Studies of ANF processing and secretion using a primary cardiocyte culture model

Area of Science:

• Cardiovascular Endocrinology
• Cellular Biology
• Peptide Hormone Processing

Background:

• Most endocrine peptides are secreted as active hormones, but atrial natriuretic peptide (ANF) is stored as a larger prohormone (pro-ANF) and processed to its active 28-amino acid form.
• The exact location and mechanism of pro-ANF conversion to ANF within the heart have been areas of investigation.

Purpose of the Study:

• To investigate the location and mechanism of pro-ANF processing to ANF in neonatal rat heart cell cultures.
• To explore the mechanisms regulating stimulated ANF secretion from cardiac myocytes.

Main Methods:

• Primary neonatal rat heart cell culture system.
• Use of protease inhibitors, pulse-chase biosynthetic labeling, and incubation with ANF-related peptides.
• Investigation of phospholipase C-coupled receptor activation and calcium signaling pathways.

Main Results:

• Pro-ANF processing to ANF requires glucocorticoid-containing serum-free medium in culture, mirroring in vivo conditions.
• Processing occurs within cardiac myocytes just before secretion.
• Stimulated ANF release via alpha 1-adrenergic and endothelin receptors depends on extracellular and intracellular calcium, with an initial phase independent of extracellular calcium.

Conclusions:

• Pro-ANF processing to ANF occurs intracellularly within cardiac myocytes prior to secretion.
• Hormonal environment influences differentiated endocrine functions of atrial myocytes, regulating ANF expression, biosynthesis, and secretion.
• Calcium influx and intracellular calcium mobilization are critical for agonist-stimulated ANF release.