Biomedical subjects

T Onaya

Publications and source records attributed to T Onaya.

At least 19 recordsLinked to original sources

PubMed2002

DNA demethylation modulates mouse leptin promoter activity during the differentiation of 3T3-L1 cells.

AIMS/HYPOTHESIS: The mouse leptin gene, a major hormonal regulator of appetite and fat cell mass, expresses during the differentiation of 3T3-L1 preadipocytes to adipocytes. To determine if DNA methylation is involved in regulating the expression of the leptin gene, we examined the methylation status and methylation-sensitive transcription factors during 3T3-L1 differentiation. METHODS: DNase I footprinting, electrophoretic mobility-shift assays, and a Southwestern analysis were carried out using nuclear extracts from preadipocytes and adipocytes. Promoter activity was measured by luciferase assays. The CpG methylation pattern was determined. RESULTS: Transient transfection of reporter constructs with the leptin promoter showed that preadipocytes that do not transcribe the leptin gene show enough transactivation, suggesting the presence of an additional regulatory mechanism. We identified eight CpG sites in the promoter up to nt -161, all of which were highly methylated ( > 92 %) in preadipocytes. Seven of these sites showed a varying degree of demethylation during differentiation, while the site at nt -54 remained methylated. In electrophoretic mobility-shift assays, DNA fragments from nt -115 to nt -70 generated a methylation-sensitive band with nuclear extracts from preadipocytes when the CpG sites were methylated. Southwestern analysis identified a 52 kMr protein that binds strongly to the methylated probes. Promoter activity was reduced by methylation of the CpG sites up to nt -115, but not up to nt -70. CONCLUSION/INTERPRETATION: These results suggest that methylation of specific CpG sites between nt -115 and nt -70 and a methylation-sensitive protein could contribute to leptin gene expression during adipocyte differentiation in 3T3-L1 cells.

3T3 Cells↗

PubMed2001

Transcriptional activation of the thyroglobulin promoter directing suicide gene expression by thyroid transcription factor-1 in thyroid cancer cells.

Gene therapy with thyroglobulin (TG) promoter and a prodrug/suicide gene combination may prove useful as a treatment for thyroid carcinoma. However, most poorly differentiated and anaplastic thyroid carcinomas have lost the ability to express the TG gene expression accompanied by loss of transcription factors [thyroid transcription factor-1 (TTF-1), TTF-2, or Pax-8] interacting with the TG promoter. In anticipation of developing transcriptionally targeted gene therapy of TG-nonproducing thyroid carcinomas, we investigated the effect of TTF-1 gene transfer on TG promoter activity and the cytotoxic effect obtained by the TG promoter-driven HSV-TK gene along with ganciclovir in thyroid carcinoma and nonthyroidal cells. Using a chimeric construct containing the 5'-flanking region of the rat TG gene between -826 and +39 bp and the luciferase gene, TG promoter activity was detected in a normal rat thyroid cell line (FRTL-5), but not in a dedifferentiated line of thyroid cells (FRT) expressing Pax-8 but not TTF-1, TTF-2, or TG [TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)], or in a human papillary thyroid carcinoma cell line [BHP15-3; TTF-1(-)/TTF-2(-)/Pax-8(-)/TG(-)], a human pulmonary cell line [H441; TTF-1(+)/TTF-2(-)/Pax-8(-)/TG(-)], or a dog kidney epithelial cell line [MDCK; TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. Cotransfection of the TTF-1 expression vector stimulated TG promoter activity in FRT and BHP15-3 dedifferentiated thyroid cells, but not in H441 pulmonary cells. Only weak activation was observed in MDCK kidney cells. We then constructed recombinant adenovirus vectors, AdTTF-1 and ADTGTK: AdTTF-1 contained cytomegalovirus promoter and rat TTF-1 cDNA; AdTGTK carried the TG promoter-driven HSV-TK gene. Infection with AdTGTK and combined with GCV treatment induced a cytotoxic effect in FRTL-5 cells but not in dedifferentiated thyroid or nonthyroid cells. Cotransduction of AdTTF-1 and AdTGTK permitted 90% cytotoxicity for BHP15-3 and >95% cytotoxicity for FRT, as well as for BHP7-13 and BHP18-21v thyroid cancer cell lines [both/TTF1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. In contrast, little cytotoxicity was seen for H441 and MDCK cell lines even with 300 microg/ml of ganciclovir. These results suggest that cotransduction of a TG promoter-controlled suicide gene and the TTF-1 gene by adenoviral vectors confers transcriptionally targeted gene-mediated cytotoxicity in poorly differentiated thyroid carcinoma cells unable to express the TG gene.

