ACTIVATION OF THE MITOGEN-ACTIVATED PROTEIN-KINASE CASCADE BY PERTUSSIS-TOXIN-SENSITIVE AND PERTUSSIS-TOXIN-INSENSITIVE PATHWAYS IN CULTURED VENTRICULAR CARDIOMYOCYTES

The involvement of pertussis toxin (PTX)-sensitive and -insensitive pa thways in the activation of the mitogen-activated protein kinase (MAPK ) cascade was examined in ventricular cardiomyocytes cultured from neo natal rats. A number of agonists that activate heterotrimeric G-protei n-coupled receptors stimulated MAPK activity after exposure for 5 min. These included foetal calf serum (FCS), endothelin-1 (these two being the most effective of the agonists examined), phenylephrine, endothel in-3, lysophosphatidic acid, carbachol, isoprenaline and angiotensin I I. Activation of MAPK and MAPK kinase (MEK) by carbachol returned to c ontrol levels within 30-60 min, whereas activation by FCS was more sus tained. FPLC on Mono Q showed that carbachol and FCS activated two pea ks of MEK and two peaks of MAPK (p42(MAPK) and p44(MAPK)). Pretreatmen t of cells with PTX for 24 h inhibited the activation of MAPK by carba chol, FCS and lysophosphatidic acid, but not that by endothelin-1, phe nylephrine or isoprenaline. Involvement of G-proteins in the activatio n of the cardiac MAPK cascade was demonstrated by the sustained (PTX-i nsensitive) activation of MAPK (and MEK) after exposure of cells to Al F4-. AlF4- activated PtdIns hydrolysis, as did endothelin-1, endotheli n-3, phenylephrine and FCS. In contrast, the effect of lysophosphatidi c acid on PtdIns hydrolysis was small and carbachol was without signif icant effect even after prolonged exposure. We conclude that PTX-sensi tive (i.e. G(i)/G(o)-linked) and PTX-insensitive (i.e. G(q)/G(s)-linke d) pathways of MAPK activation exist in neonatal ventricular myocytes. FCS may stimulate the MAPK cascade through both pathways.