The propeptide Asn1–Tyr126 is the storage form of rat atrial natriuretic factor

Abstract

Granules from rat atria were isolated by differential centrifugation and by a 53% (v/v) Percoll gradient after tissue homogenization in 0.25 M-sucrose/50 mM-Na2EDTA. About 40% of the immunoreactive ANF (atrial natriuretic factor) sedimented with the atrial granules during differential centrifugations. On the Percoll gradient, two distinct bands were observed. Cell debris, mitochondria, lysosomes, myofilaments and microsomes were mostly contained in the lightest-density (rho) (1.03-1.07 g/ml) fraction, as demonstrated by electron microscopy and by enzymic markers such as lactate dehydrogenase, monoamine oxidase, cytochrome c reductase, beta-glucuronidase and acid phosphatase. Atrial granules were mostly contained in the denser (rho 1.11-1.15 g/ml) band and were only slightly contaminated by lysosomes, as shown by beta-glucuronidase activity. Analysis of the ANF content in these isolated granules by h.p.l.c., amino acid composition and sequencing demonstrated that it was only the pro-ANF [ANF-(Asn1-Tyr126)-peptide]. The precursor was present in all granules, as demonstrated by immunocytochemistry. Since hormonal propeptides usually undergo intracellular processing, and the matured peptides are subsequently stored in the secretory granules, these results indicate that the processing pathway of ANF may be different from that of other hormonal peptides.

The heart is the center of a new endocrine, paracrine, and neuroendocrine system.

“…900 of controversy when the first amino-acid sequences of CDD/ANP from extracted cardiac polypeptides were published in 1983(see review FoRSSMANN, 1986. Extraction studies indicated that the entire prohormone is probably stored in the secretory granules of myoendocrine cells (FORSSMANN et al, 1984), as confirmed by other researchers (BLOCH et al, 1985;NOKIHARA et al, 1987;THIBAULT et al, 1987;ZISFEIN et al, 1987;HASSALL et al, 1988). It is now well accepted that the prohormone of CDD/ANP contains 126 amino acids; minor quantities of smaller molecules occur in fresh endocrine heart tissue and the release of CDD/ANP is followed by the immediate formation of CDD-99-126 (SHIELDS and GLEMBOTSKI, 1988), the circulating, biologically active cardiac hormone K.…”

Breed Differences in Natriuretic Peptides in Healthy Dogs

“…Both atrial natriuretic peptide (ANP) and B‐type natriuretic peptide (BNP) are synthesized as high‐molecular weight precursors (prohormones), which are cleaved into 2 segments21, 22; 1 biologically active peptide in the C‐terminal region described as ANP or BNP, and 1 biologically inactive N‐terminal segment described as NT‐proANP or NT‐proBNP 1, 21, 22, 23. NT‐proANP is further cleaved into 3 segments; proANP 1‐30, 31‐67, and 68‐98 24.…”

Annexins V and VI: major calcium-dependent atrial secretory granule-binding proteins.

“…These membranes were separated from granules as a low density band in a Percoll gradient as described previously. 3 The preparation consisted mainly of broken cell membranes, microsomes, mitochondria, and lysosomes with very few granules.…”

“…Granules were isolated essentially as reported earlier. 3 Batches of 30 atria (from 15 rats) were processed and pooled. They were rinsed, minced, and homogenized in 0.25 M sucrose, 10 mM tris (hydroxymethyl) aminomethane (Tris) (pH 7.4), and 50 mM ethylenediamine-tetraacetic acid (EDTA) (2 atria/ml).…”