Adenoviridae↗

PubMed2001

Pax-8 is essential for regulation of the thyroglobulin gene by transforming growth factor-beta1.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that is thought to play a major role in the regulation of growth and differentiation of thyroid cells. However, little is known of its detailed mechanisms of action in thyrocytes. We have therefore studied the molecular mechanisms of TGF-beta1 action on thyroglobulin (TG) gene expression by focusing our attention on TGF-beta1 regulation of thyroid-specific transcription factors. TGF-beta1 decreased TG messenger RNA (mRNA) expression both in the presence and in the absence of TSH in rat thyroid FRTL-5 cells. Transfected into FRTL-5 cells, the activity of reporter plasmids containing the rat TG promoter ligated to a luciferase gene was significantly suppressed by the addition of TGF-beta1. When the nuclear extracts prepared from TGF-beta1-treated FRTL-5 cells were used in gel mobility shift assays, the amount of protein-DNA complex formed by Pax-8 was reduced, both in the presence and in the absence of TSH, but protein-DNA complexes formed by thyroid transcription factor-1 (TTF-1) and TTF-2 were not. The suppressive effect of TGF-beta1 on Pax-8/DNA complex formation is in part due to the suppression of Pax-8 mRNA and protein levels by TGF-beta1. Expressions of Pax-8 mRNA and protein, which were assessed by Northern blot and Western blot analyses, respectively, were decreased by TGF-beta1 treatment of FRTL-5 cells in a concentration-dependent manner. In a transfection experiment, mutation of the Pax-8-binding site caused a loss of both TGF-beta1- and TSH-responsiveness in TG promoter activity. Overexpression of Pax-8 abolished the TGF-beta1 suppression of TG promoter activity. These results indicate that TGF-beta1 decreases Pax-8 mRNA levels as well as Pax-8 DNA-binding activity, which, at least in part, seems to be involved in the TGF-beta1-induced suppression of TG gene expression.

Animals↗

PubMed2001

Differential regulation of the human sodium/iodide symporter gene promoter in papillary thyroid carcinoma cell lines and normal thyroid cells.

The absence of TSH-stimulated radioiodide uptake in differentiated thyroid cancer is associated with a high recurrence rate and reduced survival. We studied regulation of the sodium/iodide symporter gene in human papillary thyroid cancer cell lines (BHP) and primary human thyroid cells. BHP cells expressed very low levels of sodium/iodide symporter mRNA and did not concentrate iodide, but iodide uptake was restored to levels seen in FRTL-5 rat thyroid cells by stable transfection of a sodium/iodide symporter cDNA. Sodium/iodide symporter gene expression, therefore, was necessary and sufficient for iodide uptake in BHP cells. We cloned the human sodium/iodide symporter gene 5'-flanking region and analyzed progressive 5'-deletions in transient transfections. We identified a region, -596 to -268, essential to confer full promoter activity in primary normal human thyroid cells. Sodium/iodide symporter promoter activity in four BHP cell lines, however, was markedly reduced, consistent with down-regulation of the endogenous sodium/iodide symporter gene. Nuclear extracts from BHP 2-7 cells had reduced or absent binding to regions of the sodium/iodide symporter promoter shown to be critical for expression, compared with nuclear extracts from FRTL-5 cells. Competition studies indicated that these nuclear proteins were not known thyroid transcription factors. Modifications of the sodium/iodide symporter promoter with demethylation or histone acetylation did not increase sodium/iodide symporter expression, and no deletions of the critical regulatory region were identified in the endogenous gene in BHP cells. Regulation of the sodium/iodide symporter 5'-flanking region in transient transfection paralleled endogenous sodium/iodide symporter expression. Reduced expression of potential novel nuclear factor(s) in these cell lines may contribute to reduced sodium/iodide symporter expression resulting in absence of iodide uptake in some papillary thyroid cancers.

Base Sequence↗

PubMed2001

Ligands for peroxisome proliferator-activated receptor gamma inhibit growth and induce apoptosis of human papillary thyroid carcinoma cells.

