title: Rapid increase in immunoreactivity to GFAP in astrocytes in vitro induced by acidic pH is mediated by calcium influx and calpain I.
abstract: In higher vertebrates, reactive gliosis resulting from injury to the central nervous system (CNS) is characterized by a rapid increase in immunoreactivity (IR) to glial fibrillary acidic protein (GFAP). Little is known about the extracellular signals that initiate the increase in GFAP-IR following CNS injury. We demonstrated recently [T.H. Oh, G.J. Markelonis, J.R. Von Visger, B. Baik, M.T. Shipley, Acidic pH rapidly increases immunoreactivity of glial fibrillary acidic protein in cultured astrocytes, Glia 13 (1995) 319-322] that a rapid increase in GFAP-IR can be evoked in mature astrocyte cultures by exposing the cells to an acidic medium. We investigated the intracellular pathway(s) involved in initiating increased GFAP-IR, a hallmark of reactive astrocytes. The increase in GFAP-IR produced by exposure to acidic medium was blocked by pretreatment with nickel ions, by such blockers of L-type calcium channels as nifedipine, verapamil and diltiazem, by calpain inhibitor I, or by the intracellular calcium chelator, BAPTA-AM. At physiological pH, treatment with the calcium ionophore, A23187, resulted in increased GFAP-IR which could be blocked by pretreatment with calpain inhibitor I. Astrocytes exposed to low pH exhibited a marked increase in a GFAP fragment with a molecular weight of 48 kDa. In astrocytes exposed to acidic medium, alpha-fodrin, a selective endogenous substrate of calpain, was also found to be hydrolyzed producing fragments with molecular weights of 120-150 kDa. As anticipated, pretreatment with calpain inhibitor I prevented the proteolytic degradation of both GFAP and alpha-fodrin in these samples. These results suggest that the initial increase in GFAP-IR after CNS injury appears to be linked to Ca(++) influx, and is mediated further by a proteolytic process that seemingly involves the activation of the calcium-dependent protease, calpain I.
authors.affiliation: Department of Anatomy & Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland, MD, USA. yolee@umaryland.edu
authors.forename: Y B
authors.initials: YB
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Du
authors.forename: H
authors.initials: H
authors.lastname: Rhim
authors.forename: E B
authors.initials: EB
authors.lastname: Lee
authors.forename: G J
authors.initials: GJ
authors.lastname: Markelonis
authors.forename: T H
authors.initials: TH
authors.lastname: Oh
mesh_terms: D000143:Acids / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004110:Diltiazem / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D008840:Microfilament Proteins / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D016548:Prosencephalon / Q000166:cytology; D051381:Rats; D011916:Rats, Inbred F344; D013379:Substrate Specificity; D014700:Verapamil / Q000494:pharmacology; D013049:Spectrin
chemical_list: D000143:Acids; D000906:Antibodies; D000001:Calcimycin; D002118:Calcium; D002121:Calcium Channel Blockers; D002154:Calpain; D002352:Carrier Proteins; D002614:Chelating Agents; D015853:Cysteine Proteinase Inhibitors; D004110:Diltiazem; D004533:Egtazic Acid; D005904:Glial Fibrillary Acidic Protein; D006023:Glycoproteins; D007476:Ionophores; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D009543:Nifedipine; D014700:Verapamil; D013049:Spectrin; C039304:calpain inhibitors; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester