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Biomedical subjects

R Takayanagi

Publications and source records attributed to R Takayanagi.

At least 127 records · Page 7Linked to original sources
PubMed1989

Porcine brain natriuretic peptide receptor in bovine adrenal cortex.

The action of porcine brain natriuretic peptide (pBNP) on the steroidogenesis was investigated in cultured bovine adrenocortical cells. Porcine BNP induced a significant dose-dependent inhibition of both ACTH- and A II-stimulated aldosterone secretion. 10(-8) M and 10(-7) M pBNP also significantly inhibited ACTH-stimulated cortisol and dehydroepiandrosterone (DHEA) secretions. Binding studies of [125I]-pBNP to bovine adrenocortical membrane fractions showed that adrenal cortex had high-affinity and low-capacity pBNP binding sites, with a dissociation constant (Kd) of 1.70 x 10(-10) M and a maximal binding capacity (Bmax) of 19.9 fmol/mg protein. Finally, the 135 Kd radioactive band was specially visualized in the affinity labeling of bovine adrenal cortex with disuccinimidyl suberate (DSS). These results suggest that pBNP may have receptor-mediated suppressive actions on bovine adrenal steroidogenesis, similar to that in atrial natriuretic peptide (ANP).

Adrenal Cortex↗
PubMed1989

Effect of porcine brain natriuretic peptide (pBNP) on human adrenocortical steroidogenesis.

Porcine brain natriuretic peptide (pBNP), purified from porcine brain, had a significant suppressive effect on aldosterone and cortisol secretions in ACTH-treated cultured human adrenal cells. Concomitantly, the intracellular cGMP formation was enhanced by pBNP treatment. A specific pBNP receptor was identified in the human adrenal tissues. Affinity labelling of 125I-pBNP showed two separate molecular weights of specific binding sites for pBNP of 140 and 67 kDa. A 125I-pBNP binding study of the human adrenal membrane fraction demonstrated the presence of high-affinity and low-capacity binding sites for pBNP. Moreover, these binding sites for 125I-pBNP were displaced by unlabelled alpha-rANP as well as pBNP. From these studies, we concluded that pBNP had suppressive effects on human adrenocortical steroidogensis, possibly via a receptor which may be shared with ANP.

Adrenal Cortex↗
PubMed1989

Evidence for age-related change in plasma 19-hydroxyandrostenedione.

The steroid, 19-hydroxyandrost-4-ene-3, 17-dione (19-hydroxyandrostene-dione, 19-OH-A-dione) has been known to enhance the mineralocorticoid action of aldosterone. To investigate the age-related change in the plasma 19-OH-A-dione concentration, plasma 19-OH-A-dione, androst-4-ene-3, 17-dione (A-dione), aldosterone and cortisol of 38 non-hypertensive healthy subjects (18 young men and 20 aged men) measured by specific radioimmunoassays. The basal plasma 19-OH-A-dione and A-dione concentration in aged men was significantly lower than in young men (P less than 0.01). Moreover, there was found to be a positive correlation between plasma 19-OH-A-dione and A-dione (P less than 0.01). On the other hand, plasma aldosterone and cortisol in aged men showed a tendency to decrease, but no statistical significance compared to young men was observed. This study demonstrated that there was an apparent age-related decrease not only in plasma A-dione, but also in plasma 19-OH-A-dione, an amplifier or aldosterone action.

Adolescent↗
PubMed1988

Atrioactivase, a specific peptidase in bovine atria for the processing of pro-atrial natriuretic factor. Purification and characterization.

A seryl protease which catalyzes conversion of proatrial natriuretic factor (ANF) to the active circulating form, ANF(99-126), was purified from a particulate fraction of bovine atria. The enzyme was solubilized with 1.6 M KCl. The molecular mass of the purified enzyme was 580 kDa on gel filtration, whereas by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a cluster of six bands with molecular masses around 30 kDa was observed. The purified enzyme produced ANF(99-126) from partially purified bovine pro-ANF by the selective cleavage of the arginyl peptide bond in the -Pro97-Arg98-Ser99-sequence in pro-ANF. The enzyme was localized mainly in the microsomal fraction rather than the granule fraction. It is likely that the enzyme selectively cleaves the Arg98-Ser99 peptide bond in pro-ANF during the process of secretion.

Animals↗
PubMed1988

Purification of a specific peptidase in bovine atria for the processing of pro-atrial natriuretic factor.