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) induce apoptosis and exert antiproliferative effects on several carcinoma cell lines. The present study investigates the expression of PPARgamma and the possibility that agonists for PPARgamma also inhibit the growth of human thyroid carcinoma cells. We examined this hypothesis using six cell lines, designated BHP thyroid carcinoma cells, which originated from patients with papillary thyroid carcinoma. RT-PCR analysis revealed that the thyroid carcinoma cell lines BHP2-7, 7-13, 10-3, and 18-21 express PPARgamma. More PPARgamma was expressed in carcinoma than in adjacent normal thyroid tissue in three of six samples of human papillary carcinoma of the thyroid. PPARgamma-positive thyroid carcinoma cells were treated with agonists of PPARgamma, troglitazone, BRL 49653, and 15-deoxy-12,14-prostaglandin J2. Troglitazone (10 micromol/L), BRL 49653 (10 micromol/L), and 15-deoxy-12,14-prostaglandin J2 (1 microg/mL) decreased [(3)H]thymidine incorporation and reduced cell number, respectively, in BHP carcinoma cell lines that expressed PPARgamma. Under low serum conditions, ligands for PPARgamma induced condensation of the nucleus and fragmentation of chromatin into nucleosome ladders. These findings indicate that the death of thyroid carcinoma cells is a form of apoptosis. To investigate the molecular mechanism of the apoptosis, we assessed expression of the apoptosis-regulatory genes bcl-2, bax, and c-myc. Troglitazone significantly increased the expression of c-myc messenger RNA but had no effect on the expression of bcl-2 and bax in thyroid carcinoma cells. These results suggest that, at least in part, the induction of apoptosis in human papillary thyroid carcinoma cells may be due to an increase of c-myc. Troglitazone (500 mg/kg.day) significantly inhibited tumor growth and prevented distant metastasis of BHP18-21 tumors in nude mice in vivo. Taken together, these results suggest that PPARgamma agonist inhibit cell growth of some types of human thyroid cancer.

Animals↗

PubMed2000

A patient with type 2 diabetes mellitus associated with mutations in calcium sensing receptor gene and mitochondrial DNA.

A 44-year-old female with familial hypocalciuric hypercalcemia (FHH) due to a homozygous missense mutation (Pro40Ala) in calcium sensing receptor (CaSR) gene has type 2 diabetes mellitus. The identical heterozygous mutation of CaSR gene was observed in consanguineous parents and all other family members examined except her two sisters. Many subjects with abnormal glucose tolerance were observed in this family, which is compatible with maternal inheritance. Mitochondrial function of complex I (NADH-coenzyme Q reductase) activity in cybrid cells between mitochondrial DNA (mtDNA)-deleted (rho(0)) HeLa cells and mtDNA from the proband was decreased by 35%. The proband has eight substitutions and among these 4833 A/G is a missense substitution in NADH dehydrogenase 2 gene and may probably be a major pathogenic mutation of impaired complex I activity. These results suggest that coexistence of nuclear gene and mtDNA mutations may have caused or modified the development of abnormal glucose tolerance in this family.

Adult↗

PubMed2000

Disruption of aldose reductase gene (Akr1b1) causes defect in urinary concentrating ability and divalent cation homeostasis.

Aldose reductase (AKR1B1) is the first enzyme in the polyol pathway through which glucose is converted to sorbitol, and has been implicated in the etiology of diabetic complications. However, its physiological role is still not well understood. In the kidney, AKR1B1 is quite abundant in the collecting tubule cells and thought to provide protection against hypertonic environment. We report here that the mice lacking AKR1B1 showed hypercalciuria, hypercalcemia, hypermagnesemia, and reduced ability to concentrate urine, suggesting a new physiological role of AKR1B1 in divalent cation homeostasis.

Aldehyde Reductase↗

PubMed2000

A type 2 diabetic patient with liver dysfunction due to human insulin.

A 45-year-old man had been complaining of thirst and polydypsia for the last 3 months and was diagnosed as having type 2 diabetes mellitus because his fasting blood glucose showed 221 mg/dl with positive urinary ketone. He was hospitalized to a private hospital and Penfil 30R was started. However, serum gamma-GTP and aminotransferases began to elevate after insulin treatment and exceeded 1000 IU/l. Insulin was discontinued and serum gamma-GTP and aminotransferases returned close to the normal range. Since his glycemic control became poor again, Penfil 30R was restarted and serum gamma-GTP and aminotransferases elevated again. Therefore, insulin was discontinued and the patient was referred to the Third Department of Internal Medicine, Yamanashi Medical University Hospital because of liver dysfunction. His plasma glucose decreased by diet therapy, and improved further by the administration of glibenclamide. After obtaining informed consent, Humalin R was challenged. Seven days after insulin injection, serum aminotransferases began to elevate again. Lymphocyte stimulation test was negative against three preparations (Penfil R, Penfil N and Humalin R). The present case suggests that human insulin itself can cause liver dysfunction and we need to pay more attention to liver function tests when we start insulin treatment.

