Production and Differential Endocrine Regulation of Atrial Natriuretic Peptide in Neuron-Enriched Primary Cultures*

Abstract

To identify factors that directly regulate the synthesis and secretion of atrial natriuretic peptide (ANP) in neuronal cells, we have developed a neuron-enriched primary culture system from fetal rat brains. A number of factors proved of importance in maintaining adequate levels of ANP secretion in such cultures: 1) cultures derived from diencephalon produced much more ANP than cultures derived from diencephalon produced with the distribution of ANP-containing cells in the rat brain; 2) brains from rats at gestational day 17 proved a better source of ANP-secreting cells than brains from rats at gestational day 16; 3) the presence of serum was required in the latter stages of the culture period to allow expression of the ANP gene; and 4) the cultures secreted more ANP when maintained at 39 C vs. 37 C. ANP mRNA transcripts in the neuron-enriched primary cultures were analyzed by S1 nuclease protection and shown to have a transcription start site similar to that employed by rat atrium and fetal hypothalamus in vivo. Dexamethasone and T3, in contrast to their stimulatory effect on ANP production in cardiocyte cultures, suppressed both the release of immunoreactive ANP and the levels of ANP mRNA in the neuron-enriched primary cultures. The cultures incorporated [35S]cysteine into immunoprecipitable ANP. HPLC analysis of 35S-labeled products in the medium revealed that, unlike neonatal cardiocyte cultures, the majority of secreted immunoreactive ANP migrated with the processed form(s) of ANP rather than the prohormone.