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A Hirasawa

Publications and source records attributed to A Hirasawa.

At least 19 recordsLinked to original sources
PubMed2001

Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains.

To measure pH in defined intracellular microdomains of living cells, we developed ratiometric indicators based on fusing in tandem two green fluorescent protein (GFP) variants having different pH sensitivities. The indicators function in a single-excitation/dual-emission mode involving fluorescence resonance energy transfer, as well as in a dual-excitation/single-emission mode. The fluorescence ratio from GFpH and YFpH showed pH dependency and pK(a) values were 6.1 and 6.8, respectively. Using these indicators expressed in cultured cells, we measured and visualized pH changes in the cytosol and nucleus. Furthermore, by tethering the indicator to a membrane protein (the alpha(1B) adrenergic receptor), we visualized the pH in the vicinity of the protein during internalization caused by endocytosis after agonist stimulation. These novel probes will serve as a useful tool for monitoring pH in the defined organelle and in the microenvironment of a target protein, to analyze cellular function.

Animals↗
PubMed2001

Galphaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade.

G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein betagamma subunit (Gbetagamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gq-coupled alpha1B-adrenergic receptor is mediated by the alpha subunit of Gq (Galphaq), not by Gbetagamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha1B-adrenergic receptor/Galphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the alpha1B-adrenergic receptor/Galphaq required c-Src and Rho family small GTPases. Furthermore, activation of the alpha1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the alpha1B-adrenergic receptor. Together, these results suggest that the alpha1B-adrenergic receptor/Galphaq may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.

Animals↗
PubMed2001

Molecular monitoring of bleomycin-induced pulmonary fibrosis by cDNA microarray-based gene expression profiling.

Pulmonary fibrosis is a progressive disorder whose molecular pathology is poorly understood. Here we developed an in-house cDNA microarray ("lung chip") originating from a lung-normalized cDNA library. By using this lung chip, we analyzed global gene expression in a murine model of bleomycin-induced fibrosis and selected 82 genes that differed by more than twofold intensity in at least one pairwise comparison with controls. Cluster analysis of these selected genes showed that the expression of genes associated with inflammation reached maximum levels at 5 days after bleomycin administration, while genes involved in the development of fibrosis increased gradually up to 14 days after bleomycin treatment. These changes in gene expression signature were well correlated with observed histopathological changes. The results show that microarray analysis of animal disease models is a powerful approach to understanding the gene expression programs that underlie these disorders.

Animals↗
PubMed2001

Beta2-adrenergic receptor/cyclic adenosine monophosphate (cAMP) leads to JNK activation through Rho family small GTPases.

Gi- and Gq-coupled G protein-coupled receptors (GPCRs) have been shown to activate c-Jun N-terminal kinase (JNK), a subfamily of mitogen-activated protein kinases (MAPKs), through Rho family small GTPases in mammalian cells. We investigated the signaling pathway linking the Gs-coupled beta2-adrenergic receptor with JNK, using smooth muscle DDT1 MF-2 cells, which natively express the beta2-adrenergic receptor. Stimulation of the beta2-adrenergic receptor activated JNK in a time-dependent manner, and a cell-permeable cyclic adenosine monophosphate analogue (8-Br-cAMP) activated JNK. The beta2-adrenergic receptor- or 8-Br-cAMP-induced activation of JNK required Rho family small GTPases. Also, the beta2-adrenergic receptor or 8-Br-cAMP induced activation of Rho family small GTPases. These results demonstrate that the beta2-adrenergic receptor/cAMP leads to JNK activation through Rho family small GTPases in DDT1 MF-2 cells. Activation of Rho family small GTPases may provide a common step in GPCR-mediated JNK activation.

Animals↗
PubMed2001

Regulation of subcellular localization of alpha1-adrenoceptor subtypes.

Alpha1-adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. Alpha1-AR comprise a heterogeneous family of three distinct isoforms of alpha1A, alpha1B and alpha1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of alpha1-ARs; thus, alpha1A-AR predominantly localize intracellularly, while alpha1B-AR on the cell surface. To examine the molecular mechanism for the subtype-specific differences in subcellular localization, we conducted a search for novel proteins that interact with the alpha1B-AR, specifically focusing on the carboxyl-terminal cytoplasmic domain. Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R interacts with alpha1B-AR in vitro and in vivo through the specific site, and that in cells which co-express alpha1B-AR and gC1q-R, the subcellular localization of alpha1B-AR is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of alpha1B-ARs.

