Atrial peptide inactivation by rabbit‐kidney brush‐border membranes

Abstract

Atriopeptin (AP) 24, containing amino acids Serlo3 -Tyr126 of the carboxy-terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high-performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC. The major products were purified individually and their peptide sequences determined. Results indicate that AP24 was proteolytically cleaved at three peptide bonds: Ser103-Ser'04, C y~'~' -P h e '~~ and S e~-'~~-P h e ' 24. des-Serlo3-AP24 had similar vasorelaxant activity to AP24, while AP24 cleaved at C y~'~' -P h e ' '~ was inactive. Regarding the proteolytic cleavage at Ser123-Phe'24, there was an accumulation of the C-terminal tripeptide, Phe-Arg-Tyr, only at the later time points of the incubation. Degradation experiments were repeated with an amino-and carboxy-terminal protected peptide, acetyl-AP24-amide. Peptide sequence analysis of the major degradation products of this peptide revealed that the critical peptide bond cleaved was C y~'~' -P h e '~~. We conclude that the Cys-Phe peptide bond renders atrial peptides highly susceptible to proteolysis by renal brush border membranes, resulting in inactivation.Atrial peptides are factors which have been isolated from mammalian heart atria and effect natriuresis, diuresis and vasorelaxation [I, 21. Atriopeptin 24 (also named atriopeptin 111) consists of 24 amino acids with a disulfide bridge between the cysteine residues in positions 105 and 121, the ring structure of the peptide being important for bioactivity [3]. It has been reported for rat [4], dog [5] and human [6] that exogenously administered atrial peptides have a short biological half-life, of the order of a few minutes. Recent reports have implicated the kidney as a major site for clearance of these peptides from the circulation [7,8]. A number of peptide hormones, such as angiotensin, glucagon and lutcinizinghormone-releasing hormone [9] are believed to be proteolytically degraded at the brush border of the renal proximal tubule which is rich in hydrolases [I 01.In this study, we report that atriopeptin 24 is rapidly degraded by renal brush border membranes. From peptide sequence analysis of the degradation products from AP24 and acetyl-AP24-amide, we have determined that there is a single peptide bond which renders atrial peptides susceptible to endoproteolytic cleavage, resulting in inactivation.

Dendroaspis natriuretic peptide is degraded by a metalloproteinase in the rat kidney

“…Furthermore, following incubation for 4 h, the degradation of 125 I-labeled DNP remained attenuated by phenanthroline. The present findings are consistent with previous studies (17,18) and indicate the significance of NEP in the inactivation of DNP, similar to ANP, revealing that NEP is involved in the inactivation of BNP and ANP in the plasma. However, these observations are fairly different from an earlier study, which revealed that DNP was resistant to degradation by NEP (29).…”

Degradation and clearance of atrial natriuretic factors (ANF)

“…These enzTmes are membrane bound and their active sites face the lumen of the tubule (39). Incubating ANF(99-126) with these kidney microvilli results in a major degradation product cleaved between cysteine and phenylalanine (C'ys 105-Phe 106) (37,38,(40)(41)(42). Since the intact ring structure of ANF(99-126) is essential for its biological activity (43,44), ANF is effectively inactivated by this cleavage process.…”

Inhibition of Neutral Endopeptidase Causes Vasoconstriction of Human Resistance Vessels In Vivo

“…21,22,35 ANP may also be metabolized by an aminopeptidase, which is insensitive to thiorphan. 61 Although incomplete local NEP inhibition is possible, it is highly unlikely because the doses of both candoxatrilat and thiorphan used were chosen to achieve local blood concentrations in the forearm Ͼ50-fold and Ͼ10-fold higher than the IC 50 of (ϩ)candoxatrilat and thiorphan, respectively, for ANP in vitro. 39 Consistent with earlier work, 62 systemic ACE inhibition with enalapril had no effect on plasma ET concentrations.…”