It remains probable that both (and other) pathways are necessary for the full range of transcriptional and morphological changes associated with cardiac hypertrophy. Acknowledgments We thank R.J.A. but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response. (Poole, Dorset, UK). PD98059 and GF109203X were from (Beeston, Nottinghamshire, UK) and were prepared as 25 mM and 10 mM stock solutions in DMSO. SB203580 was a gift from Dr. John C. Lee (SmithKline Beecham, King of Prussia, PA) and a 10 mM stock solution was prepared in DMSO. Lab-Tek tissue culture chamber slides were from Life Technologies (Paisley, Scotland, UK). Other tissue culture materials were from previously described sources (Bogoyevitch et al., Mouse monoclonal antibody to SMYD1 1995(and supplied by Autogen Bioclear (Devizes, Wiltshire, UK). The antibody to the dually phosphorylated form of p38-MAPK was from (UK) Ltd. (Hitchin, Herts., UK). A mouse monoclonal antibody to -myosin heavy chain (-MHC) was from Novocastra (Newcastle-upon-Tyne, Tyne and Wear, UK). Secondary antibodies (horseradish peroxidaseC linked, FITC and biotinylated antiCmouse and antiCrabbit IgG antibodies) and fluorescent mounting medium were from Dako (High Wycombe, Buckinghamshire, UK). Primary Culture of Neonatal Ventricular Myocytes and Sample Preparation Myocytes were dissociated from the ventricles of neonatal Sprague-Dawley rat hearts by a previously described adaptation (Bogoyevitch et al., 1995Axioskop microscope with a 100 oil immersion objective. Cells were photographed using Tmax 400 film with a set exposure time (30 s). Results Phosphorylation of p38-MAPK To assess the activation of p38-MAPK in cardiac myocytes, we used an antibody selective for the dually phosphorylated (activated) form of the p38-MAPKs for Western blot analysis. Hyperosmotic shock (0.5 M sorbitol) stimulated extensive phosphorylation of p38-MAPK (25-fold relative to controls; Fig. ?Fig.11 and and and and and and and and ?and1010 and ?and1010 and and and and and Diphenylpyraline hydrochloride and and and and and and and and 10, and and results not shown). These data at 48 h are essentially similar to those of Zechner et al. (1997) who only studied the effects at this time point, but in view of the absence of any effect of this inhibitor up to 24 h, suggest that the p38-MAPK pathway may be necessary to maintain the cells during hypertrophy rather than have a direct effect in stimulating the hypertrophic response. Consistent with a role for the p38-MAPK pathway in cytoprotection, activation of MAPKAPK2 by ET-1 induced the sustained phosphorylation of HSP25/27 (Fig. ?(Fig.7).7). Such phosphorylation of HSP25/ 27 is associated with cytoprotection in other cell types, and is particularly important in the maintenance of the actin cytoskeleton. Even though the activation of the p38-MAPK pathway Diphenylpyraline hydrochloride by ET-1 was only 50% of that by 0.5 M sorbitol (Figs. ?(Figs.22 and ?and4),4), the extent of phosphorylation of HSP25/27 induced by either agonist was similar (Fig. ?(Fig.7)7) and may confer equivalent degrees of cytoprotection. Experiments by others have implicated the p38-MAPK pathway in ischemic preconditioning of the heart, whereby a short period of ischemia protects the heart against a subsequent more prolonged insult (reviewed by Sumeray and Yellon, 1997). Perfusion with SB203580 attenuates this preconditioning (Weinbrenner et al., 1997) suggesting that p38-MAPK, which is activated during ischemia (Bogoyevitch et al., 1996; Yin et al., 1997) plays an essential role in the preconditioning response. Ischemic preconditioning can be mimicked by perfusion with 1-adrenergic agonists (Banerjee et al., 1993), ET-1 (Wang et al., 1996), and in the rat heart, activation of PKC may be involved (Speechly-Dick et al., 1994; Li and Diphenylpyraline hydrochloride Kloner, 1995; Mitchell.