A serine protease which catalyses the conversion of the precursor of ANF (atrial natriuretic factor) to the active circulating form, ANF-(99-126)-octacosapeptide, was purified from a particulate fraction of bovine atria. The enzyme was solubilized with a buffer containing 1.6M KCl. The molecular mass of the purified enzyme was 580 kDa on gel filtration, whereas on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a cluster of six bands with molecular masses around 30 kDa was observed. The purified enzyme produced the ANF-(99-126)-octacosapeptide from partially purified bovine pro-ANF by the selective cleavage of the arginyl peptide bond in the -Pro97-Arg98-Ser99-sequence in pro-ANF. It is likely that the enzyme selectively cleaves the Arg98-Ser99 peptide bond in pro-ANF when ANF is secreted into the circulation.

Animals↗
PubMed1987

Two distinct forms of receptors for atrial natriuretic factor in bovine adrenocortical cells. Purification, ligand binding, and peptide mapping.

The atrial natriuretic factor (ANF) receptor of bovine adrenal cortex was solubilized with Triton X-100 and purified by sequential chromatography on ANF-(99-126)-agarose, GTP-agarose, and wheat germ agglutinin-Sepharose. Two subtypes of ANF receptors were isolated, both of which showed specific ANF binding, whereas one of the ANF receptor subtypes also possessed significant cyclase activity. Both of the receptors showed high capacities (Bmax = 5.7-6.8 nmol/mg of protein) and high affinities (Kd = 54-68 pM) for ANF-(99-126). The cyclase-free receptor had high affinity (Ki = 150-220 pM) to C-terminal truncated ANF analogs, whereas the cyclase-containing receptor had a much weaker affinity (Ki = 10(6)-10(7) pM). When treated with dithiothreitol, the purified cyclase-containing and cyclase-free ANF receptors migrated as a single band at Mr 135,000 and 62,000, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cyclase-free receptor is not a product derived from the cyclase-containing receptor because (i) two proteins with Mr of 135,000 and 62,000 were specifically labeled with 4-azidobenzoyl 125I-ANF-(102-126) in nonsolubilized intact membranes; (ii) the truncated ANF analogs (10(4) pM) prevented the photolabeling of the 62,000-dalton protein but not that of the 135,000-dalton protein; and (iii) two-dimensional peptide mapping showed more than 90% difference between the profiles of the two purified ANF receptor subtypes. This study provides first direct evidence for the existence of two distinct ANF receptors which are different not only in their pharmacological properties but also in their primary structure.

Adrenal Cortex↗
PubMed1987

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.

Adrenal Cortex↗
PubMed1987

Identification of a peptidase which processes atrial natriuretic factor precursor to its active form with 28 amino acid residues in particulate fractions of rat atrial homogenate.

With the objective of identifying specific peptidase responsible for the processing of atrial natriuretic factor precursor pro-ANF to the circulating active form ANF (99-126), a fluorometric assay method was devised using synthetic fluorogenic substrate Boc-Ala-Gly-Pro-Arg-MCA(methylcoumarinamide) which contains the amino acid sequence immediately adjacent to the arginyl peptide bond which is cleaved in the natural processing of pro-ANF. A protease which selectively cleaves this bond and produces the natural circulating peptide was identified in the particulate fraction of rat atrial homogenate and was solubilized by 1.6 M KCl. It was partially purified by affinity chromatography heparin-agarose column and was shown to be a serine protease. Its reaction product with natural pro-ANF was identified as ANF (99-126) containing 28 amino acid residues.

Animals↗
PubMed1987

Synthesis and presence of atrial natriuretic factor in rat ventricle.

Rat heart ventricles contained immunoreactive atrial natriuretic factor (irANF) and mRNA for ANF. The size of ANF mRNA in the ventricle was identical with that of the atria. High performance gel filtration chromatography showed that 84% of ventricular irANF elutes at a position corresponding to the low molecular weight form of ANF (99-126) and 16% of irANF elutes at a position corresponding to the precursor form of ANF. The irANF content of the ventricles of spontaneously hypertensive rats was 3 times as much as that of Wistar Kyoto rats. These results suggest that ventricle synthesizes ANF in response to hypertension and processes in a manner different from that in atria.

Animals↗
PubMed1987

Identification of atrial natriuretic factor receptor of neuroblastoma N4TG1 cells: binding characteristics and photoaffinity labeling.

