The sites of gene expression of atrial, brain, and C-type natriuretic peptides in mouse fetal development: temporal changes in embryos and placenta.

Atrial (ANP), brain (BNP), and C-type natriuretic peptide (CNP) belong to a family of hormones important in blood pressure and sodium homeostasis. Expression of ANP has been reported in embryo hearts, but BNP and CNP expression during development has not been described. We used in situ hybridization to identify the sites of gene expression of ANP, BNP, and CNP during development in mouse embryos at daily intervals from midgestation. Very intense expression of ANP and BNP was visible in the heart from 9.5 days gestation; levels of expression of both peptides in the ventricle exceeded those in atria throughout gestation. There was a major peak of atrial and ventricular ANP and BNP expression at 12.5 days, attaining levels similar to those in adult heart and then declining until birth. Cardiac expression of CNP was undetectable. Expression of ANP and CNP was also observed in distinct sites in the brain, and all three peptides were expressed in the spinal cord. In mouse placenta, strong CNP expression was seen in the decidua basalis around the large maternal blood vessels, and BNP message was detected at the peripheral margin of the decidual layer. This pattern of expression indicates that ANP and CNP are present during development of the mouse central nervous system and suggests that CNP and BNP participate in regulating the maternal blood supply to the developing embryo.

Ovine brain natriuretic peptide in cardiac tissues and plasma: effects of cardiac hypertrophy and heart failure on tissue concentration and molecular forms.

Whereas numerous studies have examined the cardiac tissue content and secretion of atrial natriuretic peptide (ANP), the response of brain natriuretic peptide (BNP) in states of experimental cardiac overload is less well documented. Our recent partial cloning of the ovine BNP gene has enabled us to study changes in cardiac tissue concentration, together with tissue and circulating molecular forms of ANP and BNP, in response to cardiac overload induced by rapid ventricular pacing (n = 7) and aortic coarctation (n = 6). In normal sheep, although highest levels of BNP were found in atrial tissue (15-fold those of the ventricle), the BNP/ANP concentration ratio in the ventricles was 10- to 20-fold higher than the ratio calculated for atrial tissue. Compared with normal sheep, significant depletion of both ANP and BNP concentrations within the left ventricle occurred after rapid ventricular pacing. Size exclusion and reverse phase HPLC analysis of atrial and ventricular tissue extracts from normal and overloaded sheep showed a single peak of high molecular weight BNP consistent with the proBNP hormone. In contrast, immunoreactive BNP extracted from plasma drawn from the coronary sinus was all low molecular weight material. Further analysis of plasma BNP using ion exchange HPLC disclosed at least 3 distinct immunoreactive peaks consistent with ovine BNP forms 26-29 amino acid residues in length. These findings show that BNP is stored as the prohormone in sheep cardiac tissues and that complete processing to mature forms occurs at the time of secretion. The capacity to process the prohormone at secretion is not impaired by chronic heart failure.

The characterization of ovine genes for atrial, brain, and C-type natriuretic peptides.

The natriuretic peptides (NPs) play an important role in the homeostasis of blood pressure and sodium balance in all mammals studied to date. Their combined actions on the vasculature, kidneys, and adrenals reduce blood pressure and intravascular volume. In order to provide sequence information about the ovine NP genes for our physiological studies in sheep, we have determined the genomic DNA sequence of each of the NPs; atrial NP, brain NP (BNP), and C-type NP using an ovine genomic library. Strong homology with other species was found for ovine peptide and genomic sequences of atrial NP and for C-type NP. Further, despite previous reports of poor conservation of BNP across species, the peptide sequence for ovine BNP was found to be identical to both the 26 amino acid-residue porcine BNP, and the 35 amino acid peptide known as bovine aldosterone secretion-inhibitory factor. This data also revealed strong homology of BNP mature forms in dog, cow, pig, and sheep, thus permitting the use of porcine antisera to study BNP-level changes in sheep models of cardiac failure. This conservation of the BNP gene sequence suggests that BNP, like the other NPs, plays an important role in mammalian physiology.

Amino-terminal proBNP in ovine plasma: evidence for enhanced secretion in response to cardiac overload.

We have recently identified a novel amino-terminal fragment of pro-brain natriuretic peptide (NT-proBNP) in the circulation of humans, the concentration of which increases progressively as the left ventricle fails. To clarify the origins of NT-proBNP in experimental animals, we have developed an RIA for NT-proBNP based on residues 52-71 of ovine proBNP-(1-103) and used it to study cardiac processing, secretion, and metabolism of BNP in sheep with cardiac overload induced by coronary artery ligation (CAL) or rapid left ventricular pacing (rLVP). The concentration of NT-proBNP in left atrial plasma extracts drawn from normal control sheep was threefold that of mature BNP. Size-exclusion and reverse-phase HPLC analyses of plasma extracts coupled to RIA revealed a single peak of immunoreactive (ir) NT-proBNP [ approximately 8,000 relative molecular weight (Mr)], quite distinct from a single peak of ir-mature BNP ( approximately 3,000 Mr). In contrast, ovine cardiac tissue contained only a single immunoreactive peak of high-molecular-weight BNP ( approximately 11,000 Mr), consistent in size with proBNP-(1-103). Sampling from the cardiac coronary sinus in normal control sheep (n = 5) and sheep with CAL (n = 5) revealed that the molar ratio of NT-proBNP to mature BNP was similar. There was a significant gradient of both mature and NT-proBNP across the heart in normal sheep, whereas after CAL the gradient was significant for mature BNP only. In both forms of cardiac overload (CAL and rLVP), left atrial plasma levels of NT-proBNP were significantly increased above normal levels, in contrast with mature BNP levels, which were raised only in the rLVP group of animals. Blockade of natriuretic peptide metabolism in sheep with heart failure (induced by rLVP) raised mature BNP levels threefold but did not affect levels of NT-proBNP. In conclusion, these studies show that NT-proBNP is formed from proBNP stores during secretion and, compared with mature BNP, accumulates in plasma because metabolism of NT-proBNP appears to differ from that of mature BNP. Although its function, if any, remains unclear, plasma NT-proBNP may prove to be a sensitive marker of cardiac overload and/or decompensation.