β-renergic Receptor Agonist Mediated Signal Transduction Mechanism Of Na⁺, K⁺-ATPase α₂ Subunit In Astrocyte

Na⁺,K⁺-ATPaseα₂,which is loacated in central nerve system through hybridization in situ and immunohistochemistry experiements is a subunit of Na⁺, K⁺-ATPase.And it is only expressed in astrocyte and a few immature neuron of ripe mice.Na⁺,K⁺-ATPase constituted byαandβsubunit not only maintains the concentration gradient of sodium ion and kalium ion of external and internal cells but also has specific function.Recently,it has been demonstrated that amygdala and piriform cortex would degenerate if Na⁺,K⁺-ATPaseα₂ is deprived from the mice.At the same time,a research demenstrated the mice can display more fear and anxious.Glial cells can be classified astrocytes,oligodendrocytes and microglias.Among that,astrocytes are the most widely contributed ones in the brains of mammals. Astrocytes extend a large number of processes and filopodias and they cover most of the perimeter of microvessels.They are coupled to each other by gap junctions,which is narrow,allowing passage of low molecular constituents(ions,metabolites) from cell to cell.In contrast to neurons,astrocytes are non-excitable cells,meaning that they do not generate action potentials.They were previously regarded as only structurally supportive cells,but within the last decades it has been found that they exert a number of functions of key importance in the central nervous system.Transactivation of Epithelial growth factor receptor(EGFR) is a novel signaling parthway,which is discoved recent years.It is a kind of ingenious style in which the cells regulate their functions according to external stimulations.External stimulations or G-protein-coupled receptor(GRCP) activated lead to release('shedding') of a kind of growth factor,which in turn stimulates receptor tyrosine-kinase(RTK) itself and adjacent cells,which evoked simillar action as RTK is directly stimulated.It is a novel signaling pathway which plays a major role on regulating cell proliferation, differentiation and survival.After activated by the ligands,EGFR initiate introcellular signal transduction direct the migration,adhesion,proliferation,differention and apoptosis through the cascade reaction of adapter protein and enzymes in cytosol.β-adrenergic receptor agonist isoproterenol has cellular specificity in cell regulation.In our study,we have studied its effect in primary cultured astrocytes.The expression of Na⁺,K⁺-ATPaseα₂ was increased by chronic treatment with pharmacologically relevant concentration ofβ-adrenergic receptor agonist isoproterenol.β-adrenergic receptor agonist isoproterenol-induced expression of Na⁺, K⁺-ATPaseα₂ could be inhibited by AG1478,a specific EGFR tyrosine kinase inhibitor, and inhibited by GM6001,an inhibitor of Zn²⁺-dependent metalloproteinase.Also,the expression of Na⁺,K⁺-ATPaseα₂ could be inhibited by PP1,a Src inhibitor,and also by U0126,a MEK inhibitor.Objective1.Whetherβ-adrenergic receptor agonist isoproterenol has effect on expression of Na⁺,K.+-ATPaseα₂ mRNA and change of enzymatic activity in primary cultures of mouse astrocytes.2.Whether EGFR is involved in the parthway.3.Whether Src is involved in the parthway.MethodsPrimary cultures of astrocytes were from cerebal hemisphere of new born mice of either sex.The neopallia of the cerebral hemispheres were aseptically isolated,vortexed to dissociate the tissue,filtered through nylon meshes with pore sizes of 100μM and subsequently 70μM,diluted in culture medium,and planted in Falcon Primaria culture dishes.The culture medium was a Dulbecco's Medium with 7.5 mM glucose,initially containing 20%horse serum,and the cultures were incubated at 37℃in a humidified atmosphere of 5%CO₂.The culturing medium was exchanged with fresh medium of similar composition on Day 3,and subsequently every 3-4 days.From the third time, the serum concentration was reduced to 10%,and after the age of 2 weeks,0.25 mM dBcAMP was included in the medium.The cultures were used after at least three weeks of culturing.For determination of Na⁺,K⁺-ATPaseα₂ subunit expression,the cells were incubated in corresponding medium without serum at 37℃for specific time periods in the absence or presence ofisoproterenol(1nM,10nM,100nM,1μM,10μM), AG1478(1μM),PP1(10μM),GM6001(10μM) and U0126(10μM).Na⁺, K⁺-ATPaseα₂ subunit mRNA expression was detected by reversetranscriptionpolymerase chain reaction(RT-PCR),The PCR products were separated by 1.5% agarose gel electrophoresis,and captured by Fluorchem 5500.Band density was measured with Window AlphaEaseTM FC32-bit software.The enzymatic activity of Na⁺,K⁺-ATPaseα₂ subunit was detected by UV spectrophotometer.The differences between multiple groups were analyzed by one-way analysis of variance(ANOVA) followed by Fisher's LSD multiple comparison test for unequal replications.The level of significance was set at P＜0.05.Results1.Effects of Isoproterenol on Na⁺,K⁺-ATPaseα₂ subunitThe stimulation of Na⁺,K⁺-ATPaseα₂ subunit mRNA expression by isoproterenol was concentration-dependent in astrocytes.There was no significant change at 1nM, 10nM.At 100nM,1μM,10μM,a pharmacologically relevant concentration,the level of Na+,K+-ATPaseα₂ mRNA was increased with isoproterenol and concentrationdependent, which was statistically significant(P＜0.05). 2.Effects of inhibitors of EGFR and of SrcThe effect of AG1478,a specific EGFR tyrosine kinase inhibitor,was involved.In the presence of 1μM AG1478,the increase of isoproterenol-induced Na⁺,K⁺-ATPaseα₂ subunit by 100 nM isoproterenol was inhibited.10μM PP1,a Src inhibitor,can inhibit the increase of 100 nM isoproterenol induced mRNA and enzymatic activity of Na⁺,K⁺-ATPaseα₂ subunit,which was statistically significant(P＜0.05).3.Effects of inhibitor of metalloproteinase10μM GM6001,an inhibitor of Zn²⁺-dependent metalloproteinase,affected the response of exposure to 100 nM isoproterenol in exactly the same manner as PP1, which was statistically significant(P＜0.05).4.Effects of inhibitors of MEKThe effect of U0126,a specific MEK inhibitor,was involved.In the presence of 10μM U0126 of isoproterenol-induced the increase of enzymatic activity of Na⁺, K⁺-ATPaseα₂ subunit by 100 nM isoproterenol was inhibited,which was statistically significant(P＜0.05).ConclusionIn astrocytes,the expression of Na⁺,K⁺-ATPaseα₂ subunit mRNA and the enzymatic activity of Na⁺,K⁺-ATPaseα₂ subunit were increased by chronic treatment with a pharmacologically relevant concentration of isoproterenol.In the pathway, EGFR was activated directly and Transactivated.At the same time,Src and MEK involved the parthway.