Biomedical subjects

M Iwashina

Publications and source records attributed to M Iwashina.

14 recordsLinked to original sources

PubMed2001

Co-expression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues.

BACKGROUND: Urotensin-II (UII), a cyclic dodecapeptide originally isolated from fish urophysis that has potent cardiovascular effects, has recently been identified as an endogenous ligand for the orphan G protein-coupled receptor, GPR14. The physiological roles of endogenous UII and its receptor in humans remain unknown. OBJECTIVE: To investigate the presence of human (h) UII-like immunoreactivity (hUII-LI) in human biological fluids, and the expression of hUII and GPR14 genes in human tissues. METHODS: We have established a specific radioimmunoassay for hUII and the real-time quantitative reverse transcriptase polymerase chain reaction method using LightCycler for the quantification of hUII and GPR14 mRNAs. RESULTS: Gel filtration and reverse-phase high performance liquid chromatography of human urine extracts revealed a single major peak of hUII-LI co-eluting with known hUII. The concentrations of hUII-LI in urine from normal individuals were 7.4 +/- 0.9 microg/g creatinine, whereas its plasma concentration was undetectable (< 50 pg/ml). Urinary hUII concentrations from patients with essential hypertension and those with renal tubular abnormality, but not with glomerular diseases, were significantly greater than those from normal individuals. The resulting fractional excretion of hUII, exceeding the glomerular filtration rate, suggests a renal origin of urinary UII-LI. hUII mRNAs were abundantly expressed in the kidney and the right atrium, but far less so in the vasculature, whereas GPR14 mRNAs were equally and abundantly expressed in both cardiovascular and renal tissues. CONCLUSIONS: These data suggest that urinary hUII is derived mainly from a renal source, and that hUII functions as an autocrine/paracrine vasoactive factor not only in the cardiovascular system, but also in the kidney, with an as yet unspecified function.

Adult↗

PubMed1999

Natriuretic peptides and nitric oxide induce endothelial apoptosis via a cGMP-dependent mechanism.

Apoptosis is a mode of cell death in which the cell participates in its own demise. We studied whether endothelium-derived relaxing factor, nitric oxide (NO), and natriuretic peptides affect apoptosis of rat vascular endothelial cells via a cGMP-dependent pathway and whether such effects are antagonized by an endothelium-derived vasoconstrictor, endothelin-1 (ET-1). Three natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide) induced endothelial apoptosis as demonstrated by nucleosomal laddering on agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling method. This dose-dependent relation was assessed by quantifying the fragmented and intact DNA contents by the diphenylamine method. The atrial natriuretic peptide-induced endothelial apoptosis was completely blocked by a guanylate cyclase-coupled receptor antagonist (HS-142-1) and an inhibitor of cGMP-dependent protein kinase (KT5823). An NO donor, NOR3 ((+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide; FK409) also induced endothelial apoptosis; the effect of this compound was abrogated by KT5823 and an inhibitor of soluble guanylate cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). A cGMP derivative, 8-bromo-cGMP, but not the cAMP derivative 8-bromo-cAMP, caused endothelial apoptosis; the effect of ODQ was also abrogated by KT5823. Endothelial apoptosis induced by ANP, NOR3, and 8-bromo-cGMP was similarly antagonized by ET-1. ANP, NOR3, and 8-bromo-cGMP caused marked accumulations of the tumor suppressor gene product p53 but not of bcl-2, as determined by Western blot analysis. These results demonstrate for the first time that endothelium-derived NO and natriuretic peptides are proapoptotic factors for endothelial cells, whereas the endothelium-derived vasoconstrictor ET-1 is an antiapoptotic factor, suggesting that the countervailing balance between these vasodilators and vasoconstrictors, in addition to regulation of vascular tonus, may contribute to endothelial cell integrity.

Animals↗

PubMed1998

Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells.

BACKGROUND: Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in a variety of physiological processes including vascular remodeling. To elucidate whether endogenous NO generated by iNOS is involved in the programmed cell death (apoptosis) of the vasculature, iNOS cDNA- expressing construct was transfected into rat and human vascular smooth muscle cells (VSMCs) by lipofection. METHODS AND RESULTS: VSMCs transiently transfected with iNOS cDNA functionally expressed 130 kd iNOS protein with full catalytic activity to generate massive NO in proportion to the doses of cDNA used; its enzymatic activity as well as NO production was completely blocked by an NOS inhibitor, NG-monomethyl-L-arginine (LNMMA). Overexpression of iNOS led to a marked inhibition of DNA synthesis as well as induction of apoptosis in VSMCs. Evidence for apoptotic cell death was provided by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry analysis. Apoptosis after transfection with iNOS cDNA was abrogated by LNMMA. Transfection of iNOS cDNA caused accumulation of the tumor suppressor gene p53 but not of bcl-2, which was also blocked by LNMMA. CONCLUSIONS: These results demonstrate that massive generation of endogenous NO derived from iNOS overexpression leads to a marked apoptosis in VSMCs, thus suggesting an important role of NO as a proapoptotic factor for VSMCs in the process of vascular remodeling.

