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NEUROPHARMACOLOGY

Subnanomolar Concentrations of Thrombin Enhance the Volume-Sensitive Efflux of Taurine from Human 1321N1 Astrocytoma Cells

Molecular and Behavioral Neuroscience Institute (T.A.C., C.E.W., S.K.F.) and Department of Pharmacology (T.A.C., S.K.F.), University of Michigan, Ann Arbor, Michigan

The ability of subnanomolar concentrations of thrombin to protect both neurons and glia from ischemia and other metabolic insults has recently been reported. In this study, we demonstrate an additional neuroprotective property of thrombin; its ability to promote the release of the organic osmolyte, taurine, in response to hypoosmotic stress. Incubation of human 1321N1 astrocytoma cells with hypo-osmolar buffers (320-227 mOsM) resulted in a time-dependent release of taurine. Inclusion of thrombin (EC₅₀ = 60 pM) resulted in a marked increase in taurine efflux that, although evident under isotonic conditions (340 mOsM), was maximal at an osmolarity of 270 mOsM (3-4-fold stimulation). Thrombin-stimulated taurine efflux was dependent upon its protease activity and could be mimicked by addition of the peptide SFLLRN, a proteinase activated receptor-1 (PAR-1) subtype-specific ligand. Inclusion of anion channel blockers known to inhibit the volume-sensitive organic osmolyte anion channel attenuated thrombin-stimulated taurine release. Depletion of intracellular Ca²⁺ with either 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or thapsigargin, or alternatively, inhibition of protein kinase C (PKC) with bisindolylmaleimide or chelerythrine resulted in a 30 to 50% inhibition of thrombin-stimulated taurine efflux. Under conditions in which intracellular Ca²⁺ was depleted and PKC activity inhibited, thrombin-stimulated taurine efflux was reduced by >85%. The results indicate that activation of PAR-1 receptors by thrombin facilitates the ability of 1321N1 astrocytoma cells to release osmolytes in response to a reduction in osmolarity via a mechanism that is dependent on intracellular Ca²⁺ and PKC activity.

Address correspondence to: Dr. Stephen K. Fisher, University of Michigan, Molecular and Behavioral Neuroscience Institute Laboratories at MSRB II, 1150 West Medical Center Drive, C560, MSRB II, Ann Arbor, MI 48109-0669. E-mail: skfisher{at}umich.edu

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