Norio Ohnuma

C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells.

C-type natriuretic peptide (CNP) which potently stimulates particulate guanylate cyclase activity in cultured rat vascular smooth muscle cells (VSMC) inhibited serum-induced DNA synthesis of the cells 10-fold more effectively than alpha-human atrial natriuretic peptide (alpha-hANP). The inhibitory effect of CNP was mimicked by 8-bromo-cGMP. The proliferation of VSMC was also suppressed by CNP more potently than alpha-hANP, while the peptide was less active for cGMP augmentation and for vasorelaxation than alpha-hANP in isolated rat aorta. These results suggest that CNP may be a growth regulating factor of VSMC rather than a vasodilator.

Novel natriuretic peptide, CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cells.

The newly identified peptide C-type natriuretic peptide (CNP) caused only a slight elevation of cGMP in rat renal glomeruli. In contrast, CNP potently increased cGMP levels in cultured rat vascular smooth muscle cells (VSMC) and stimulated guanylate cyclase activity in the particulate fraction of the cells. The extent of maximum activation of the enzyme induced by CNP was 4-fold higher than that by human atrial natriuretic peptide (alpha-hANP) while CNP was 4- and 16-fold weaker than alpha-hANP in binding affinity for the putative receptors on VSMC and vasorelaxant activity for rat aorta, respectively. These results indicate that CNP is a potent stimulator of cGMP formation in VSMC but not in glomeruli and pharmacological feature of CNP is distinct from that of ANP.

Isolation of a new metabolite of vitamin D produced in vivo, 1α,25-dihydroxyvitamin D3-26,23-lactone☆

Abstract A new vitamin D metabolite was isolated in pure form from the blood of rats given oral doses of 50 μg/kg of 1α-hydroxyvitamin D 3 . The isolation involved methanol-chloroform extraction and four successive column chromatographic procedures. A tentative structure of the metabolite is proposed on the basis of its column chromatographic behavior via mass spectrometry, ultraviolet absorption spectrophotometry, and as 1α,3β,25-trihydroxy-9,10 (19)-cholestatrieno-26,23-lactone. The trivial name 1α,25-dihydroxyvitamin D 3 -26,23-lactone is suggested for this compound.

Studies on the mode of action of calciferol XIX. A 24R-hydroxyl-group can replace the 25-hydroxyl-group of 1α,25-dihydroxyvitamin D3 for optimal binding to the chick intestinal receptor☆

Abstract Various 1α-hydroxylated side chain analogs of vitamin D3 have been studied for their ability to compete with 1α,25-dihydroxy[3H]vitamin D3 for binding to the chick intestinal receptor. Of the analogs examined, 1α,24R-dihydroxyvitamin D3 was found to be nearly equivalent to 1α,25-dihydroxyvitamin D3 in its ability to compete for receptor binding. However, this near equivalence was not shared by its stereoisomer, 1α,24S-dihydroxyvitamin D3, which was only 10% as effective a competitor. It is proposed that the ability of a 24R-hydroxyl group to mimic the 25-hydroxyl group is not due to a lack of side chain specificity on the part of the receptor, but is instead due to the similar orientation of the 25-hydroxyl and the 24R-hydroxyl such that they can be accommodated equivalently by the receptor.

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.

Production in vitro of 1α,25-dihydroxyvitamin D3-26,23-lactone from 1α,25-dihydroxyvitamin D3 by rat small intestinal mucosa homogenates

Abstract The metabolism of 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] in the rat has been studied under both in vivo and in vitro conditions. A time course study of the appearance of 1α,25-dihydroxyvitamin D 3 -26,23-lactone in the plasma following intravenous or oral administration of 1α,25(OH) 2 D 3 suggests that the small intestine may take part in production of the 1α,25(OH) 2 D 3 -26,23-lactone. In an in vitro study using a homogenate of rat small intestinal mucosa, 1α,25(OH) 2 D 3 undergoes further metabolism to give more polar metabolite(s) which comigrate with authentic 1α,24,25-trihydroxyvitamin D 3 [1α,24,25(OH) 3 D 3 ] on Sephadex LH-20 column chromatography. The metabolic profile obtained after high-pressure liquid chromatography reveals two major classes of metabolites, designated Peaks X and Y. Peak X is an unidentified metabolite of 1α,25(OH) 2 D 3 . Peak Y is chromatographically identical with 1α,25-dihydroxyvitamin D 3 -26,23-lactone which has been recently isolated from the plasma of rats and dogs as a major metabolite produced in vivo from either 1α,25(OH) 2 D 3 or 1α-hydroxyvitamin D 3 ( N. Ohnuma, K. Bannai, H. Yamaguchi, Y. Hashimoto, and A. W. Norman, 1980, Arch. Biochem. Biophys. 204, 387 ). The enzyme activity which produces metabolites X and Y in the rat intestinal homogenates is induced in vitamin D-replete rats by pretreatment of the animals with intravenous 1.25 μg/kg doses of 1α,25-dihydroxyvitamin D 3 , 6 to 8 h previously.

A new strategy for metabolic stabilization of motilin using the C-terminal part of ghrelin.

Ghrelin consists of 28 amino acid residues with an octanoyl modification at the third serine residue. Recently we have found that the C-terminal part of ghrelin protects the ester bond of 3-octanoyled serine from plasma esterases and plays the essential role to prolong the plasma half-life and to show its biological activity in vivo. In the present study, we researched whether the C-terminal part of ghrelin has a potential to prolong the plasma half-life of motilin, by comparing the pharmacokinetics of various chimeric peptides of ghrelin and motilin. Motilin is another gastro-intestinal peptide hormone related with ghrelin structurally, binding to the same family of G protein-coupled receptors. Chimeric peptides were designed to be composed of motilin(1-12) fragment, the active core binding to the motilin receptor, GPR38, and C-terminal part of ghrelin. The modification of motilin(1-12) fragment by C-terminal part of ghrelin hardly influenced its agonist activity to GPR38 and almost all these chimeric peptides showed more than two times longer plasma half-lives than motilin in rats. From the relationship between structures of chimeric peptides and their corresponding plasma half-lives, the mid-region of ghrelin rich in basic amino acids ((15)RKESKK(20)) was considered to be the most important in prolonging the plasma half-life of motilin. The deletion of these fragments or replacement of 17th glutamic acid with a neutral amino acid resulted in short plasma half-lives. In conclusion, our data suggested that the C-terminal part of ghrelin has a potential to improve the biokinetics of motilin probably by a metabolic stabilizing effect.