Abstract
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.
Footnotes
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This work was supported by National Institutes of Health Grant NS23831 (to S.K.F.) and F31 NS053020-01 (to T.A.C.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.090787.
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ABBREVIATIONS: PAR, proteinase-activated receptor; RVD, regulatory volume decrease; VSOAC, volume-sensitive organic osmolyte anion channel; DDF, 1,9-dideoxyforskolin; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; DIDS, 4,4′diisothiocyanatostilbene-2,2′-disulfonic acid; SITS, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; KN-93, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-(methylbenzylamine); PD 98059, 2′-amino-3′-methoxyflavone; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance; PPACK, d-Phe-Pro-Arg chloromethyl ketone; PLC, phospholipase C; CNS, central nervous system; mAChR, muscarinic cholinergic receptor; BIM, bisindolylmaleimide; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; AM, tetra(acetoxymethyl)ester.
- Received June 9, 2005.
- Accepted July 25, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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