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T Inagami

Publications and source records attributed to T Inagami.

At least 289 records · Page 16Linked to original sources
PubMed1988

Suppression of angiotensin II release by prostaglandin synthesis inhibitors in hind legs.

Previously we reported that immunoreactive angiotensin II (Ang II) release from isolated perfused rat mesenteric arteries was mediated by beta-adrenergic receptor activation. However, the precise mechanism of regulation of vascular renin-angiotensin is not completely understood. In this study, we examined the effect of indomethacin and meclofenamate on immunoreactive angiotensin I (Ang I) and immunoreactive Ang II release from perfused rat hind leg vasculature to delineate the possible relevance of prostaglandins to the vascular renin-angiotensin system in vitro. We also examined the effects of isoproterenol and propranolol on the immunoreactive Ang I and II release. Isolated rat hind legs were perfused with Krebs-Ringer solution, and immunoreactive Ang I and II released into the perfusate were measured directly by using a Sep-Pak C18 cartridge connected to the perfusion system. Indomethacin and meclofenamate (10(-8) to 2 X 10(-6) M) added to the perfusion medium suppressed immunoreactive Ang I and II release to similar extents in a dose-dependent manner (p less than 0.001); the maximal percent inhibition of immunoreactive Ang II release evoked by these inhibitors (2 X 10(-6) M) was 60 +/- 6% (p less than 0.001) for indomethacin and 50 +/- 4% (p less than 0.001) for meclofenamate. Isoproterenol (10(-6) M) failed to cause a change in the release of both peptides, but propranolol (10(-6) M) slightly decreased the release of immunoreactive Ang I and II by 28 +/- 4% (p less than 0.001) and 32 +/- 4% (p less than 0.001), respectively. There was a highly significant positive correlation between the released amount of immunoreactive Ang I and that of immunoreactive Ang II altered by indomethacin (r = 0.91), meclofenamate (r = 0.94), or propranolol administration (r = 0.90). These results suggest that the renin-angiotensin in the hind legs is modulated by prostaglandins and that a difference exists in the beta-adrenergic receptor-mediated release of Ang II among diverse vascular beds.

Angiotensin I↗
PubMed1988

Presence of renin secretory granules in rat adrenal gland and stimulation of renin secretion by angiotensin II but not by adrenocorticotropin.

Renin has been identified biochemically and immunohistochemically in the adrenal gland. We examined the subcellular distribution and behavior of adrenal renin. By differential centrifugation of adrenal capsules, we found renin mainly in mitochondrial fractions. By Percoll density gradient centrifugation of this fraction, dense granules were separated from mitochondria and microsomes. The renin activity in the dense granules from the capsules of nephrectomized rats was 15 times greater than that of the intact rat. Immunohistochemical studies revealed that the dense granules increased in number after bilateral nephrectomy. Immunogold staining of these granules showed unequivocally the presence of renin in these granules. Adrenal capsules in organ culture were found to release renin at a steady rate. Renin release from bilaterally nephrectomized rat adrenals was 46 times faster than from the organs of intact animals. The mechanism of the control of renin secretion from the adrenal gland was different from the kidney in that the secretion was stimulated by potassium chloride (10 mM) or angiotensin II (10(-9)-10(-7) M) but not by ACTH (10(-9)-10(-7) M), suggesting stimulation by intracellular calcium. These results provide evidence that the adrenal synthesizes renin, stores it in specific secretory granules and secretes it in a regulated manner.

Adrenal Cortex↗
PubMed1988

Immunohistochemical localization of atrial natriuretic peptide binding sites in juxtaglomerular cells and vascular walls of rat kidney.