Animals↗
PubMed2001

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation.

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.

Adrenergic alpha-Agonists↗
PubMed2001

Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF-EDG5 cascade.

The molecular mechanism of immunoglobulin A nephropathy (IgAN), the most common primary renal glomerular disease worldwide, is unknown. HIGA (high serum IgA) mouse is a valid model of IgAN showing almost all of the pathological features, including mesangial cell proliferation. Here we elucidate a pattern of gene expression associated with IgAN by analyzing the diseased kidneys on cDNA microarrays. In particular, we showed an enhanced expression of several genes regulating the cell cycle and proliferation, including growth factors and their receptors, as well as endothelial differentiation gene-5 (EDG5), a receptor for sphingosine 1-phosphate (SPP). One of the growth factors, platelet-derived growth factor (PDGF) induces a marked upregulation of EDG5 in proliferative mesangial cells, and promotes cell proliferation synergistically with SPP. The genomic approach allows us to identify families of genes involved in a process, and can indicate that enhanced PDGF-EDG5 signaling plays an important role in the progression of IgAN.

Animals↗
PubMed2001

Functional genomic research of alpha 1-adrenoceptors.

The Human Genome Project is now almost completed, and we are about to move into the post-genome sequence era of functional genomics. The advent of genome science has markedly changed the way life science research including pharmacological study is conducted; thus, systematic and integrated 'genome-wide' survey is feasible. The stream of 'Genome-->Transcriptome--> Proteomics' is logical and, in each aspect, approaches for functional genomics are now pursued at a high pace. We have recently developed a standardized technical platform (in various levels, such as transcription, cell and whole animal levels, etc.), and applied these techniques to the study of functional genomics of G-protein-coupled receptors, particularly alpha1-adrenoceptors as a model. Combining the genome information and technology, future pharmacological studies would become the genome-based search and research.

Animals↗
PubMed2001

In vivo antiarrhythmic profile of AP-792 assessed in different canine arrhythmia models.

The antiarrhythmic effects of a novel antiarrhythmic drug AP-792, 4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-1-[4-cyclohexylbutyl]piperidine hydrochloride, were analyzed using the epinephrine-, digitalis- and two-stage coronary ligation-induced canine ventricular arrhythmia models. Intravenous administration of AP-792 (0.3 or 1.0 mg/kg) effectively suppressed each of the ventricular arrhythmias, an action that resembles that of a typical cardioselective Ca2+ channel blocker, AH-1058. The antiarrhythmic action of AP-792 was slow in onset and longer-lasting than those in our previous studies using more than 50 antiarrhythmic drugs, including Na+ and Ca2+ channel blockers. These results suggest that AP-792 can become a unique long-acting antiarrhythmic drug.

Animals↗
PubMed2001

[Primary renal non-Hodgkin's lymphoma presenting as massive macrohematuria and bladder tamponade].

A 43-year-old man with macrohematuria and anuresis was admitted, and diagnosed as having bladder tamponade due to coagulates. Abdominal ultrasonography and computed tomography revealed bilateral renal tumors. Bilateral renal arteriography showed hypovascular lesions. Percutaneous needle biopsy of the left renal tumor was performed, and the final diagnosis was non-Hodgkin's lymphoma (diffuse mixed, B cell type, CSIIA). After six courses of chemotherapy, the tumor lesions were markedly reduced, and at present there is no evidence of recurrence.

Adult↗
PubMed2000

Key amino acids of vasopressin V1a receptor responsible for the species difference in the affinity of OPC-21268.

A non-peptide, vasopressin V1a receptor-selective antagonist, OPC-21268, exhibited a markedly higher affinity for the rat V1a receptor (Ki = 380 nM) than for the human V1a receptor (Ki = 140 microM). To delineate the region responsible for the high affinity binding of OPC-21268 for the rat V1a receptor, we have constructed a series of chimeric human and rat V1a receptors, and examined the chimeric and point-mutated receptors by competitive radioligand binding analysis. The results showed that the transmembrane domain (TMD) VI-VII of the vasopressin V1a receptor, in particular the amino acid residue Ala-342 in TMD VII, is the major component conferring the rat-selective binding of OPC-21268 to the V1a receptor.

