Roles of protein kinase C and Ca2+-dependent signaling in angiotensin II-induced adrenomedullin production in rat cardiac myocytes

OBJECTIVES: We showed that angiotensin II stimulates adrenomedullin production in cultured neonatal rat cardiac myocytes, and that the secreted adrenomedullin inhibits hypertrophy of the myocytes, although the intracellular mechanisms of adrenomedullin production are still unknown. Since protein kinase C (PKC) and the Ca2+ signaling system are involved in cardiac hypertrophy, we examined the roles of these intracellular signaling systems in the production of adrenomedullin by myocytes. METHODS: Cultured neonatal rat cardiac myocytes were incubated with agonists or antagonists of PKC and Ca2+ signaling systems for 24 h. Adrenomedullin secreted into the medium and adrenomedullin mRNA expression were measured by radioimmunoassay and quantitative polymerase chain reaction, respectively. RESULTS: Both phorbol-12-myristate-13-acetate (PMA), a PKC activator and A23187, a calcium ionophore, significantly increased adrenomedullin mRNA expression and secretion from the myocytes. The induction of adrenomedullin secretion by PMA was abolished by H7, a PKC inhibitor, and by downregulation of PKC induced by pre-incubation with PMA. Similarly, the stimulation of adrenomedullin secretion by 10(-6) mol/l angiotensin II was significantly reduced following the inhibition or downregulation of PKC activity in the myocytes. Blockade of the L-type Ca2+ channel and chelation of intracellular Ca2+ both resulted in a significant reduction of the stimulation of adrenomedullin secretion by angiotensin II. In addition, the secretion was significantly attenuated by inhibitors of calmodulin (W-7) and calmodulin kinase II (KN-62), and slightly attenuated by FK506, a calcineurin inhibitor. CONCLUSIONS: These results suggest that PKC and the Ca2+/calmodulin signaling systems are involved in angiotensin II-induced adrenomedullin secretion from rat cardiac myocytes.