The Singnaling Pathways Of ERK Phosphorylation By β-adrenergic Agonist Isoproterenol In Astrocytes

IntroductionCompared to glutamatergic and GABAergic neurons the CNS contains very few noradrenergic and even fewer adrenergic neurons.Their cell bodies are located in the brain stem within medulla and pons.One can distinguish between two groups of adrenergic and noradrenergic neurons.One group primarily functions in the supraspinal regulation of the entire autonomic nervous system.The second group consists of a single nucleus,the locus coeruleus,and it provides virtually the entire noradrenergic innervation of the cerebral hemispheres(cerebral cortex,hippocampus and cerebellum) from its rostral and medium parts and part of the noradrenergic innervation of the spinal cord from its caudal part.Recently it has been demonstrated that adrenergic stimulation has an inhibitory effect on immune reactions in the CNS.This effect may play a major role in multiple sclerosis(MS),where a disappearance of astrocyticβ₂-adrenergic receptors in white matter has been observed.Astrocytes constitute one of the two groups of macroglial cells(astrocytes and oligodendrocytes)that in the brain cortex by far outnumber neurons.Astrocytes extend a large number of processes and filopodia and they cover most of the perimeter of microvessels.They are coupled to each other by gap junctions,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 become obvious that they exert a number of functions of key importance for the function of the central nervous system.Epithelial growth factor receptor(EGFR)transactivation in which signaling to a G-protein-coupled receptor(GRCP)leads to release('shedding')of a growth factor, which in turn stimulates receptor tyrosine-kinase(RTK)on the same and adjacent cells. This leads to the recruitment of Grb2-Sos1 complexes to the activated receptor-tyrosine kinase and the exchange of GDP for GTP on the Ras.Activation of Ras,in turn, initiates the phosphorylation cascade onsisting of Raf,MEK,and the MAP kinases (MAPK)ERK.It is a novel signaling pathway which plays a majoy role on regulating cell proliferation,differentiation and survival.β-adrenergic receptor agonist isoproterenol have cell specificity in cell regulation. In the present study we have studied in astrocytes(1)Whetherβ-adrenergic receptor agonist isoproterenol caused EGFR transactivation;(2)The singnaling pathways of ERK phosphorylation induced byβ-adrenergic agonist isoproterenol.MethodsPrimary cultures of astrocytes,from newborn CD-1 mice of either sex,were prepared.The neopallia of the cerebral hemispheres,which roughly corresponds to the forebrains,were aseptically isolated,vortexed to dissociate the tissue,filtered through nylon neshes with pore sizes of 100μm and 70μm,diluted in culture medium and planted in Falon 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 CO₂/air(5%:95%).For determination of ERK_1/2phosphorylation,the culturing medium was removed and the cells were incubated in corresponding medium without serum at 37℃for certain time periods in the absence or presence of isoproterenol or/and specific inhibitors.The reaction was stopped by washing with ice cold phosphate-buffered saline(PBS) containing 7.5 mM glucose,and the cells were scraped off the dishes.Protein content was determined by the Lowry method,using bovine serum albumin as the standard. Samples containing 50μg protein were applied on slab gels of 5%and 10% polyacrylamide and electrophoresed.After transfering to nitrocellulose membranes,the samples were blocked by 5%skim milk powder in TBS-T for 1 h.The nitrocellulose membranes were incubated with the specific antibody.Staining was visualized by ECL detection reagents,followed by exposure to film.The results were collected by Flurchem imaging system.Band density was measured with Window AlphaEaseTM FC32-bit software.Results 1.Isoproterenol-induced ERK_1/2phosphorylationIn astrocytes,Exposure to isoproterenol for 20 min caused increase of p-ERK_1/2, whereas there was no significant change in total content of ERK_1/2.A small response of ERK_1/2appeared to occur at a isoproterenol concentration of 1 nM and statistically significant increase was seen at 10 nM,100 nM,1μM and 10μM.After 10 min,20 min and 40 min of exposure to 100 nM isoproterenol,p-ERK₁ and p-ERK₂ increased statistically significant.2.Effects of inhibitors of Gi proteinThe effect of pertussis toxin(PTX),a specific Gi protein inhibitor,was tested in order to establish if Gi proteinβγ,subunit was involved.0.2μg/ml PTX did not inhibit 100 nM isoproterenol-induced ERK_1/2phosphorylation,suggesting that it was not induced byβγ,subunit of Gi protein.3.Effects of inhibitors of PKAThe effect of H-89,a specific PKA inhibitor,was tested in order to establish if PKA was involved.5μM H-89 did not inhibit isoproterenol-induced ERK_1/2 phosphorylation,suggesting that it was independent on PKA activity.4.Epac-induced ERK_1/2phosphorylationIn astrocytes,10μM 8-pCPT-2'-O-Me-cAMP,Epac1/2 specific agonist,induced ERK_1/2phosphorylation,suggesting that Epacs and their downstream regulatory mechanism took part inβ-adrenergic receptor induced ERK phosphorylation.5.Effects of inhibitors of metalloproteinase and of heparin10μM GM6001,an inhibitor of Zn²⁺-dependent metalloproteinase and 1 mg/ml of heparin,an inhibitor of HB-EGF did not inhibit isoproterenol-induced ERK_1/2 phosphorylation,suggesting that it was independent on metalloprotease,and there was no HB-EGF releasing.6.Effects of inhibitors of EGFR and of Src1μM AG1478,EGFR tyrosine kinase inhibitor,partially inhibited isoproterenol induced ERK phosphorylation and 10μM PP1,Src PTK inhibitor,completely inhibit it, suggesting that Src was involved in EGFR phosphorylation and/or ERK_1/2 phosphorylation. DiscussionStandard cAMP-protien kinase pathway is an increase of intracellular cAMP concentration which in turn stimulates PKA.PKA can phosphorylate many proteins at serine and/or threonine residue to regulate cell metabolism and gene expression.Recent study suggests that in mammalian cells,cAMP can directly activate PKA and/or Epac1/2.Epac is directly activated by cAMP.The downstream signal of it are small GTPases Rap1 and Rap2.Different from EGFR transactivation by Gi/o and Gq protein-coupled receptor,Gs protein-coupled receptor activated ERK is more complicated.Different G protein-coupled receptor activating EGFR may have distinct physiologic function probably because 1)the intensity and duration of ERK phosphorylation are different. ERK phosphorylation by the activity of Gi/o or Gq protein-coupled receptor occurs quickly and lasts for only short period of time(mins).But ERK phosphorylation by cAMP persists long time.2)when ligand binds to the receptor,dimerization occur. EGFR can form homodimer or heterodimer with HER₂,HER₃,HER₄.The transactivation of EGFR not only depends on the type of G protein-coupled receptor but also cell category.It has been found that activation of Gs protein-coupled receptor can activate EGFR in some cells,such as PC12 cells,but not in other cells such as fibroma cell and vascular smooth muscle cell.The increase of cAMP may inhibit ERK phosphorylation.More interesting,ERK phosphorylation byβ₂-adrenergic receptor in COS cell is mediated by Gi protein and can not be substituted by cAMP.But in lymphoma cell,ERK phosphorylation is by the activity of Epac1/Rap1 signal pathways.ConclusionIn astrocytes,β-adrenergic receptor agonist isoproterenol induced ERK phosphorylation by Gs protein,cAMP,Epacs,Src and EGFR signal pathways.