Effects Of Compounds YZG-330 And YZG-331 On Chloride Ion Flow In Neurons

Insomnia is an extremely common symptom in modern society. Three generation of hypnotics are based on GABAA receptor-mediated inhibitory processes. The first generation of hypnotics (barbiturates) prolonged the opening times of the chloride channel, the second generation (benzodiazepines) improved the frequency of the chloride channel opening, and the third (imidiazepines and cyclopyrrolones) can both improved the frequency and prolonged the opening times. We have demonstrated that YZG-330 and YZG-331 which are new modifiers of N6-(4-hydroxybenzyl) adenine riboside that can enhance pentobarbital-induced sleep behaviors. The content of the study include two parts:Part 1:The effects of YZG-330 and YZG-331 on chloride (Cl-) exflux in primary cultured cerebellar granule cells. Part 2:The effects of GAB A, pentobarbital sodium, diazepam, clonazepam, YZG-330 and YZG-331 on chloride (Cl-) influx in mouse cortex.Part 1:We used the chloride-sensitive fluorescent probe N-(ethoxycarbonylmethyl)-methoxyquinolinium bromide (MQAE) as a tool to estimate the chloride exflux in cells. GABA can enhance the rate of chloride exflux in a concentration-dependent manner, the chloride exflux by YZG-330 (3μM,10 μM and 30 μM) was increased to 149%(P<0.05),152%(P<0.05)and 185% (P<0.01) of control, respectively, and the chloride exflux by YZG-331 (3 μM,10 μM and 30 μM) was increased to 129%(P<0.01)、147%(P<0.01) and 156%(P<0.001) of control, respectively. The GABAAR antagonists Picrotoxin, bicuculline, and flumazenil partly blocked YZG-330 -enhanced chloride exflux. Bicuculline and flumazenil partly blocked YZG-331-enhanced chloride exflux. These data indicate that the effect of chloride exflux stimulated by YZG-330 and YZG-331 may be mediated by GABAA receptor.Part 2:The chloride-sensitive fluorescent indicator MQAE (N-(6-methoxyquinolyl) acetoacetyl ester) has been used for determination of the chloride influxes in cells from mice cortex. Loading of the mice cortex cells with MQAE occurs by transmembrane diffusion. In physiological conditions, the extracellular chloride ion concentration is higher than the intracellular, when the chloride channel is open, the chloride ion fluxs from extracellular to intracellular. GABA, pentobarbital sodium, diazepam and clonazepam can enhance chloride influx into cortex cells in a concentration-dependent manner. And the onset time of GABA is the shortest during the four drugs that target the r-aminobutyric acid GABAA receptor, the effect was shown when at the time GABA was given. The duration of the effect of pentobarbital sodium was the longest among the four drugs, and the effect can last for 40 min. YZG-330 show the same manner as GABA, and onset time of YZG-331 shows at the 10th min.