Amino Acid Sequence↗
PubMed2000

Both alpha(1A)- and alpha(1B)-adrenergic receptor subtypes couple to the transient outward current (I(To)) in rat ventricular myocytes.

1. Regulation of transient outward current (I(To)) by alpha(1)-adrenergic (alpha(1)AR) plays a key role in cardiac repolarization. alpha(1)ARs comprise a heterogeneous family; two natively expressed subtypes (alpha(1A) and alpha(1B)) and three cloned subtypes (alpha(1a), alpha(1b) and alpha(1d)) can be distinguished. We have examined the electrophysiological role of each alpha(1)AR subtype in regulating I(To) in isolated rat ventricular myocytes. 2. Reverse transcription-PCR study revealed the presence of three subtype mRNAs (alpha(1a), alpha(1b) and alpha(1d)) in rat myocytes. 3. Radioligand binding assay using [(125)I]-HEAT showed that the inhibition curves for alpha(1A)AR-selective antagonists (WB4101, 5-methylurapidil, (+)-niguldipine and KMD-3213) in rat ventricles best fit a two-site model, with 30% high and 70% low affinity binding sites. The high affinity sites were resistant to 100 microM chloroethylclonidine (CEC), while the low affinity sites were highly inactivated by CEC. 4. Whole cell voltage clamp study revealed that methoxamine reduced a 4-aminopyridine(4-AP)-sensitive component of I(To) in the isolated rat ventricle myocytes. Lower concentrations of KMD-3213 (1 nM) or 5-MU (10 nM) did not affect the methoxamine-induced reduction of I(To). On the other hand, CEC treatment (100 microM) of isolated myocytes reduced the methoxamine-induced reduction of I(To) by 46%, and the remaining response was abolished by lower concentrations of KMD-3213 or 5-MU. 5. The results indicate that rat ventricular myocytes express transcripts of the three alpha(1)AR subtypes (alpha(1a), alpha(1b) and alpha(1d)); however, two pharmacologically distinct alpha(1)AR subtypes (alpha(1A) and alpha(1B)) are predominating in receptor populations, with approximately 30% alpha(1A)AR and 70% alpha(1B)AR. Although both alpha(1A) and alpha(1B)AR subtypes are coupled to the cardiac I(To), alpha(1B)ARs predominantly mediate alpha(1)AR-induced effect.

Adrenergic alpha-Agonists↗
PubMed2000

Neutrophilia associated with anaplastic carcinoma of the thyroid: production of macrophage colony-stimulating factor (M-CSF) and interleukin-6.

Cancers associated with marked neutrophilia are relatively rare. We report here two cases of anaplastic thyroid carcinoma associated with neutrophilia. We measured the concentrations of granulocyte colony-stimulating factor (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in sera, pleural effusion, cyst fluid of the thyroid carcinoma region, or culture supernatants of carcinoma cells. Maximum levels of elevated white blood cell counts reached 106.1 x 10(9)/L (neutrophils 103.0 x 10(9)/L) in case 1 and 62.3 x 10(9)/L (neutrophils 57.9 x 10(9)/L) in case 2. Acute-phase reactants were elevated to various degrees, and hypercalcemia was found in both cases. IL-6, G-CSF, and M-CSF seemed to play the principal roles in neutrophilia in case 1, and the elevated levels of IL-6 and M-CSF seemed to mainly contribute to neutrophilia in case 2. Immunohistochemical staining revealed that carcinoma cells themselves produce IL-6 regardless of the types of carcinoma cells. To our knowledge, this is the first report describing the contribution of M-CSF to neutrophilia in patients with thyroid carcinoma.

Adenocarcinoma, Papillary↗
PubMed2000

Cellular expressions and serum concentrations of Fas ligand and Fas receptor in patients with infectious mononucleosis.