Animals↗

PubMed1998

Secretion of adrenomedullin by renal tubular cell lines.

Adrenomedullin (AM) is a novel and potent vasodilator peptide originally isolated from human pheochromocytoma. The present study was designed to study whether AM is produced by and secreted from renal tubular cell lines and whether arginine vasopressin (AVP) affects AM secretion from these cell lines. Three renal tubular cell lines derived from different species (LLCPK1, MDCK, and MDBK) secrete AM-like immunoreactivity (AM-LI) into culture medium, the immunological and physicochemical properties of which are similar to that of synthetic human AM as evaluated by reverse-phase high-performance liquid chromatography. Among the three cell lines, AVP in combination with a phosphodiesterase inhibitor (isobutylmethylxanthine) stimulated AM-LI secretion most potently from MDCK cells in a time- and dose-dependent manner. In MDCK cells, a V2 receptor agonist (deamino-D-Arg8-vasopressin) dose-dependently stimulated AM-LI secretion in the same manner as AVP. Furthermore, the AVP-induced AM-LI secretion was blocked by a V2 receptor antagonist (OPC31260), but not by a V1 receptor antagonist (OPC21268). These data indicate that AM is secreted from renal tubular cell lines and that AVP stimulates AM secretion via V2 receptors, suggesting its autocrine/paracrine role in renal function.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Abnormal FSH hypersecretion as an endocrinological manifestation of POEMS syndrome.

Two patients with POEMS syndrome had high basal levels of FSH in the absence of primary hypogonadism. They were fertile eugonadal men with normal serum testosterone and estradiol levels. Provocation with LH-releasing hormone revealed blunted response of FSH secretion, but normal response of LH secretion in both patients, and one had pituitary microadenoma on brain MRI. This report adds primary FSH hypersecretion in the absence of primary testicular failure to the list of endocrinopathy of POEMS syndrome.

Adenoma↗

PubMed1997

[Endothelin and nitric oxide].

Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor peptide, whereas nitric oxide (NO) is a potent endothelium-derived vasorelaxing factor synthesized from L-arginine by NO synthase (NOS). ET-1 and NO exert many biological actions in a counter-regulatory manner in vascular remodeling and vascular tonus. Furthermore, there exists a close interaction between ET-1 and NO:ET-1 stimulates NO generation, while NO down-regulates the expression of ET-1 gene.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of endothelial, inducible nitric oxide synthase gene from rat aortic endothelial cell.

We have isolated and sequenced clones of an inducible nitric oxide synthase (iNOS) from cDNA library of interleukin-1 beta-treated rat aortic endothelial cells (EC) completely free from other cell types. The cloned cDNA contains an ORF consisting of 3441 bp, which encodes 1147 amino acid residues. The deduced amino acid sequence contains putative binding sites for NADPH, FMN, FAD, calmodulin and heme. By comparison with amino acid sequences of other isoforms, rat EC iNOS is very similar (92% similarity) to mouse macrophage iNOS. There are four AUUUA motifs, potentially responsible for the instability of the mRNA, in 3'non-coding region of rat EC iNOS cDNA. Transient transfection of cultured rat vascular smooth-muscle cells with a full-length rat EC iNOS cDNA/SR alpha 296 construct by electroporation resulted in massive NO production in proportion to the doses of cDNA used. Northern blot analysis using rat EC iNOS cDNA as a probe revealed that cycloheximide treatment led to a marked accumulation of iNOS mRNA in the presence and absence of interleukin-1 beta. No appreciable decay in the cycloheximide-induced iNOS mRNA accumulation was observed, suggesting that blockade of de novo protein synthesis stabilizes mRNA. These results demonstrate that rat EC iNOS is identical (or very similar) to macrophage iNOS, and suggest that the EC iNOS gene is also regulated at the post-transcriptional level.

Amino Acid Sequence↗

PubMed1996

Specific binding sites for proadrenomedullin N-terminal 20 peptide (PAMP) in the rat.

