Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardio-myocyte PAM (Pam(Myh6-cKO/cKO)) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult Pam(Myh6-cKO/cKO) atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in Pam(Myh6-cKO/cKO) atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by Pam(Myh6-cKO/cKO) myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by Pam(Myh6-cKO/cKO) myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.