Kunio S. Misono

Atrial natriuretic factor in rat hypothalamus, atria and plasma: determination by specific radioimmunoassay.

A rapid and reproducible radioimmunoassay method was developed for rat atrial natriuretic factor (ANF)-IV. The method is also applicable to human atrial peptide. ANF was detected in rat hypothalamus (5.03 pmoles/g tissue), right (86.8 pmoles/mg tissue) and left atria (52.5 pmoles/mg tissue), and plasma (156 fmoles/ml). After high salt intake immunoreactive ANF in atria and plasma increased significantly, while a significant decrease was observed in hypothalamus. Gel chromatography revealed high and low molecular weight ANF in atria and hypothalamus while only a low molecular weight form was found in plasma.

Rat atrial natriuretic factor: Complete amino acid sequence and disulfide linkage essential for biological activity

The complete amino acid sequence of an atrial natriuretic peptide from rat possessing both natriuretic and smooth muscle relaxant activity has been determined. The peptide has the structure (sequence in text) and a calculated molecular weight of 2,706. The ring structure formed by the disulfide linkage between the two half-cystine residues was found essential for both the natriuretic activity and smooth muscle relaxant activity. The purified peptide caused 50% relaxation of norepinephrine (5 X 10(-8)M) induced contraction of rabbit thoracic aorta at the concentration of 2 X 10(-9)M and complete relaxation at 6 X 10(-9)M.

Rat atrial natriuretic factor. Purification and vasorelaxant activity.

The atrial natriuretic activity of rat heart has been found to exist in multiple forms. One of these factors has been purified to apparent homogeneity by a combination of gel filtration and high pressure liquid chromatography in two different systems and its amino acid composition determined. The purified active peptide is shown to have a molecular weight of approximately 3800. In addition, the vasorelaxant activity of rat atrium has been purified and found to co-chromatograph with the natriuretic activity in all chromatographic systems employed. Thus, the vasorelaxant activity resides in the natriuretic factor. The existence of this new multifunctional peptide implies a higher level of complexity for cardiovascular control of blood volume and pressure.

Rat atrial natriuretic factor: Isolation, structure and biological activities of four major peptides

Four peptides possessing both natriuretic activity and smooth muscle relaxant activity were isolated from rat atrium and their amino acid sequences determined. The four peptides designated ANF-I, ANF-II, ANF-III and ANF-IV containing 35, 31, 30 and 25 amino acid residues, respectively, were obtained in a molar ratio of 4:60:20:16. The predominant species ANF-II, which may represent the native form of ANF, has the following sequence: (H2N)-G-P-R-S-L-R-R-S-S-C-F-G-G-R-I-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y-(COO H) in which Cys-10 and Cys-26 are disulfide linked. Cleavage of the aspartyl linkage at position 16 by staphylococcal protease caused complete inactivation, indicating that the ring conformation is essential. The dose-response relationships determined for the four peptides in relaxing norepinephrine-induced contraction of rabbit thoracic aorta showed half-maximum relaxation at concentrations ranging from 1.5 X 10(-9) to 2.5 X 10(-9) M. Comparable dose-response relationships were observed in relaxation of carbacol-induced contraction of chick rectum strips as tested with ANF-II and ANF-IV.

Purification and characterization of two types of atrial natriuretic factor receptors from bovine adrenal cortex: Guanylate cyclase-linked and cyclase-free receptors

Atrial natriuretic factor (ANF) receptors with and without guanylate cyclase activity were simultaneously purified to apparent homogeneity from bovine adrenal zona glomerulosa cell membrane fractions. The particulate guanylate cyclase which co-purified with the ANF receptor showed one of the highest specific activity reported. The receptors with or without the guanylate cyclase activity showed high affinities to ANF (99-126). The receptor without the cyclase showed a high affinity to truncated ANF analogs, ANF (103-123) and ANF (105-121), whereas the cyclase-linked receptor had a much lower affinity to these analogs. Both of the receptors migrated as a single band with a molecular weight of 135,000 daltons on SDS-gel electrophoresis under non-reducing conditions. The 135,000 daltons band of the receptor without the cyclase was shifted to a 62,000 daltons band under reducing conditions, but the band for the cyclase-linked receptor was not shifted. These results demonstrated the presence of two subtypes of ANF receptor in bovine adrenal cortex and indicate two different modes of intracellular action of ANF.

