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

Abstract

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.

Keywords: Dorsal raphe nucleus; Melatonin; Nitric oxide; Sodium potassium chloride cotransporter; WNK.

Fig. 1. Distribution of melatonin receptors (MT) and tryptophan hydroxylase 2 (TPH2)-containing serotonergic neurons in the dorsal raphe nucleus (DRN).

Fluorescent images of melatonin receptors (including both MT1 and MT2, red) and TPH2-containing neurons (TPH, green). The bottom right images was taken from the rat brain atlas. The red square indicates the location of DRN. Scale bars: 200 μm.

Fig. 2. Effect of melatonin on neuronal nitric oxide synthase (nNOS) expression in dorsal raphe nucleus (DRN) neurons.

(A) Western blots showing nNOS expression in DRN serotonergic neurons following melatonin treatment (0.1, 1, and 10 μM) for 4 h. (B) Averaged optical density of nNOS expressions after 4 h melatonin incubation, expressed relative to control. Each column represents the mean and SEM of data from 12 experiments. *p < 0.05 compared with control.

Fig. 3. Effect of melatonin on expression of with-no-lysine kinases (WNKs) in dorsal raphe nucleus (DRN) neurons.

(A) Western blots showing changes in WNK1, 2, 3, and 4 expression in DRN slices after melatonin treatment (0.1, 1, and 10 μM) for 4 h. (B–E) Averaged optical density of WNK 1–4 expressions after 4 h melatonin incubation, expressed relative to control. Each column represents the mean and SEM of data from 13–15 experiments. *p < 0.05 compared with control.

Fig. 4. Effect of melatonin on Ste20-related proline alanine rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1) expression in dorsal raphe nucleus (DRN) neurons.

(A) Western blots showing phospho- and total SPAK and OSR1 expression in DRN slices after melatonin treatment (0.1, 1, and 10 μM) for 4 h. (B–E) Averaged optical density of phospho- and total-forms of SPAK/OSR1 expressions after 4 h melatonin incubation, expressed relative to control. Each column represents the mean and SEM of data from 14–15 experiments. **p < 0.01, *p < 0.05 compared with control.

Fig. 5. Effect of melatonin on the expression of sodium-potassium-chloride co-transporter 1 (NKCC1) and potassium-chloride co-transporter 2 (KCC2) in dorsal raphe nucleus (DRN) neurons.

(A) Western blots showing NKCC1 and KCC2 expression in DRN slices with melatonin treatment (0.1, 1, and 10 μM) for 4 h. Averaged optical density of NKCC1 (B) and KCC2 (C) expression after 4 h melatonin incubation, expressed relative to each control level. Each column represents the mean and SEM of data from 10–12 experiments. ***p < 0.001, *p < 0.05 compared with control.

Fig. 6. Effect of melatonin on tryptophan hydroxylase 2 (TPH2) expression in dorsal raphe nucleus (DRN) neurons.

(A) Western blots showing TPH2 expression in DRN neurons with melatonin treatment (0.1, 1, and 10 μM) for 4 h. (B) Averaged optical density of TPH2 expression, at 4 h melatonin incubation, expressed relative to control level of TPH2. Each column represents the mean and SEM of data from 15 experiments. *p < 0.05 compared with control.

Fig. 7. Schematic diagram showing a possible suggestion of the signaling pathways by which melatonin modulates NO-regulated WNK-SPAK/OSR1-CCC signaling during the sleep-wake cycle in rat DRN serotonergic neurons.

NO, nitric oxide; WNK, with-no-lysine kinase; SPAK, Ste20-related proline alanine rich kinase; OSR1, oxidative stress response kinase 1; CCC, cation-chloride co-transporters; DRN, dorsal raphe nucleus; nNOS, neuronal nitric oxide synthase; NKCC1, sodium-potassium-chloride co-transporter 1; KCC2, potassium-chloride co-transporter 2.