Cytosolic Ca 2+ during atrial natriuretic peptide secretion from cultured neonatal cardiomyocytes

Abstract

Mechanisms of atrial natriuretic peptide (ANP) release were studied in neonatal rat heart atrial and ventricular myocytes cultured on Cytodex 3 microcarriers. For simultaneous observations of cytosolic free calcium concentration ([Ca 2+] f) and ANP secretion, the culture was packed in a chromatography column, inserted into the cell holder of a spectrofluorometer was perifused with a buffer solution. [Ca 2+] f was measured by the fluorescent calcium indicator Fura-2 and ANP in the effluent perifusate by radioimmunoassay. No cell damage was observed and the basal ANP secretion rate and [Ca 2+] f were comparable with values obtained by other methods. K +-induced depolarization raised [Ca 2+] f by 50%, but it rapidly declined again to a steady level 10–20% above the baseline. The calcium channel agonist Bay k8644 elicited a similar temporal pattern of [Ca 2+] f changes and 1 μM ionomycin induced a 100-fold increase in [Ca 2+] f with a slow re-establishment of the orginal baseline. None of these stimuli increased the ANP secretion rate of the atrial or ventricular myocytes. Protein kinase C activation by 12- O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated ANP secretion from the atrial myocytes, while the ventricular myocytes were unresponsive to TPA. It is concluded that Ca 2+ is not the main mediator in the regulation of ANP release in cultured neonatal heart cells.