Alterations in atrial and plasma atrial natriuretic factor (ANF) content during development of hypoxia-induced pulmonary hypertension in the rat

Abstract Distension of the atrial wall has been proposed as a signal for the increased release of atrial natriuretic factor (ANF) from atrial myocytes in response to perceived volume overload. To determine whether pressure changes resulting from hypertension in the pulmonary circulation may stimulate release of ANF, rats were exposed to chronic hypobaric hypoxia for 3 or 21 days and the ANF concentration in the atria and plasma were determined by specific radioimmunoassay. Exposure to chronic hypoxia resulted in significant increases in hematocrit at both 3 (p < 0.025) and 21 days (p < 0.005) and in the development of right ventricular hypertrophy (RVH) expressed as the ratio of the weight of the right ventricle to the weight of the left ventricle and septum (RV/LV+S) at both 3 (RV/LV+S = 0.278±0.005) and 21 days (RV/LV+S = 0.536±0.021). After 21 days, left atrial (LA) ANF content was significantly increased in hypoxic rats compared to controls (508±70 ng/mg tissue vs 302±37 ng/mg), while right atrial (RA) ANF content was significantly reduced (440±45 vs 601±58 ng/mg). At this time, plasma ANF concentration was significantly elevated compared to controls (238±107 pg/ml vs 101±10 pg/ml). These results suggest that the development of pulmonary hypertension following chronic hypobaric exposure induces altered atrial ANF content and increased plasma ANF concentration as a result of altered distension of the atrial wall.

Synthesis of atrial natriuretic peptides and studies on structural factors in tissue specificity

Two atrial natriuretic peptides, containing 25 amino acid residues, ANF IV, and 21 amino acid residues, ANF V, were synthesized by a solid phase method and oxidized with K3Fe(CN)6 to form a disulfide bridge. Synthetic ANF IV exhibited a natriuretic activity with an ED50 70 times higher than that of synthetic ANF V, whereas the longer peptide was only 2.5 times more potent in chick rectal smooth muscle relaxant activity. Both peptides inhibited norepinephrine-induced contraction of rabbit aorta. The shorter peptide, ANF V, was 300 times less efficient than the longer peptide, ANF IV. It is proposed that the carboxy-terminal of ANF IV seems to have a modulating effect on receptor affinity in kidney and vascular tissue.

Evidence for intracellular formation of angiotensins: Coexistence of renin and angiotensin-converting enzyme in Leydig cells of rat testis

Leydig cells were purified from rat testes by discontinuous metrizamide density gradient and were shown to contain renin (EC 3.4.99.1), angiotensin-converting enzyme (dipeptidyl carboxypeptidase, (EC 3.4.15.1), and the peptide hormone angiotensins I, II and III as determined by the combined HPLC and radioimmunoassay. In germinal cells only angiotensin II (AII) was found at a significant level. These findings provide evidence for intracellular formation of AII in testicular cells and demonstrate that an intracellular renin-angiotensin system exists in normal non-transformed cells.

Photoaffinity labeling of atrial natriuretic factor receptor in bovine and rat adrenal cortical membranes

Radioiodinated synthetic atrial natriuretic factor (ANF) bound to a single class of high affinity binding sites in the plasma membrane from bovine adrenal cortex with a KD of 7.4 X 10(-10) M. The binding affinities of related peptides showed close parallelism to their potencies in natriuretic and vasorelaxant activities. Incubation of adrenal membranes with radioiodinated 4-azidobenzoyl ANF or a similar derivative of its analogue followed by photolysis resulted in specific radiolabeling of a protein band in SDS gel electrophoresis with an apparent Mr of 124,000 in bovine or Mr of 126,000 in rat, which was abolished by inclusion of unmodified ANF in the incubation. Prevention of the labeling was dependent on the concentration of ANF and was not observed with atriopeptin I or with unrelated peptides, angiotensin II, ACTH or [Arg8] vasopressin. These results indicate specific covalent labeling of ANF-receptor or its subunit by the photoaffinity ligands.

Rapid and large-scale purification and characterization of renin from mouse submaxillary gland

Abstract Little is known about the structural basis for the highly restricted substrate specificity of renin, whose only function known to date is to hydrolyze the unique leucyl peptide bond in the prohormone angiotensinogen to form angiotensin I. Our lack of knowledge is due to our inability to purify this enzyme in a large quantity sufficient for structural studies. A two-step column chromatographic method for rapid and large-scale purification of renin from mouse submaxillary gland has been developed. It allows isolation of the enzyme in a hundreds-of-milligrams quantity at an overall yield of 60%. Single bands obtained by polyacrylamide gel electrophoresis, isoelectric focusing, and the result of ultracentrifugal studies indicated homogeneity of the product. The moelcular weight of renin was estimated to be 36,000 by sedimentation equilibrium studies in 6 m guanidine · HCl. Sedimentation velocity study gave a single sedimenting boundary with an s 20,w of 2.58 × 10 −13 s. A Stokes radius of 27 A was obtained by gel filtration. The far ultraviolet-circular dichroism spectrum indicated a high content of β-structure (46%). In contrast to renal renin, submaxillary gland renin does not contain amino sugars or neutral sugars. No renin activity was retained by concanavalin Aagarose gels. Results of amino acid analysis, isoelectric focusing, and determination of amino-terminal residues by the dansylchloride reaction, together indicated that this protein is identical with renin A isolated previously in a much smaller quantity.