Dieter Hock

Isolation and structural analysis of “Urodilatin”, a new peptide of the cardiodilatin-(ANP)-family, extracted from human urine

SummaryTwo major forms of cardiac peptides have been established in the last few years: (a) a prohormone of 126 amino acids (CDD/ANP-1-126) in the endocrine heart and (b) the circulating CDD/ANP-99-126 (=alpha ANP) in blood plasma. The method we applied earlier to isolate the circulating form of cardiodilatin from human blood was used to detect and analyze the biologically active, predominant form of the same polypeptide family excreted by the kidneys. Each step of the isolation procedure was followed up by a bioassay using an in vitro vascular smooth muscle relaxation test and a highly specific RIA against cardiodilatin (CDD-99-126) for the initial purification steps. The polypeptides excreted in 1000 1 of normal human urine were adsorbed to 2.5 kg of alginic acid, and after elution and lyophilization processed on a G-25 Sephadex column. The obtained crude polypeptide fractions were applied to ion-exchange chromatography. Thereafter four steps of HPLC were carried out to purify the polypeptide which was the suggested form of cardiodilatin (CDD) in human urine. The amino acid analysis and gas phase sequence analysis showed that the main form of urinary cardiodilatin is a 32 amino acid residue containing molecule, cardiodilatin-95–126. The molecule is N-terminally extended compared to the circulating CDD-99-126. This suggests that the analyzed urinary peptide is not the residual plasma form, filtrated and renally cleared from blood, but probably a polypeptide produced and processed in the kidney tubules and cleaved by a different postranslational process. Therefore, this vasorelaxant polypeptide is called urodilatin.

Urodilatin (CDD/ANP‐95‐126) is not biologically inactivated by a peptidase from dog kidney cortex membranes in contrast to atrial natriuretic peptide/cardiodilatin (α‐hANP/CDD‐99‐126)

Atrial natriuretic peptide (CDD/ANP‐99‐126) is rapidly inactivated by a membrane preparation from dog kidney cortex. Inactivation occurs by cleavage of the ring structure in the position between Cys‐105 and Phe‐106. A unique proteolytic product separated by HPLC on reverse‐phase column appears as a single peak which elutes prior the intact peptide. In contrast, CDD/ANP‐95‐126 (urodilatin) which is released from the kidney is not destroyed by proteolysis using an identical membrane preparation.

Isolation and structural analysis of the circulating human cardiodilatin (alpha ANP)

SummaryA new method was applied to isolate a polypeptide hormone from human blood. The polypeptides from 1,000 1 of hemofiltrate with a molecular weight lower than 20 kDaltons were adsorbed to 2.5 kg alginic acid, then eluted, precipitated, and desalted on a G-25 Sephadex column, thus obtaining a crude lyophilised plasma polypeptide extract. These polypeptides were further submitted to ion-exchange chromatography. Thereafter, two steps of HPLC were carried out to purify a distinct polypeptide which was the circulating form of cardiodilatin (CDD) in this case. The amino acid analysis, C-terminal enzymatic cleavage by carboxypeptidase A, and sequence analysis showed that the only form of circulating cardiodilatin is the 28 amino acid residue containing molecule, cardiodilatin-99-126 cleaved from the C-terminus of cardiodilatin-126 and identical with alpha-ANP (alpha atrial natriuretic polypeptide). Other bioactive molecular forms of the polypeptide hormones of the cardiodilatin family were not detected in the hemofiltrate. The isolation procedure was followed up by a bioassay using in vitro vascular smooth muscle relaxation.

Degradation of porcine brain natriuretic peptide (pBNP-26) by endoprotease-24.11 from kidney cortical membranes.

Porcine brain natriuretic peptide of 26 amino acid residues (pBNP-26) is inactivated by endoprotease-24.11 (EC 3.4.24.11) of kidney cortical membranes. In contrast to human alpha atrial natriuretic peptide/cardiodilatin (ANP/CDD) showing a single major cleavage within the disulfide-linked loop between Cys and Phe in position 7 and 8, pBNP-26 is cleaved at several sites. Although both pBNP-26 and ANP/CDD exhibit Cys-Phe peptide bonds at the corresponding positions this bond is not cleaved in BNP-26.

Isolation and characterization of the bioactive circulating human parathyroid hormone, hPTH-1–37

The occurrence of hPTH‐1–37 as the native bioactive circulating form of PTH‐1–84 has now been obtained using a specific purification procedure for circulating parathyroid hormone, which involves a newly developed immunoenzymetric assay for N‐terminally intact hPTH. In combination with two different methods of mass spectrometry, the molecular weight of the isolated immunoreactive peptide was shown to be 4401 Da, which corresponds to hPTH‐1–37. Synthetic hPTH‐1–37 material was tested in the chick bioassay and produced a clear‐cut increase in serum calcium concentration. We conclude that hPTH‐1–37 is the native bioactive fragment of hPTH‐1–84 in circulation.

