Rapid increase in immunoreactivity to GFAP in astrocytes in vitro induced by acidic pH is mediated by calcium influx and calpain I

In higher vertebrates, reactive gliosis resulting from injury to the centra l nervous system (CNS) is characterized by a rapid increase in immunoreacti vity (IR) to glial fibrillary acidic protein (GFAP). Little is known about the extracellular signals that initiate the increase in GFAP-IR following C NS injury. We demonstrated recently [T.H. Oh, G.J. Markelonis, J.R. Von Vis ger, B. Balk, 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 cu ltures by exposing the cells to an acidic medium. We investigated the intra cellular pathway(s) involved in initiating increased GFAP-IR, a hallmark of reactive astrocytes. The increase in GFAP-IR produced by exposure to acidi c 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 physiol ogical pH, treatment with the calcium ionophore, A23187, resulted in increa sed GFAP-LR which could be blocked by pretreatment with calpain inhibitor I . Astrocytes exposed to low pH exhibited a marked increase in a GFAP fragme nt with a molecular weight of 48 kDa. In astrocytes exposed to acidic mediu m, cr-fodrin, a selective endogenous substrate of calpain, was also found t o be hydrolyzed producing fragments with molecular weights of 120-158 kDa. As anticipated, pretreatment with calpain inhibitor I prevented the proteol ytic degradation of both GFAP and oc-fodrin in these samples. These results suggest that the initial increase in GFAP-IR after CNS injury appears to b e linked to Ca++ influx, and is mediated further by a proteolytic process t hat seemingly involves the activation of the calcium-dependent protease, ca lpain I. (C) 2000 Elsevier Science BN. All rights reserved.