Sedative And Hypnotic Mechanisms Of NHBA

The rhizomes of Gastrodia elata have been extensively used since ancient time in China for treatment of insomnia, pain and convulsive illnesses. Collaborated with Professor Shi Jiangong in department of Phytochemistry, we found compound N6-(4-hydroxybenzyl) adenine riboside (NHBA), isolated from Gastrodia elata. Previous study showed that NHBA significantly decreases spontaneous locomotor activity and potentiates hypnotic effect of sodium pentobarbital in mice; NHBA significantly decreases wakefulness time and increases NREM sleep time, which suggests that NHBA exerts sedative and hypnotic effects. Previous study on the mechanisms of NHBA inducing sedative and hypnotic effects mainly included:NHBA showed affinities and intrinsic activity with adenosine A1 and A2A receptors by radioligand binding studies; NHBA increases the contents of adenosine in striatum, brain stem and thalamus by a HPLC-UV analysis; Immunohistochemical studies showed that NHBA increases c-Fos expression in the ventrolateral preoptic area (VLPO); GABAA receptor antagonist picrotoxin has no antagonism effect on hypnotic activity of NHBA. This study aimed to further explore possible sedative and hypnotic mechanisms of NHBA.Ca2+/calmodulin-dependent protein kinase â…¡ (CaMKâ…¡) is a serine/threonine kinase found in essentially all neuronal compartments. Activation of intracellular CaMKII signaling in the PPT promotes wake and suppresses sleep. The phosphorylation of CaMKII was examined by Western blotting. The results showed that p-CaMKII expression significantly decreased in mouse hypothalamus after the intraperitoneal administration of NHBA (5 mg/kg). The sedative activity of KN93, an inhibitor of CaMKII phosphorylation, was investigated by recording the spontaneous locomotor activity in mice. The results showed that KN93 (0.1 nmol/mouse, i.c.v.) could inhibit the locomotor activity of mice by 58.8%. The result of Western blot analysis showed that KN93 (0.1 nmol/mouse, i.c.v.) and NHBA (0.1 nmol/mouse, i.c.v.) significantly decrease the p-CaMKII expression in mouse hypothalamus. These findings indicate NHBA might exert sedative and hypnotic effects by inhibiting the phosphorylation of CaMKII in mouse hypothalamus. The activities of phosphatase PP1/PP2B and the expression of cAMP-dependent protein kinase (PKA) and Synapsin I which were related to CaMKII signaling were then determined by phosphatase activity assay and Western blotting. The results showed that NHBA (5 mg/kg, i.p.) significantly increased PP1 activity by 16% in mouse hypothalamus, but did not affect PP2B activity. NHBA (5 mg/kg, i.p.) reduced p-PKA expression level in mouse hypothalamus and NHBA (0.1 nmol/mouse, i.c.v.) significantly suppressed the phosphorylation of Synapsin I in mouse hypothalamus.GABA is the most important inhibitory amino acid in the CNS, whereas Glu is the most abundant excitatory neurotransmitter in the CNS. The balance of GABA and Glu plays a role in sleep-wake regulation. The contents of extracellular GABA and Glu in different mouse brain slices were measured by HPLC-EC after pre-column derivatization with OPA. The results showed that NHBA (10-6 and 10-5 mol/L) significantly decreased the extracellular Glu level by 32.8% and 34.8% in mouse hypothalamus slices, respectively. NHBA (10-6 and 10-5 mol/L) significantly decreased the extracellular Glu level by 24.1% and 27.6% in mouse cortex slices, respectively. But NHBA (10-6 and 10-5 mol/L) had no significant effect on extracellular GABA level in mouse hypothalamus or cortex slices. Based on these findings, it suggests that decreased Glu contents may involved in sedative and hypnotic effects of NHBA.Monoamine neurotransmitters play an important role in sleep-wake regulation. The contents of 5-HT, DA and their metabolites in different mouse brain slices were detected by HPLC-EC. The results showed that NHBA (10-6 and 10-5 mol/L) decreased the extracellular 5-HT level by 24.0% and 27.4%, and increased the extracellular 5-HIAA level by 21.9% and 24.1% in mouse cortex slices, respectively, but there was no statistically significant compared with vehicle control. There was a decreasing trend in the contents of 5-HT in mouse hypothalamus slices after treatment of NHBA (10-5 mol/L), but the 5-HIAA contents were not significantly affected by NHBA.In conclusion, NHBA could significantly reduce phosphorylation of PKA expression, increase PP1 activity, then significantly inhibit the phosphorylation of CaMKII, further decrease the expression of p-Synapsin I and reduce extracellular Glu contents in mouse hypothalamus, which might the mechanism of NHBA exerting sedative and hypnotic effects. Further studies are needed to confirm this mechanism.