BIOGENESIS OF CARDIAC SECRETORY VESICLES .

V. Labrador, A. Roatti, A.J. Baertschi
Department of Physiology, C.M.U., University of Geneva.

Atrial natriuretic peptide (ANP) is a diuretic vasorelaxant hormone stored in specific secretory vesicles of heart and brain. ANP is secreted from the heart in response to overload and oxygen deficiency. Our recent study (Circ Res. 2001;89(3):E23-9) provides new insights about formation and docking of secretory vesicles in cardiomyocytes: amino acid residues in proANP, that favor the calcium-dependent cargo aggregation, are critically important for shaping the secretory vesicles and determining their fate - docking or not docking - at the plasma membrane. We propose that surface motifs of aggregated proANP recruit Golgi transmembrane proteins essential for shaping and docking the secretory vesicles. In the present study, we test this hypothesis by focusing on PAM (peptidylglycine alpha-monooxygenase), a family of abundant vesicle transmembrane proteins (PAM-1, PAM-2) of as yet unknown cardiac function. First, we examined on 435 secretory vesicles the effects of mutations in proANP on its co-localization with PAM-1. These vesicles were visualized by expressing fusion proteins of proANP with green fluorescent protein (EGFP), and by PAM-1-specific immunolabeling. Atrial myocytes expressing intact proANP-EGFP display two populations of green fluorescent vesicles: a majority (87%) where EGFP is co-localized with PAM-1, and a minority (13%) where EGFP is expressed without PAM-1. Point mutations (glu23,24 to gln23,24) in proANP or deletion of the calcium-binding N-terminal 44 amino acids reduce the proportion of double-labelled vesicles to 59% and 33%, respectively. Second, we investigated, on 800 ProANP-EGFP vesicles, the role of PAM in shaping the secretory vesicle, by inhibiting the enzymatic activity of PAM with PBA (4-Phenyl-3-butenoic acid, 10microM). PBA dramatically changes the shape from spherical balls to irregular floppy vesicles: the proportion of spherical vesicles drops to 51% (2 days), 20% (3 days) and 29% (4 days). Corresponding controls are 60%, 53% and 51%, respectively. Third, the shapes of 712 vesicles were analyzed in proANP-EGFP expressing myocytes transfected with an antibody directed to the cytosolic tail of PAM-1 and PAM-2. After 2 days these myocytes contain a significantly smaller proportion of spherical vesicles (39%) relative to the mock-transfected cells (53%). The results support the hypothesis that aggregation of proANP in Golgi recruits PAM to the vesicle membrane, and suggest that the PAM protein family is critically involved in the biogenesis of ANP-containing spherical vesicles. Thus PAMs are trafficking-proteins in the ANP secretion pathway.

Supported by SUK and SNSF