor endothall (100 nmol/l), whereas the PP1 blocker tautomycin (3 nmol/l) was less effective. However, none of the inhibitors used was able to significantly change the basal amplitude of the inward sodium current. Changes appeared only after administration of the oxidant, meaning that a preconditioning of the channels or regulatory proteins, e.g. an oxidation of thiol groups by H2O2, is necessary for this action. This was confirmed by the observation that in the presence of the reduced form of GSH (3 mmol/l), H2O2 was unable to inhibit inward sodium currents. These results suggest that H2O2 might act via a shift of the equilibrium between protein phosphorylation and dephosphorylation involved in the regulation of Na+ currents in rat myenteric neurones. The consequence is an inhibition of the sodium currents responsible for the generation of action potentials. Together with the hyperpolarization, H2O2 should likely reduce the ongoing inhibitory tone of the ENS on the gastrointestinal muscle, which might result in a hypercontractility leading to diarrhea.
Verknüpfung zu Publikationen oder weiteren Datensätzen
