Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage.

Abstract

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 ...

CCR3-Blocking Antibody Inhibits Allergen-Induced Eosinophil Recruitment in Human Skin Xenografts from Allergic Patients

“…In situ hybridization was performed as previously described (Hamid et al, 1987;Tsicopoulos et al, 1998). Briefly, sections were hybridized with 35 S-labeled riboprobes (2 ϫ 10 6 cpm per section) in hybridization buffer containing 50 mM dithiothreitol (Promega, Madison, Wisconsin).…”

Growth and partial differentiation of presumptive human cardiac myoblasts in culture.

“…Atrial natriuretic factor is expressed at very high levels in fetal atrium and ventricle (Block et al, 1986;Kikuchi et al, 1987), and is restricted to myocardial cells in the heart (Hamid et al, 1987). Induction of ANF expression was identified in this study as a marker for the differentiation of cardiac myoblasts in culture.…”

“…Expression of ANF has been observed at high levels in fetal atrium and ventricle (Bloch et al, 1986), and in situ hybridization experiments (Hamid et al, 1987) have revealed ANF transcripts in atrial and ventricular myocytes and not in other types of cells in the heart. Expression of ANF therefore represents a useful marker of cardiac myogenesis.…”

Mr-Proanp Rises during Exercise Even after Surgical Closure of the Left Atrial Appendage: A Sub-Study of the Laacs Randomized Study

“…It has been reported that the most intensely staining granules are localized in the atrial appendages [13,14]. There is still a need for better mapping of the ANP producing areas, [8] and it is claimed that 30 % of the ANP in the heart is contained in the LAA [15].…”

“…Therefore resting levels of natriuretic peptides are higher in chronic heart failure and atrial fibrillation compared with normal [1,11,12]. ANP gene transcripts have been shown in both atrial and ventricular myocytes; however, the level of ventricular ANP transcription is generally lower than that found in the atria [1,9,13].…”