The distributions of atrial natriuretic peptide (ANP) in human hearts during the developmental stage and in adult pathological states was examined with an antibody specific to human cr-ANP. With immunoblotting and immunofluorescence methods, we found that a 17-kDa protein, which is a pro ANP, was expressed in human fetal ventricles, in which the numbers of myofibers containing ANP granules were more abundant in the subendocardial region than the subepicardial region. As determined by radioimmunoassay, the content of immunoreactive ANP (per milligram protein) in the developing heart was greatest in the left atrium and occurred decreasingly in the right atrium, right ventricle, and left ventricle, respectively. Because ANP content in the left ventricle declined during the progress of gestation in developing hearts and because it was very low, if ever detectable, in normal adult hearts, ventricular ANP expression appears to be developmentally regulated from the early gestational stage. However, it was reexpressed in the ventricles of patients who had suffered from severe congestive heart failure. In this situation, we found that the ventricular ANP expression was more marked in patients with dilated cardiomyopathy than in patients with severe valvular disease. Interestingly, in the ventricles of patients with dilated cardiomyopathy, ANP contents were higher in the left ventricular free wall than in the right ventricular free wall, although the left ventricular subendocardium contained more ANP than the subepicardium, showing a transmural gradient similar to that expressed in fetal ventricles. Thus, the expression of ANP in human ventricles is developmentally regulated from the early gestational stage, and even adult ventricular myofibers can synthesize ANP during severe congestive heart failure. Considering the ventricular site that is subjected to an overload in fetal and adult pathological states and considering that myofibers in the subendocardial region normally sulfer from a greater wall stress than do those in the epicardial region, it is reasonable to hypothesize that focal factors, such as wall stress, contribute to the expression of ventricular ANP. (Circulation 1988;78:920-927)
RadioimmunoassaySamples were homogenized in 10 volumes extraction buffer and diluted to appropriate concentrations in radioimmunoassay (RIA) buffer (100 mM phosphate-buffered solution at pH 7.2, 0.1% bovine serum albumin, 0.05% Tween 20, 0.1% NaN3, and 500 units/ml aprotinin). Then, 100 g1 diluted extract or various amounts of unlabeled ha-ANP were placed in tubes, together with 100 gl RIA buffer, and 50 ,ud diluted anti-ANP antibody, and the mixtures were incubated at 40 C for 24 hours, after which '25I-labeled ha-ANP (about 2,500 cpm) in 50 gl RIA buffer was added to each sample. After incubation at 4°C overnight, free and bound ANP fractions were separated by precipitation with a sufficient amount of anti-rabbit immunoglobulin G in 12.5% polyethylene glycol 6000. After centrifugation, the supernatant was decanted, and the ...