We have found specific receptors for atrial natriuretic factor (ANF) in cultured neuroblastoma cells (N4TG1) of peripheral ganglionic origin. Scatchard analysis of the displacement binding revealed noninteracting, single-class binding sites with a KD of 1 X 10(-10) M and a density (Bmax) of 110,000-150,000 sites/cell. The cell-bound 125I-ANF was displaced by unlabeled ANF in a dose-dependent manner. Hormones unrelated to ANF such as angiotensins, adrenocorticotropic hormone, or arginine vasopressin were ineffective in displacing the cell-bound radioactivity. Using azidobenzoyl-125I-ANF as a photoaffinity ligand, an ANF receptor with an apparent Mr of 138,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The addition of unlabeled ANF (1 microM) to the incubation medium completely abolished the labeling of this protein band, but atriopeptin I (1 microM) or angiotensins I, II, and III (each 1 microM) were not effective in inhibiting the affinity labeling. The treatment of the neuroblastoma cells with ANF stimulated intracellular cyclic GMP levels in a dose-dependent manner with an EC50 of 5 nM. ANF (1 X 10(-7) M) stimulated cyclic GMP accumulation in less than 5 min by 30-fold as compared to the controls.

Affinity Labels↗
PubMed1987

Structure and physiological actions of rat atrial natriuretic factor.

Natriuretic substances were purified from rat atrium (atrial natriuretic factor, ANF) and were shown to be identical with the inhibitor of norepinephrine-induced contraction of smooth muscle. Four native forms were isolated and their amino acid sequences were determined. The presence of a high-molecular-weight prohormone was shown. Complementary DNA (cDNA) encoding for the precursor was cloned and used to deduce the amino acid sequence of the prohormone. Genomic DNA for ANF was cloned and two introns were found. Several ANF peptides were synthesized. Structure-function studies showed that the ring structure was essential for the activity. Antibodies produced against the synthetic 25-amino acid residue ANF were used to develop a radioimmunoassay. The presence of ANF in rat plasma demonstrated that ANF is a circulating hormone. ANF was also found in the hypothalamus of rats. The ANF in plasma was found to be a low-molecular form, whereas that in atria and hypothalamus consisted of both the high-molecular-weight precursor and low-molecular-weight active ANF. The presence of messenger RNA for ANF was determined using ANF cDNA as a probe and was considered as evidence for ANF synthesis in the brain, atrium, and ventricles. ANF was shown to be released from the brain. ANF administered intracerebroventricularly was shown to inhibit angiotensin II and thirst-induced dipsogenesis. In vitro and in vivo experiments showed ANF inhibits release of vasopressin from posterior pituitary and renin from the kidneys. The hypotensive effect of ANF was examined at various doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Regional increase of cyclic GMP by atrial natriuretic factor in rat brain: markedly elevated response in spontaneously hypertensive rats.

Atrial natriuretic factor (ANF)-responsive areas in rat brain were examined by measuring ANF-stimulated cyclic GMP production in rat brain slice preparations. The medulla oblongata, thalamus, and pituitary gland responded most sensitively, the septum, hypothalamus, pons, midbrain and olfactory bulb responded moderately, but neocortex, cerebellum, striatum and hippocampus were unresponsive to ANF. The most responsive regions in spontaneously hypertensive rats brains showed 2 to 5 times higher cyclic GMP production than those from the control Wistar-Kyoto rats. These findings provide evidence for biological action of ANF on brain tissues, and indicate the action of ANF produced in the brain.

Animals↗
PubMed1986

Relative inactivation of steroidogenic enzyme activities of in vitro vitamin E-depleted human adrenal microsomes by lipid peroxidation.

The effects of lipid peroxidation and vitamin E on the steroidogenic activities of human adrenal microsomes were studied. The vitamin E content in the microsomes could be varied by treating the lyophilized microsomes with n-pentane, without affecting the steroidogenic enzyme activities. When the level of microsomal vitamin E in the adrenal was reduced to that in other tissues such as liver and kidney, NADPH-supported lipid peroxidation increased about 200-fold, and concomitantly, the steroidogenic enzyme activities decreased. After 5 min of the lipid peroxidation reaction, 17 alpha-hydroxylase and C17,20-lyase activities were inactivated to 13% and 18%, respectively, of the original activities. 3 beta-Hydroxysteroid dehydrogenase-isomerase and 21-hydroxylase, however, retained 89% and 84%, respectively, of the original activities. When vitamin E was reincorporated into the original activities. When vitamin E was reincorporated into the extracted microsomes, neither the lipid peroxidation reaction nor the inactivation of the enzyme activities was observed. These results indicate that the high concentration of vitamin E in adrenal protects the enzymes from oxidative damage, and that the microsomal C19 steroidogenic cytochrome P-450 activities are highly sensitive to lipid peroxidation. This suggests an association between adrenal lipid peroxidation and a decrease in C19 steroid synthesis with advancing age.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Atrial natriuretic factor in spontaneously hypertensive rats: concentration changes with the progression of hypertension and elevated formation of cyclic GMP.