Relaxation of smooth muscle by cardiodilatin/atrial natriuretic peptide is inhibited by cAMP-dependent phosphorylation

Cardiodilatins/atrial natriuretic peptides (CDD/ANP) exhibit a common amino acid sequence: Arg101‐Arg102‐Ser103‐Ser104. Cyclic AMP‐dependent phosphorylation of Ser104 of atrial peptides with [γ‐32P]ATP enables rapid identification of cardiac hormones. The biological activity of in vitro phosphorylated cardiodilatin (CDD‐28/α‐hANP) is dramatically altered compared to the unphosphorylated peptide: the vasorelaxant effect of cardiodilatin 28 is inhibited upon phosphorylation.

Isolation of bovine cardiodilatin by fast protein liquid chromatography and reversed-phase high-performance liquid chromatography

Cardiodilatin (CDD), a polypeptide exhibiting vasorelaxant and diuretic natriuretic bioactivity, was isolated from bovine atria. The isolation procedure reported here is different from that originally used for the purification of porcine and bovine CDD. Instead of cation-exchange chromatography on Fractogel TSK-CM 650 S and several purification steps on different high-performance liquid chromatographic (HPLC) columns, it is now possible to obtain CDD-88 by an automated fast-protein liquid chromatography system for repeated injections and a motor valve as fraction collector and only one final step of reversed-phase HPLC on a TSK-ODS-120T column.

Biochemistry of the Differential Release, Processing and Degradation of Cardiac and Related Peptide Hormones

Investigations during the last few years supported the existence of two major forms of cardiac peptides. In the heart, a prohormone of 126 amino acid residues (CDD-1-126) is stored, while CDD-99-126/ANP (= αANP) is the circulating form in the blood plasma. By use of a similar isolation procedure, as used for the isolation of circulating CDD-99-126/ANP, a biological active peptide, strongly related to CDD-99126/ANP, was purified from urine, indicating excretion from the kidneys. Sequence analysis shows that the main form of urinary cardiodilatin is a 32 amino acid residue containing molecule (urodilatin-95-126) similar to CDD-99-126/ANP except N-terminal extension by four amino acid residues. The prolongation first of all indicates that urodilatin is not derived from a plasma form and obtained by filtration and renal clearance. Secondly, it demonstrates that urodilatin undergoes a different posttranslational process during excretion than CDD-99-126/ANP. Both, synthetic human cardiodilatin (CDD-99-126)/atrial natriuretic peptide (ANP) and urodilatin (95–126) are phosphorylated by the catalytic subunit of cAMP-dependent protein kinase at a serine residue in position 104. Phosphorylated peptides exhibit decreased relaxing potency compared to unphosphorylated peptides. CDD-99-126/ANP is rapidly removed from the circulation by clearance and proteolytic degradation. Inactivation of CDD-99-126/ANP in the kidneys occurs by an endoprotease-24.11 present in the cortical membranes. Due to the enzymatic reaction a major cleavage occurs within the ring structure between Cys105 and Phe106. In contrast, N-terminal extension by four amino acid residues (Thr-Ala-Pro-Arg) and cAMP-dependent phosphorylation of CDD-99-126/ANP inhibit proteolysis by kidney cortical membranes.

Pharmacokinetic and Pharmacodynamic Characteristics of Subcutaneously Applied PTH-1-37

Background/Aims: Parathyroid hormone (PTH) derivatives exert pronounced renal and osteoanabolic properties when given intermittently. The current study was performed to assess the pharmacokinetic and pharmacodynamic properties as well as safety of subcutaneously applied PTH-1-37 after repeated dosing in healthy subjects. Methods: This randomized, double-blind, dose-escalating, placebo and active comparator controlled study was conducted in 33 healthy postmenopausal women. Subjects were allocated to one of five treatment options: 10, 20, or 40 µg PTH-1-37, 20 µg PTH-1-34 or placebo, administered as once daily subcutaneous doses for three days. Plasma drug concentrations and serum levels of endogenous PTH-1-84, and calcium as markers of biological activity were monitored during the treatment. Results: PTH was absorbed rapidly from the subcutaneous tissue with a median tmax of 30 minutes for 20 and 40 µg of PTH-1-37. tmax was 45 minutes for 20 µg PTH-1-34. Elimination half-lives were estimated as 76 ± 34 min and 70 ± 13 min for 20 µg and 40 µg PTH-1-37 (mean ± SD), and 78 ± 34 for 20 µg PTH-1-34. Both PTH fragments (PTH-1-37 and PTH-1-34) increased serum calcium. For PTH-1-37 the effect on serum calcium was dose-dependent. Suppression of endogenous PTH-1-84 was seen after the application of both PTH-1-37 and PTH-1-34. During the study period, the subjects experienced no unexpected or serious adverse events. Conclusions: PTH-1-37 is rapidly absorbed after s.c. injection, has a short plasma elimination half-life, and does not accumulate during multiple dosing. Biological activity was demonstrated by rising serum calcium and decreasing endogenous PTH-1-84 in blood plasma. The study drugs were well tolerated and safe. Our investigation presents data that PTH-1-37 is an excellent drug candidate for intervening with syndromes of dysregulation of calcium metabolism.