Pharmacological characterization of protease-activated receptor (PAR-1) in rat astrocytes

Abstract The proteolytic action of thrombin on its receptor (protease-activated receptor-1 or PAR-1) results in a conformational change in which the new N-terminal sequence auto-activates the receptor. Peptide analogs of this N-terminal sequence (TRAPs) are able to mimic the effect of thrombin and an extensive search has led to the definition of the structural requirement for the agonist and antagonist activity on thrombin receptors in several peripheral systems. Thrombin plays an important role in central and peripheral nervous system development and PAR-1 is present in neurons and astrocytes. We have now characterized thrombin receptors pharmacologically in cultured rat astrocytes by using [ 3 H]thymidine incorporation and reversal of stellation induced by Bt 2 cAMP as end-points. Thrombin increased [ 3 H]thymidine incorporation into DNA with an EC 50 of 1 nM and induced a complete reversion of cell stellation. The effects of thrombin on [ 3 H]thymidine incorporation were mimicked by TRAP-14 (EC 50 =3 μM) and a peptide containing non-natural amino acids Ala-Phe( p -F)-Arg-Cha-HArg-Tyr-NH 2 (A6Y; EC 50 =0.8 μM). Similarly, these two peptides reversed Bt 2 cAMP-induced stellation. The effect of thrombin, TRAP-14 and A6Y on [ 3 H]thymidine incorporation into DNA was significantly prevented by L9R, a 9-amino-acid peptide (Leu-Val-Arg- d -Cys-Gly-Lys-His-Ser-Arg; IC 50 =180 μM against thrombin and TRAP-14 and 800 μM against A6Y) previously described as an antagonist in human platelet aggregation. L9R antagonized also thrombin effects on astrocyte morphology. These results demonstrate that rat astrocytes express PAR-1 receptors which are pharmacologically similar to those previously characterized in human platelets. © 1997 Elsevier Science B.V. All rights reserved.