Phosphorylation c-Jun and ATF2 in ventricular myocytes by endothelin and phenylephrine.

The cardiac myocyte is a terminally differentiated cell. In response to increased myocardial workload or damage, myocytes undergo hypertrophic (as opposed to hyperplastic) growth. This response includes transient upregulation of immediate early genes such as c-jun [ 1,2]. Expression of c-jun is partly controlled by cJun/ATF2 dimers bound to AP-1-like sites in the c-jun promoter [3,4]. Phosphorylation of c-Jun and ATM by stress-activated protein kinases (SAPKs) increases their transactivating activities [5-81. The hypertrophic response in vivo can be simulated in myocytes cultured from neonatal rat heart ventricles exposed to agonists such as the a-adrenergic agonist phenylephrine (PE) or the vasoconstrictor peptide endothelin-1 (ET-1) [2], which activate the extracellular-regulated kinases (ERKs) and, to a lesser extent, the SAPKs [9-121. We have studied the effects of ET-1 and PE on the induction and phosphorylation of c-Jun and ATFZ in cultured ventricular myocytes using Western blot analysis. Ventricular myocytes, isolated and cultured by standard methods [11,12], were stimulated with ET-1 (0.1 pM) or PE (50 pM). Nuclear extracts were prepared [ 131 and proteins (100 pg) were separated by SDS-PAGE using 10 96 and 8 96 acrylamide gels to study c-Jun and ATF2 respectively. After transfer to nitrocellulose, specific proteins were identified using primary antibodies to c-Jun and ATFZ (Santa Cruz Biotechnology) and horseradish peroxidase-linked secondary antibodies. Bands were detected by the ECL method. Phosphorylated forms of c-Jun and ATF2 were identified as reduced mobility bands. ET-1 stimulated a small apparent increase in total c-Jun protein over 30 min (Fig. 1). The protein synthesis inhibitor, cycloheximide (20 p M ) did not inhibit this increase. This inhibitor itself activated the SAPKs and induced an increase in cJun immunoreactivity. The apparent increase in c-Jun probably represents a change in its phosphorylation state as is seen in cell stress [13]. ET-1 stimulated a second phase of increase in total c-Jun immunoreactivity. This was maximal at approximately 1 h