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The localization of mRNA encoding preproatrial natriuretic peptide was investigated in tissue sections and cultures of rat heart and in sections of human right atrial appendage using the technique of in situ hybridization with 32P-and 35S-labeled RNA probes. Rat atrial natriuretic peptide (ANP) transcripts were demonstrated in numerous atrial myocytes and, to a lesser extent, in ventricular myocytes in both tissue sections and newborn rat heart cultures. These rmdings are consistent with those obtained by RNA blot analysis of rat heart total RNA, indicating that a single prepro-ANP transcript of ""900 nucleotides was present in the ventricles as well as the atria. Using a 35S-labeled RNA probe for human ANP mRNA, ANP transcripts were also localized to the majority of myocytes in the human right atrial appendage. Only background levels of autoradiographic labeling were obtained when RNA probes identical to the coding sequence of rat or human ANP mRNA were used. A close correlation was found between the distribution of ANP immunoreactivity and prepro-ANP mRNA in these preparations. These results provide unequivocal evidence for the expression of the ANP gene in the rat ventricles, as well as the atria, because myocytes in these tissues have been established as the sites of both ANP localization and precursor biosynthesis. The combined use of cardiac cultures and in situ hybridization may be of value in future studies investigating the regulation of ANP synthesis in cardiac myocytes.Cloning and sequence analysis of the complementary DNA (cDNA) encoding rat and human atrial natriuretic peptide (ANP) mRNA has revealed the structure of the prepro-ANP molecule, which consists of 151 and 152 amino acids in the human and rat, respectively (1-5). The nucleotide sequence of the genomic DNA encoding prepro-ANP has also been determined (6-11) and in man the gene is known to consist of 2 kilobase pairs, comprising three exons and two introns, located on the distal short arm of chromosome 1 (12). The ANP gene is expressed mainly in atrial tissues that contain both ANP transcripts (6, 13-16) and ANP immunoreactivity, the latter being localized to secretory granules in the atrial myocytes (17-19).However, in nonmammalian species ANP-like immunoreactivity occurs in the ventricles as well as the atria (18), and the results of mRNA blot analysis (6, 16) and radioimmunoassay (15, 19) together indicate at least some expression of the ANP gene in the ventricles of the rat, although normally at a lower level than in the atria. While the presence of atrial natriuretic peptides in ventricular cells might result from their synthesis at these sites, the possible uptake of circulating ANP cannot be excluded (15). Furthermore, immunocytochemical techniques alone are not very useful for examining the regulation of prepro-ANP production because it is not possible to distinguish between changes in synthesis and secretion. In situ hybridization provides a method for demonstrating gene expression at the cellular level and may be of value ...

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The nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity of intracardiac neurones in the rat and guinea-pig was studied at the ultrastructural level. While some nitric oxide synthase-containing intracardiac neurones were very heavily labelled, with electron-dense immunoprecipitate distributed throughout the neuronal cell bodies and their processes, most of the labelled neurones exhibited a lighter and more patchy distribution of nitric oxide synthase-immunoreactive material. Synapses made by nitric oxide synthase-negative nerve fibres with labelled intracardiac neurones were seen. Conversely, many nitric oxide synthase-containing nerve fibres that made synaptic contacts with unlabelled intracardiac neurones were also observed. Some small granule-containing cells were nitric oxide synthase-immunoreactive and were associated with unlabelled nerve terminals, while non-immunoreactive small granule-containing cells that were innervated by nitric oxide synthase-immunoreactive nerves were also seen. Small patches of osmiophilic electron-dense material were observed in the cytoplasm of NADPH-diaphorase-positive intracardiac neurones. This is the first description of the ultrastructural distribution of nitric oxide synthase-immunoreactivity and NADPH-diaphorase activity in a subpopulation of intracardiac neurones of rat and guinea-pig heart and provides further evidence in support of a role for nitric oxide in the local control of the heart by intrinsic neurones.

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