Clearance and early hydrolysis of atrial natriuretic factor in vivo. Structural analysis of cleavage sites and design of an analogue that inhibits hormone cleavage.

Abstract

This study examines the clearance and early hydrolysis of atrial natriuretic factor (ANF) in vivo. Radiolabeled ANF was cleared from the circulation of the rat with biphasic kinetics; the majority (90%) of ANF cleared with a tl/2 of 15 s, the remaining peptide was cleared with a t1/2 of 5 min. Microsequence analysis of ANF peptides recovered from the circulation of rats revealed five major degradation products of the intact hormone. The first cleavage occurred between amino acids 12 and 13 of the hormone and would inactivate ANF. Over time, additional fragments of the hormone were generated, including fragments of 6, 7, 21, and 24 amino acids in length.Whole body radioautography of rats injected with I123II-ANF revealed the kidney as a predominant organ involved in clearance of ANF. Subsequent amino acid sequence analyses of radiolabeled ANF exposed to the kidney in vivo indicated that this organ generated four of the five major hydrolysis products observed in circulation, namely, the 6, 7, 16, and 21 amino acid fragments of the hormone. In an attempt to stabilize ANF in vivo, a synthetic analogue of the hormone was prepared that contained the amino acid analogue, aminoisobutyric acid, substituted at position 13. This analogue completely abolished the in vivo cleavage of ANF at this site. These studies demonstrate the usefulness of a protein chemistry approach in characterizing hormone metabolism in vivo and designing analogues with enhanced in vivo stability to cleavage.

Protection of atrial natriuretic factor against degradation: diuretic and natriuretic responses after in vivo inhibition of enkephalinase (EC 3.4.24.11) by acetorphan.

“…This rapid degradation is consistent with the rapid clearance of the hormone and appearance of various fragments in plasma (14,26).…”

“…Radioiodinated ANF (125I-ANF), which retains the biological activity of the parent hormone, is cleared from rat plasma with a tl,2 of less than 1 min (14,26), but no attempt has been made to identify the responsible peptidase(s).…”

Oligopeptidase B from Trypanosoma evansi

“…7, A and G)). These data are consistent with reports that ANF is very stable in whole plasma in vitro (50) since ANF is removed from the circulation by clearance receptors (51) and is broken down by hydrolysis by membrane-bound neutral endopeptidase (51), a process that takes place primarily in the kidney (50). Since both the ANF clearance receptors and neutral endopeptidase are membrane-bound entities, plasma has little or no intrinsic ANF-degrading activity.…”

Respective roles of kallikrein and endopeptidase 24.11 in the metabolic pathway of atrial natriuretic peptide in the rat

“…In plasma, Gros et al (1989) observed a marked increase in circulating intact 125I-ANP after administration of acetorphan; however, they gave no further indication of the identity of the remaining circulating radioactivity. Condra et al (1988) identified by microsequence analysis five circulating labelled fragments appearing after a bolus injection of radiolabelled ANP in rats. In contrast to our findings, the first and major metabolite identified was a 16-amino-acid fragment corresponding to In the present study, degradation of peptides by plasma enzymes after sampling was inhibited by the use of EDTA together with 1,10-phenanthroline instead of soybean trypsin inhibitor and aprotinin.…”

“…In contrast, a constant infusion technique is independent of the sampling time and offers the possibility of identifying the labelled fragments resulting from the initial degradation steps. It is noteworthy that three of the five cleavage sites observed by Condra et al (1988) have never been identified as peptide bonds at which ANP is cleaved when incubated with purified E-24. 11.…”