Adrenomedullin (AM), a potent and novel vasodilator 52-residue peptide originally isolated from pheochromocytoma, is processed from a precursor molecule (preproAM) in which another unique 20-residue sequence, termed proadrenomedullin N-terminal 20 peptide (PAMP), exists. Using [125I Tyr0] rat PAMP as a radioligand, we have examined PAMP binding sites in various rat tissues and cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding sites for rat PAMP, although very low, were widely distributed in various rat tissues examined. The relatively more abundant sites were present in aorta and adrenal glands, followed by lung, kidney, brain, spleen, and heart. An equilibrium binding study using cultured rat VSMC revealed the presence of a single class of high-affinity [dissociation constant (Kd): 3.5 x 10(-8) M] binding sites for rat PAMP with a maximal binding capacity of 4.5 x 10(6) sites per cell. Binding studies revealed that synthetic rat PAMP(1-19)-NH2 was about 10-fold less potent, and rat PAMP(1-20)-OH and human PAMP were about 20-fold less potent than rat PAMP(1-20)-NH2. SDS-polyacylamide gel electrophoresis after affinity-labeling of membranes from various rat tissues (aorta, adrenal glands, lung) and VSMC revealed a distinct labeled band with the apparent molecular mass of 90 kDa, which was diminished by excess unlabeled rat PAMP. A nonhydrolyzable GTP analog (GTP-gammaS) dose-dependently reduced binding of [125I] rat PAMP to VSMC membranes, while ATP-gammaS had no effect. Neither cyclic AMP nor inositol-1,4,5-triphosphate formation was affected by rat PAMP in rat VSMC. The present study demonstrates for the first time that PAMP receptors are widely distributed in various rat tissues, among which aorta and adrenal glands have the most abundant sites. Our data suggest that PAMP receptors are functionally coupled to G-proteins, although its signal transduction remains obscure. The present study also shows that amidation of C-terminal residue of PAMP is critical for receptor binding. The physiological function of PAMP remains undetermined.

Adenosine Triphosphate↗

PubMed1995

Characterization of immunoreactive adrenomedullin in human plasma and urine.

Adrenomedullin(AM) is a novel vasodilator peptide recently isolated from pheochromocytoma. Using a specific and sensitive radioimmunoassay for human AM, we have characterized immunoreactive AM in human plasma and urine. Patients with chronic renal failure had about five-fold higher plasma immunoreactive AM levels than normal subjects, which did not change before and after hemodialysis. Immunoreactive AM was present in normal human urine, whose concentrations were about six-fold greater than those in human plasma. Reverse-phase HPLC of human plasma and urine revealed that immunoreactive AM emerged as a single peak at a position identical to that of authentic human AM(1-52). These data suggest that circulating AM is cleared by the kidney and urinary excretion of AM may be derived from glomerular filtration and/or its renal production.

Adrenomedullin↗

PubMed1995

Hormonal regulation of rat adrenomedullin gene in vasculature.

Adrenomedullin (AM), a novel vasodilatory peptide originally isolated from human pheochromocytoma, has subsequently been shown to be expressed in a variety of tissues, such as adrenal gland, lung, heart, kidney, and aorta. To determine whether vascular AM gene expression is under hormonal control, the effects of dexamethasone (DEX) and T3 on AM messenger RNA (mRNA) expression were tested in cultured rat vascular endothelial cells and smooth muscle cells (VSMCs) by Northern blot analysis using polymerase chain reaction-cloned rat AM complementary DNA as a probe. DEX dose and time dependently increased steady-state AM mRNA levels in both endothelial cells and VSMC. T3 modestly induced AM mRNA expression in both cells, whereas rT3, a biologically inactive isomer of T3, failed to affect AM mRNA expression. A glucocorticoid receptor antagonist (RU 38486) blocked DEX-induced AM mRNA expression in both cells, whereas neither estradiol nor testosterone affected AM mRNA expression. Actinomycin D abolished basal as well as stimulated AM mRNA expression by T3, whereas cycloheximide markedly increased steady state mRNA levels (superinduction). The approximate half-lives of basal and stimulated expression of AM mRNA by DEX in VSMC were within 1 h. In contrast, there was little, if any, decay in cycloheximide-induced AM mRNA expression over 6 h. Our study demonstrates that the AM gene expressed by vascular endothelial and smooth muscle cells is similarly regulated by glucocorticoid and possibly by thyroid hormone, and that superinduction of AM mRNA is most likely due to increased mRNA stability.

Adrenomedullin↗

PubMed1994

Mutational analysis of disulfide bridges in the type C atrial natriuretic peptide receptor.

