Determinants of Atrial Natriuretic Peptide Secretion in Cultured Atrial Myocytes

Atrial natriuretic peptide (ANP) secretion by atrial myocytes in 7- to 8-day primary cultures from adult rats was measured by radioimmunoassay under conditions designed to separate primary effects on secretion from effects caused by contractions. Abolishing contraction with 10 microM tetrodotoxin significantly reduced ANP accumulated in 2 h. Raising external Ca2+ concentration from 0.2 to 1.2 mM in the presence of tetrodotoxin did not increase ANP secretion. Substantial ANP secretion persisted in a nominally Ca2+-free medium containing 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and was not diminished by 100 microM ryanodine. In the presence of EGTA, 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) significantly increased ANP secretion; this increment was unaffected by 100 microM ryanodine. These experiments suggest 1) that in absence of contractions, ANP secretion requires neither transplasmalemmal Ca2+ influx nor ryanodine-inhibitable Ca2+ release by sarcoplasmic reticulum (SR) and 2) that TPA stimulates ANP secretion without requiring Ca influx or ryanodine-inhibitable SR Ca release.