As hypertension developed in spontaneously hypertensive rats (SHR), the plasma concentration of atrial natriuretic factor (ANF) increased whereas its tissue concentration in the atria decreased. These observations suggest that ANF is secreted from the atria in response to hypertension. Atrial natriuretic factor contents in the hypothalamus and pons decreased with ageing in Wistar-Kyoto rats (WKY) but not in SHR. The responses of various SHR tissues to the hypotensive, vasorelaxant and cyclic GMP generating effects of ANF were more pronounced than in corresponding WKY tissues. The number of ANF receptors was reduced without change in the affinity in aortic smooth muscle and adrenals of SHR with established hypertension. These findings suggest that the elevated sensitivity to ANF of blood pressure of SHR can be in part explained by the increased sensitivity to ANF of guanylate cyclase in the vascular wall of SHR.

Animals↗
PubMed1985

Changes in the content of atrial natriuretic factor with the progression of hypertension in spontaneously hypertensive rats.

In order to assess possible roles of atrial natriuretic factor (ANF) in spontaneously hypertensive rats (SHR), we examined the content of immunoreactive-ANF in plasma, the atria, hypothalamus and pons of SHR and Wister Kyoto (WKY) rats by radioimmunoassay at different stages of age. With the progression of hypertension, plasma concentration of ANF increased whereas it decreased in the atria in SHR. This suggests ANF is secreted in response to hypertension. On the other hand, at hypothalamus and pons, ANF content was significantly higher in SHR than in WKY rats. This finding suggests possible involvement of ANF in the central regulation of blood pressure.

Aging↗
PubMed1985

Effects of changes in water-sodium balance on levels of atrial natriuretic factor messenger RNA and peptide in rats.

Responses of atrial mRNA, atrial peptide and plasma peptide of atrial natriuretic factor (ANF) to treatments to alter fluid volume were studied in rats using RNA dot hybridization assay and radioimmunoassay. Specific changes in the level of ANF mRNA relative to total atrial RNA were observed in atria from sodium restricted rats and water deprived then sodium loaded rats, demonstrating an association of change in water-sodium balance with the expression of ANF gene. The levels of mRNA and the immunoreactive ANF in plasma decreased to 30% and 15% of controls, respectively, on water-deprivation and then increased again to control levels after administering 1.8% NaCl solution, whereas atrial immunoreactive ANF increased to about twice the control on water-deprivation and decreased again after supplying NaCl solution, in parallel with the level of the hematocrit. These findings suggest that atrial ANF content is dependent more on ANF release than on biosynthesis.

Animals↗
PubMed1985

Mechanism of dissociation of cortisol and adrenal androgen secretion after removal of adrenocortical adenoma in patients with Cushing's syndrome.

We investigated the mechanism of dissociation of cortisol and dehydroepiandrosterone sulfate (DHEA-S) secretion by the adrenal glands after the removal of an adrenal gland containing an adrenocortical adenoma in a patient with Cushing's syndrome. After removal of the adrenocortical adenoma, the serum cortisol rapidly decreased from 24.6 +/- 6.4 micrograms/dl (mean +/- SD, n = 6) to 0.7 +/- 0.5 micrograms/dl. Serum DHEA-S levels were 15 +/- 14 micrograms/dl and 6 +/- 9 micrograms/dl before and after surgery, respectively, and significantly lower than the control values. Serum cortisol levels reverted to normal levels 1.5 to 3 years after the surgery. On the other hand, DHEA-S levels reverted to normal 5 to 7 years after the serum cortisol levels had normalized. Monolayer cultures of normal human adrenal cells obtained at adrenalectomy in patients with advanced breast cancer and atrophic adrenal cells adjacent to the adrenocortical adenoma in patients with Cushing's syndrome were used to study the mechanism of the dissociation of cortisol and DHEA-S secretion. ACTH caused significant increases in the productions of pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OH-P5), 17-hydroxyprogesterone (17-OH-P4), DHEA, DHEA-S, androstenedione (delta 4-A), and cortisol. The amounts of 17-OH-P5 and 17-OH-P4 produced by ACTH in atrophic adrenal cells were significantly greater than those in normal adrenal cells. The amounts of DHEA, DHEA-S and delta 4-A produced by ACTH in atrophic adrenal cells were significantly smaller than those of normal adrenal cells. The conversion rate of 17-OH-[3H]P5 to 17-OH-[3H]P4 and 11-deoxy-[3H] cortisol was higher in atrophic adrenal cells than in normal adrenal cells, but the conversion rate to [3H]DHEA, [3H]DHEA-S and [3H]delta 4-A was significantly lower in atrophic adrenal cells than in normal adrenal cells. These results suggest that the dissociation of cortisol from DHEA-S after the removal of adrenocortical adenoma is a probably due to diminished C17,20-lyase activity in the remaining atrophic adrenal gland.

Adenoma↗