The type C receptor (ANP-C or NPR-C) for the natriuretic peptides was demonstrated, by site-directed mutagenesis, to have an immunoglobulin-like disulfide bonding pattern that is very similar to that of the cytokine receptor superfamily. The mature form of ANP-C has a disulfide-linked homodimeric structure and contains 5 conserved cysteine residues per subunit, all in the extracellular domain. To identify the cysteine residue involved in the dimerization and further to determine the intramolecular disulfide bridges and their functional roles, cysteine to serine mutations of the 5 cysteine residues were constructed. An analysis of the mutant receptors expressed in COS-1 cells by 125I-ANP binding assay and by measuring difference in their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels indicated that 1) the first 4 cysteine residues are joined sequentially, forming the Cys104-Cys132 and Cys209-Cys257 loops of 29 and 49 residues, respectively; 2) the two disulfide-linked loops are essential for the ligand binding activity; 3) the 5th cysteine residue Cys469 is used in the formation of covalently linked dimers; and 4) the covalent association of the subunit through the disulfide bond involving Cys469 has no apparent influence on ligand-receptor interactions. The intramolecular disulfide bond Cys104-Cys132 was also confirmed by direct protein sequencing of tryptic fragments of purified ANP-C receptor. The secondary structural features revealed here will be useful in understanding the structure and function relationships of not only the dimeric ANP-C receptor, which has only a short cytoplasmic tail, but also the ANP-A (GC-A) and ANP-B (GC-B) receptor subtypes, which have a guanylate cyclase domain in their long cytoplasmic tail and have recently been shown to possess an oligomeric structure, since they have similarly spaced cysteine residues in their extracellular domains.

Amino Acid Sequence↗

PubMed1994

His145-Trp146 residues and the disulfide-linked loops in atrial natriuretic peptide receptor are critical for the ligand-binding activity.

To define the ligand-binding site of natriuretic peptide receptor (NPR), amino acid substitutions were carried out by site-directed mutagenesis at selected residues of bovine NPR-C. The mutant receptors were expressed in COS-1 cells and the effect of the mutations on the binding of ligand was analyzed by binding assay, affinity labeling, and immunoblotting. The replacement of His145-Trp146 (HW) by Leu145-Leu146 in the extracellular domain markedly reduced binding affinity, whereas mutations at charged amino acid residues in the vicinity of HW, such as Glu133 and Asp147, had little effect. Mutation of cysteine residues forming the Cys104-Cys132 and Cys209-Cys257 loops to Ser caused a reduction in the affinity of the receptor to bind ANP. These results suggest that HW residues and the two disulfide-linked loops in the extracellular domain contribute significantly to the ligand binding to NPR.

Amino Acid Sequence↗

PubMed1994

Regulation of inducible nitric oxide synthase gene by interleukin-1 beta in rat vascular endothelial cells.

To elucidate the regulation of endothelial inducible nitric oxide synthase (iNOS), we studied the effects of interleukin (IL)-1 beta on production of nitric oxide (NO) and expression of iNOS mRNA and iNOS protein in cultured rat aortic endothelial cells (ECs) by measurement of NO2-/NO3- (NOx) and Northern blot and Western blot analyses. Among several cytokines and bacterial lipopolysaccharide tested, IL-1 beta most effectively stimulated NOx production. IL-1 beta dose and time dependently stimulated NOx production. Northern blot analysis using cDNA for mouse liver iNOS as a probe showed that IL-1 beta induced expression of iNOS mRNA and stimulated NOx production in a dose- and time-dependent manner. Transforming growth factor (TGF)-beta and dexamethasone blocked the IL-1 beta-induced NOx production as well as expression of iNOS mRNA and protein. TGF-beta dose dependently inhibited the IL-1 beta-induced NOx production and iNOS mRNA expression. Northern blot analysis for the decay of the IL-1 beta-induced iNOS mRNA revealed the approximate half-life of 4 h. These data indicate that IL-1 beta induces iNOS gene expression and de novo synthesis of iNOS and subsequent NO generation in vascular endothelium and that TGF-beta and glucocorticoid block iNOS gene expression at the transcriptional level.

Amino Acid Oxidoreductases↗

PubMed1993

A variant form of the type C atrial natriuretic peptide receptor generated by alternative RNA splicing.

A novel form of type C atrial natriuretic peptide receptor (ANP-C) was identified, characterized, and shown to be a product of alternative RNA splicing. Sequencing of several clones of ANP-C cDNA isolated from a bovine lung cDNA library revealed the presence of a new clone encoding a 536-amino acid ANP-C. The nucleotide sequence of this novel clone is very similar to that of the previously cloned ANP-C which consists of 537 amino acids. The sole difference between the two clones is the presence or absence of a very short segment of only 3 base pairs (bp), by which the sequence Ser472-Gly473 of the longer form is changed to Cys472 in the variant form; the other regions are identical. Alignment of the cDNA sequences with that of the ANP-C gene indicated that the two forms of ANP-C receptor arise from a single gene by alternative splicing using one donor and two closely located acceptor sites. Biochemical and pharmacological characterization using a mammalian cDNA expression system indicated that the two variants are similar in their subunit structures, in ligand-binding properties, and even in internalization kinetics. Polymerase chain reaction amplification of the transcripts demonstrated that 1) although the novel form is a minor species, it is present in various tissues such as the lung, kidney, adrenal, and vascular smooth muscle and endothelial cells and 2) the ratio of the novel variant form to the original longer form remains almost constant in these tissues. The alternative splicing reported here contributes to the diversity of ANP receptors; however, its physiological significance remains to be clarified.

Affinity Labels↗