Since the kidney is one of the major sites of action for atrial natriuretic peptide (ANP) and immunoreactive ANP has been detected in tissue extract by radioimmunoassay, we have applied the immunohistochemical technique by using the avidin-biotin complex method to investigate ANP binding sites in the rat kidney. Although no immunostaining was observed in the kidney of control rats, immunoreactive ANP was present in the juxtaglomerular cells, the vascular walls of interlobular arteries, arcuate arteries, arterioles including vas afferens and vas efferens, and the medullary peritubular capillary of ANP-pretreated rats. In contrast, no tubular structure was stained. These results suggest that ANP may affect renin secretion via its direct action on the juxtaglomerular cells and that it predominantly induces natriuresis by its effects on renal hemodynamics.

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

Specific endogenous Na, K-ATPase inhibitor purified from bovine adrenal.

The endogenous inhibitor of Na, K-ATPase has been implicated in the pathogenesis of salt-induced hypertension. In spite of an intensive search the inhibitor has long remained elusive. We have been able to purify such an inhibitor from methanol extract of bovine adrenal glands by multiple steps of high-performance liquid chromatography (HPLC). This compound with a molecular mass of 336 showed striking similarity to the cardiac glycoside ouabain in its dose dependency in the inhibition of Na, K-ATPase and Na-pump activity, competitive binding to the ouabain-binding site, and dependence of these effects on K+ concentration. These inhibitory activities were potentiated with reduced K+ concentration in parallel with ouabain. These results indicates that vertebrate animals possess a regulator of Na, K-ATPase and suggests the possibility that it may be the mediator of salt-induced high blood pressure.

Adrenal Glands↗
PubMed1987

Direct release of angiotensins I and II from isolated rat kidney perfused with angiotensinogen-free medium.

A direct measurement of both angiotensins I and II immunoreactive substances was made in the perfusate from isolated rat kidney perfused with Krebs-Ringer solution which was free of any component of the renin-angiotensin system. The identity of the immunoreactive peptides was confirmed as angiotensin I and angiotensin II by high-pressure liquid chromatography in reference to standard compounds. The rate of release of angiotensins was as high as 1313.5 +/- 184.5 and 772.4 +/- 82.5 pg for angiotensins I and II, respectively, during the first perfusion period of 20 min, and it remained stable at least for 2 hours. There was a good relationship between the angiotensin I secretion rate and renin secretion rate simultaneously determined in the perfusate, and also between the angiotensin I secretion rate and angiotensin II secretion rate. These results taken together with the previous observations of the coexistence of renin and angiotensins I and II in juxtaglomerular cells of the kidney provide evidence for intrarenal formation and release of angiotensin II. It does not agree with the notion that these peptides are internalized from circulation. Angiotensin II secreted from the kidney may play diverse functions in intrarenal regulation.

Angiotensin I↗
PubMed1987

New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms.

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.

Adrenal Cortex↗
PubMed1987

Three distinct forms of atrial natriuretic factor receptors: kidney tubular epithelium cells and vascular smooth muscle cells contain different types of receptors.

Three distinct ANF receptor subtypes have been identified and characterized from cultured canine kidney tubular (MDCK) cells and rat thoracic aortic smooth muscle (RTASM) cells. These three ANF receptor subtypes include; (1) a disulfide-linked 140 kDa protein found in RTASM cells which was reduced by sulfhydryl reagent dithiothreitol (DTT) to a 70 kDa band, (2) a disulfide-unlinked 120 kDa protein, specific to MDCK cells whose Mr was not reduced by DTT and (3) a 68-70 kDa protein prevalent in both RTASM and MDCK cells whose Mr was not reduced by DTT. The non-reducible 68-70 kDa and the reducible 140 kDa proteins showed strong affinities to the full-length ANF (99-126) and truncated ANF (103-123) peptides, however, non-reducible 120 kDa protein showed strong affinity only to the full length ANF (99-126) but negligible or very weak affinity to truncated ANF (103-123). These findings suggest that distinct ANF receptor subtypes are present in renal and vascular cells which might be linked to diverse physiological functions of ANF such as natriuresis and diuresis in kidney and vasorelaxation in vascular smooth muscle cells.

