Melatonin modulates nitric oxide-regulated WNK-SPAK/OSR1-NKCC1 signaling in dorsal raphe nucleus of rats.

The sleep-wake cycle is regulated by the alternating activity of sleep- and wake-promoting neurons. The dorsal raphe nucleus (DRN) secretes 5-hydroxytryptamine (5-HT, serotonin), promoting wakefulness. Melatonin secreted from the pineal gland also promotes wakefulness in rats. Our laboratory recently demonstrated that daily changes in nitric oxide (NO) production regulates a signaling pathway involving with-no-lysine kinase (WNK), Ste20-related proline alanine rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1), and cation-chloride co-transporters (CCC) in rat DRN serotonergic neurons. This study was designed to investigate the effect of melatonin on NO-regulated WNK-SPAK/OSR1-CCC signaling in wake-inducing DRN neurons to elucidate the mechanism underlying melatonin's wake-promoting actions in rats. Ex vivo treatment of DRN slices with melatonin suppressed neuronal nitric oxide synthase (nNOS) expression and increased WNK4 expression without altering WNK1, 2, or 3. Melatonin increased phosphorylation of OSR1 and the expression of sodium-potassium-chloride co-transporter 1 (NKCC1), while potassium-chloride cotransporter 2 (KCC2) remained unchanged. Melatonin increased the expression of tryptophan hydroxylase 2 (TPH2, serotonin-synthesizing enzyme). The present study suggests that melatonin may promote its wakefulness by modulating NO-regulated WNK-SPAK/OSR1-KNCC1 signaling in rat DRN serotonergic neurons.