Although lymphocytosis and neutropenia are commonly associated with infectious mononucleosis (IM), the precise mechanisms involved remain unclear. Accumulated evidence has revealed that the apoptosis-mediating system, Fas receptor/Fas ligand (Fas-R/Fas-L), plays an important role in this disease. Recently, lymphocytes, monocytes, and neutrophils have been reported to constitutively express Fas-R, and the Epstein-Barr virus (EBV) has been shown to activate, in addition to B cells, peripheral blood CD8+ T cells, monocytes, and neutrophils. We elucidated cell surface expression and serum concentrations of Fas-R and Fas-L in patients with IM in an effort to more clearly define the role and contribution of apoptosis in this disease. The expression of lymphocyte surface Fas-L and Fas-R was significantly increased in patients with IM (P < .005 and P < .001, respectively), and among lymphocytes, CD4+ or CD8+ populations contained Fas-R+ as well as Fas-R- subpopulations. The constitutive Fas-R expression levels of monocytes and neutrophils were also increased in IM. Moreover, serum levels of both soluble Fas-L and Fas-R were significantly higher in patients with IM than in healthy volunteers (P < .001 and P < .0001, respectively). Positive relationships between the number of peripheral blood CD95+ lymphocytes and white blood cell count, number of lymphocytes, or number of CD4+ or CD8+ lymphocytes were observed. Our results suggest that the Fas-R/Fas-L system might play a role in eliminating EBV-infected or -activated peripheral blood cells by cell-to-cell contact or in an autocrine and/or paracrine fashion in patients with IM.

Adult↗
PubMed1999

Interaction of the alpha(1B)-adrenergic receptor with gC1q-R, a multifunctional protein.

gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the alpha(1B)-adrenergic receptor (173 amino acids, amino acids 344-516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver. In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369-378) were identified as the site of interaction. The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length alpha(1B)-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells. Interestingly, the alpha(1B)-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the alpha(1B)-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed alpha(1B)-adrenergic receptors and gC1q-R, most of the alpha(1B)-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed. These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the alpha(1B)-adrenergic receptors.

Animals↗
PubMed1999

[Diaminodiphenyl sulfone therapy for refractory idiopathic thrombocytopenic purpura--long-term effectiveness and possible mechanisms].

Eight patients with idiopathic thrombocytopenic purpura (ITP) refractory to corticosteroids were given oral diaminodiphenyl sulfone (DDS) (75 mg to 150 mg/day). Responses to DDS were observed in 5 patients, and their platelet counts significantly increased to more than 10 x 10(4)/microliter. The duration of treatment required to obtain platelet counts of more than 10 x 10(4)/microliter ranged from 35 to 64 days. In 3 of the 5 responsive patients, platelet counts stabilized at over 10 x 10(4)/microliter during DDS therapy for 3 to 6 years. Although adverse effects including local skin rash and itching were observed in 3 patients, they disappeared after suspension of DDS or in response to medication with antihistamines. Although the exact mechanism by which it increases the platelet counts in ITP patients is still obscure, DDS may inhibit the reticuloendothelial system by excessive red blood cell destruction. We concluded that DDS therapy is well-tolerated and seems to be an effective alternative treatment for patients with ITP that is refractory to other conventional therapies.

Administration, Oral↗
PubMed1998

Effects of quinidine and verapamil on human cardiovascular alpha1-adrenoceptors.

BACKGROUND: The antiarrhythmic drugs quinidine and verapamil are known to block alpha1-adrenoceptors (alpha1ARs). Alpha1ARs are a heterogeneous family of three subtypes (alpha1A, alpha1B, and alpha1D), and little is known about the effects of quinidine and verapamil on the different human alpha1AR subtypes. METHODS AND RESULTS: Reverse transcriptase-polymerase chain reaction showed that all alpha1AR subtypes are expressed in both human heart (atrium and ventricle) and the mesenteric artery. Pharmacological profiles of quinidine and verapamil actions on the alpha1AR subtypes were characterized with Chinese hamster ovary cells stably expressing cloned human alpha1AR subtypes. Radioligand binding studies showed that quinidine and verapamil had high affinities for all alpha1AR subtypes. Also, both drugs synergistically inhibited alpha1AR-mediated inositol 1,4,5-triphosphate production at the clinical effective concentration range (1 micromol/L quinidine and 0.1 micromol/L verapamil). CONCLUSIONS: The results show that all alpha1AR subtypes are expressed in the human cardiovascular system and that quinidine and verapamil may have a potent, synergistic inhibitory effect on the alpha1ARs. Clinically observed hypotension after quinidine plus verapamil can be explained by their synergistic inhibitory effects on human alpha1ARs.

Adrenergic alpha-1 Receptor Antagonists↗