Affinity Labels↗
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

Participation of adenosine 5'-triphosphate in the activation of membrane-bound guanylate cyclase by the atrial natriuretic factor.

The addition of ANF to Percoll-purified liver plasma membranes produced a slight activation of guanylate cyclase; the ANF-stimulated cyclase activity was further increased upon the addition of ATP to the enzyme assay mixture. The effect of ATP to potentiate the cyclase activation was concentration-dependent, required Mg2+ as a divalent cation, and was seen with membranes from various tissues and cells. ATP increased the maximal velocity of the cyclase without a change in the affinity for GTP or ANF. Phosphorylation by ATP might not be involved since ANF-stimulated guanylate cyclase was enhanced by non-phosphorylating ATP analogues as well. Thus, an allosteric ATP binding site is suggested to participate in ANF-induced regulation of membrane-bound guanylate cyclase.

Adenine Nucleotides↗
PubMed1987

Coronary hemodynamics and cardiac beating modulate atrial natriuretic factor release from isolated Langendorff-perfused rat hearts.

The role of coronary hemodynamics and cardiac beating on atrial natriuretic factor (ANF) release was studied in the isolated Langendorff-perfused rat heart. ANF release was measured by radioimmunoassay. When the coronary flow rate was changed, ANF release decreased or increased in a flow-dependent manner. When the perfusion pressure was changed, ANF release also increased or decreased, respectively, with concomitant changes in coronary flow rate. Furthermore, perfusion with 50 mM potassium chloride showed immediate cardiac arrest and a decrease of ANF release to an undetectable level with a significant decrease in coronary flow. However, low but readily detectable amounts of ANF were released when coronary flow rate was maintained. These results may suggest that coronary hemodynamics and cardiac beating could be factors modulating ANF secretion from the atrium.

Animals↗
PubMed1987

Lysophosphatidylcholines containing polyunsaturated fatty acids were found as Na+, K+-ATPase inhibitors in acutely volume-expanded hog.

Na+, K+-ATPase inhibitors possessing inhibitory activities against the specific binding of ouabain to Na+, K+-ATPase and 86Rb uptake into hog erythrocytes have been purified from the plasma of acutely saline-infused hog. The purifications were performed by a combination of Amberlite XAD-2 adsorption chromatography and four steps of high-performance liquid chromatography with four different types of columns. Fast atom bombardment (FAB) mass and proton NMR spectrometric studies identified the purified substances as gamma-arachidoyl- [LPCA(gamma), 34%], beta-arachidoyl- [LPCA(beta), 4%], gamma-linoleoyl- (LPCL, 33%), and gamma-oleoyl- (LPCO, 25%) lysophosphatidylcholine, expressed in molar ratio in the plasma. Small amounts of gamma-docosapentaenoyl-, gamma-eicosatrienoyl-, and gamma-palmitoyllysophosphatidylcholine were also detected by both FAB mass and 1H NMR spectrometric studies. Only gamma-acyl-LPC's showed inhibitory activities on Na+,K+-ATPase and ouabain-binding activities. These LPC's were effective at 100 microM levels in attaining 50% inhibition of the enzyme activity. The inhibition of Na+,K+-ATPase activity due to these compounds was always more sensitive than that of both ouabain-binding and 86Rb uptake activities. The ouabain-displacing activity in plasma due to these compounds increased with time during saline infusion. The maximal plasma level was approximately 10 times higher than that in the preinfusion plasma sample. Although these results suggest the gamma-acyl-LPC's with long-chain polyunsaturated fatty acids are not simple competitive inhibitors to Na+, K+ -ATPase, these compounds could be implicated in the pathogenesis of the circulation abnormality through the modulation of membrane enzyme.

Animals↗
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

Characterization of pure human renal renin. Evidence for a subunit structure.

Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.

Amino Acid Sequence↗
PubMed1987

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri.

ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102-126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled. Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.

Adrenal Medulla↗