The Effect Of The High-dose GC On [Ca²⁺]_i In The Hypothalamus Neurons And The Study Of Its Mechanism

Glucocorticoid (GC), a steroid hormone mainly secreted by adrenal cortex, is an important component element of the hypothalamus-apendix-adrenal gland axle (HPA) and one of the most important stress reaction hormones. In previous study our group found that the 10-6M DEX, but not 10-9M DEX, induced a rapid change of the [Ca²⁺]_i in the cultured hypothalamus neurons. Further, in sepecial condition DEX was able to induce a rapid and significant decrease of the [Ca²⁺]_i in the hypothalamus neurons, but the mechanism was still unknown.ObjectIn this study we will investigate the pattern and feature of the [Ca²⁺]_i chang as the cultured hypothalamus neurons were exposed to the high-dose DEX; and illuminate the mechanism of [Ca²⁺]_i decrease in the neurons when it exposed to high-dose DEX, by blocking three main calcium-extruded molecules—PMCA, NCX and SERCA.Methods and procedure1. Neurons culture and identificationThe hypothalamus neurons were grown as decribed by Velasco, and some adjustment was made. During the course of neuron growth we took photoes for the neurons at different time, measured the axis length and the cell body area of the cultured neurons on the photoes with the software of Image-Pro Plus6.0, and then identified the cultured neurons with NF-200, calculated the percentage of the neuron.2. Decrease of the [Ca²⁺]_i induced by High-dose DEX in the hypothalamus neurons.There were 5 groups in the test, including 10-6M DEX stimulation, 10-9M DEX stimulation, 10-6M DEX stimulation after pretreatment with RU-486, D'hanks stimulation, DMSO stimulation (all groups in Ca²⁺-free circumstance). Instrumentation for [Ca²⁺]_i recording from a cluster of hypothalamic neurons using Fluo-3 based on the laser confocal scanning microscophy (LCSM). The fluorescence of Ca²⁺ in the neurons was recorded with the LCSM for 20-30 seconds before the neurons were exposed to D'hanks, DMSO, 10-6M DEX, 10-9M DEX; In the group of 10-6M DEX stimulation after pretreatment with RU-486, the neurons were pretreated with RU-486 for 60min before the neurons were exposed to 10-6M DEX, the fluorescence of Ca²⁺ was recorded with LCSM.3. Pathway of [Ca²⁺]_i decrease induced by high-dose DEX in the hypothalamus neurons.There were 5 groups in the test, including 10-6M DEX stimulation, 10-9M DEX stimulation, 10-6M DEX stimulation after pretreatment with alkaline external solution(pH 8.8), 10-6M DEX stimulation after pretreatment with KB-R7943 and 10-6M DEX stimulation after pretreatment with the thapsigargin. By comparison of the amplitude of [Ca²⁺]_i decrease induced by 10-6M DEX among the groups we could identify the effect of the inhibitors to the DEX-induced decease of [Ca²⁺]_i. In room temperature, the neurons were treated with 2μM thapsigargin for 5min, 20μM KB–R7943 for 10min, pH8.8 HBSS for 10min, then exposed to 10-6M DEX. The Ca²⁺ in the hypothalamus neurons were marked with Fluo-3/AM, and the change of fluorescence in the neurons were observed with LCSM.4. The endocellular signal pathway of [Ca²⁺]_i decrease induced by high-dose DEX in the hypothalamus neurons.There were 4 groups in the test, including 10-6M DEX, pretreatment of the neurons with pH8.8 HBSS+10-6M DEX stimulation, chelerythrine chloride(a inhibitor of PKC)+ 10-6M DEX sitmulation, MDL-12330A(a inhibitor of PKA ) + 10-6M DEX stimulation. The Ca²⁺ in the hypothalamus neurons were marked with Fluo-3/AM, and the change of fluorescence in the neurons were observed with LCSM.Result1. In the neurons culture we used the methods as described by Velasco and made some adjustments. The results demonstated that the hypothalamus neurons grew rapidly, the percentage of colloid was low; About 3-7 days after culture, the axis length and the cell body area of the neurons both got to the peak, and the synapse linkage among the neurons had not formed completely, the proportion of other kinds of cells was lower. So the nerons in this time were fit for observation and test. 2. In this study we used the Fluo-3/AM to mark the endocellular free Ca²⁺, successfully reproduced the previous study results, approved that in extracellular Ca²⁺-free circumstance 10-6M DEX could cause a rapid decrease of the [Ca²⁺]_i in the hypothalamus neurons, and measuered quantitively that the amplitude of the [Ca²⁺]_i decrease was 20.5±9.9% of the basal value. The DEX-induced decrease of [Ca²⁺]_i could be blocked by the blocking of the iGR—RU486, The result showed that iGR was involved in the decrease of [Ca²⁺]_i induced by 10-6M DEX in the hypothalamus neurons.3. Exposure of neurons to the alkaline external solution (pH=8.8) for 10 min at room temperature, the DEX-induced decrease of [Ca²⁺]_i was completely abolished, indicating that GC mediated the decrease of [Ca²⁺]_i by activating the PMCA. Meanwhile, inhibition of SERCA and NCA has no effect on this phenomena, indicating that NCX and SERCA did not involve in GC-induced decrease of [Ca²⁺]_i .4. In this study we use selectly chelerythrine chloride and MDL-12330A to block PKA and PKC. After PKC was blocked but not PKA, the DEX-induced [Ca²⁺]_i decrease was completely abolished, indicating the endocellular signal pathway of PKC involved in this course.ConclusionThe result demonstrate that in extracellular Ca²⁺-free circumstance 10-6M DEX could cause a rapid [Ca²⁺]_i decrease of 20% in the hypothalamus neurons, which was mediated by iGR, by activating the PMCA to extrude the Ca²⁺ from the endochylema, and PKC involved in this course.