Regulation of cardiovascular system activity involves complex interactions amongst numerous factors. Three of these vasoactive factors are adrenomedullin, C-type natriuretic peptide (CNP) and endothelin-1 (ET-1), each of which is claimed to have important local effects. To investigate paracrine/autocrine regulation of the secretion of these peptides we used a cell immunoblot method. We postulated that basal release of adrenomedullin and CNP by endothelial cells is modulated by ET-1. Dispersed human aortic endothelial cells were attached to a protein binding membrane and incubated for 1 or 4 h with control medium or with ET-1, endothelin receptor antagonists or antibody to ET-1, and then submitted to immunohistochemical staining. Peptides (adrenomedullin, CNP and ET-1) within individual cells were stained, as was peptide secreted and adjacent to the cell. It was demonstrated that adrenomedullin, CNP and ET-1 can be contained within the same cell. In addition, we observed that individual endothelial cells can secrete all three peptides. The endothelin ET-A/ET-B receptor antagonist, bosentan, the ET-B receptor antagonist, BQ-788, and anti-ET-1 serum decreased the percentage of endothelial cells that secreted adrenomedullin and CNP relative to control. Conversely, the addition of ET-1 induced an increase in the number of endothelial cells that secreted adrenomedullin and CNP. These results provide strong evidence that endogenous ET-1, from human vascular endothelial cells, acts in a paracrine/autocrine manner to modulate the basal release of adrenomedullin and CNP. Our observations of this modulation suggest that vascular endothelial cells of humans constitute an important component of a self-responsive vasoregulatory system.

Metabolism of natriuretic peptides is regulated by two degradative pathways: uptake by the clearance receptor (natriuretic peptide receptor C--NPR-C) and hydrolysis by neutral endopeptidase (NEP). Affinity studies favour a dominant role of NPR-C in hormone degradation in several species but do not account for the efficacy of NEP inhibitors in vivo, nor the uniquely prolonged half life (t((1/2))) of human brain natriuretic peptide (hBNP). Postulating that (1) delayed metabolism of hBNP reflects resistance to NEP and (2) interactions between NPR-C and NEP increase enzyme activity, we have used purified ovine and human NEP, plus ovine lung plasma membranes to study the relative importance of receptor and enzyme pathways. We have also related the findings to hormone metabolism in vivo. Binding affinities of atrial natriuretic peptide (ANP), hBNP and ovine BNP (oBNP) to oNPR-C were similar (K(d)=8-16 pM). In contrast, unlike ANP and oBNP, hBNP was not significantly degraded by purified oNEP or plasma membranes. Despite similar (and high) affinity of oNPR-C for oBNP and hBNP, the t((1/2)) of hBNP (12.7 min) was more than fourfold that of oBNP (2.6 min). Although we found no evidence for receptor-enzyme interaction, our results show that the delayed metabolism of hBNP reflects resistance to NEP. These findings have important implications for future treatment strategies in human disease.

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.