Expression of the thrombin receptor (PAR-1,) during rat skeletal muscle differentiation

The serine protease thrombin: has been proposed to be involved in neuromusc ular plasticity. its specific receptor "protease activated receptor-1" (PAR -1), a G protein-coupled receptor, has been shown to be expressed in myobla sts but not after fusion (Suidan et al., 1996 J Biol Chem 271:29162-29169). In the present work we have investigated the expression of PAR-1 during ra t skeletal muscle differentiation both in vitro and in vivo. Primary cultur es of rat foetal skeletal muscle, characterized by their spontaneous contra ctile activity, were used for exploration of PAR-1 by RT-PCR, immunocytoche mistry and Western blotting. Our results show that PAR-1 mRNA and protein a re both present in myoblasts and myotubes. incubation of myotubes loaded wi th fluo-3-AM in presence of thrombin (200 nM) or PAR-1 agonist peptide (SFL LRN, 500 muM), induced the intracellular release of calcium indicating the activation of PAR-1. Blockade of contractile activity by tetrodotoxin (TTX, 6 nM) did not modify either PAR-I synthesis or its cellular localization. Investigation of PAR-I on rat muscle cryostat sections at Day 18 of embryog enesis and postnatal Days 1, 5, and 10 indicated that this protein is first expressed in the cytoplasm and that it later localizes to the membrane. Mo reover, its expression correlates with myosin heavy chain transitions occur ring during post-natal period and is restricted to primary fibers. Taken to gether, these results suggest that PAR-1 expression is not related to contr actile activity but to myogenic differentiation. (C) 2001 Wiley-Liss, Inc.