Alanine Transaminase↗

PubMed2000

Detection of binding and blocking autoantibodies to the human sodium-iodide symporter in patients with autoimmune thyroid disease.

The sodium iodide symporter (NIS) is a novel autoantigen in autoimmune thyroid disease (ATD). A recent study has described the development of a bioassay for human (h) NIS antibody detection, but this will not detect antibodies that bind the symporter without modulating its activity. Therefore, the establishment of a binding assay is of importance to determine the overall prevalence of hNIS antibodies in ATD patients. An in vitro transcription and translation system was used to produce [35S]-labeled hNIS. The radiolabeled ligand reacted specifically in immunoprecipitation experiments with rabbit antiserum raised against a peptide fragment of hNIS. Subsequently, the reactivity of control and ATD sera to translated [35S]hNIS was determined using RIAs. A significant difference in the frequency of hNIS antibody-positive sera was found when patients with either Graves' disease (GD) or autoimmune hypothyroidism (AH) were compared with normal controls (P = 0.01 and P = 0.03, respectively). Of 49 GD and 29 AH sera tested, 11 (22%) and 7 (24%), respectively, were found to contain hNIS antibodies. Differences were also significant when the antibody-binding indices of the control group of sera were compared with those of both the GD and the AH patient sera (P < 0.0001 and P = 0.001, respectively). In contrast, sera from 10 patients with Addison's disease and 10 patients with vitiligo (without associated ATD) were all negative for antibody reactivity to the symporter. No differences were detected when the antibody binding indices of either the Addison's disease or the vitiligo sera were compared with those of the normal sera group (P = 0.9 and P = 0.6, respectively). Eight of the 11 (73%) GD and 3 of the 7 (43%) AH sera, which were positive for hNIS antibodies in the immunoprecipitation assay, were also found to inhibit iodide uptake in hNIS-transfected CHO-K1 cells, suggesting the existence of antibodies in some serum samples that bind to the symporter without modulating its function. Overall, a significant correlation was found between the iodide uptake inhibition and the binding assays for hNIS antibody detection (r = 0.49, P < 0.0001). In summary, we have developed a specific and quantitative assay for the detection of hNIS binding antibodies in sera of patients with ATD. This system offers the advantage of studying antibody reactivity against conformational epitopes and will be useful in understanding the role of NIS autoreactivity in the pathogenesis of ATD.

Addison Disease↗

PubMed2000

Screening for genetic mutations. A review.

A point mutation of a nucleotide within a single gene can have a profound effect on a specific organ and/or the entire human body. DNA sequences associated with human diseases may differ from the corresponding normal sequences by single nucleotide mutations or by large alterations such as deletions, insertions, duplications, or translocations of DNA segments or entire chromosomes. As a result of the heterogeneity of DNA alterations and genetic mutations, various screening approaches are required to detect these alterations. However, methods which facilitate the detection of large mutations in the genome are typically insensitive to point mutations, whereas methods which detect point mutations are not appropriate to detect large alterations within the genome. Since there is no single perfect method to screen for unknown mutations, combinations of these methods may be necessary for accurate genetic diagnosis. The applications of polymerase chain reaction (PCR) technology to genomic screening have made rapid and accurate genetical diagnosis possible. Furthermore, recent developments in the technology of DNA microarrays have opened the way for high throughput sequence analysis by hybridization, which shows great potential in both molecular biology and medicine in the near future.

Genetic Testing↗

PubMed2000

A new mitochondrial DNA mutation at 14577 T/C is probably a major pathogenic mutation for maternally inherited type 2 diabetes.

From a family of 16 diabetic patients with typical maternal inheritance, we investigated a 69-year-old woman with type 2 diabetes. The proband showed no major deletions in the mitochondrial DNA (mtDNA). Direct sequencing revealed 7 missense and 5 ribosomal RNA homoplasmic nucleotide substitutions when compared with the Cambridge Sequence and its recent revision. When compared with the control cybrid cells, the proband cybrid cells showed 6 nucleotide substitutions. Among these, 14577 T/C, which turned out to be 98.9% heteroplasmic, is a new missense substitution in the NADH dehydrogenase 6 gene. We also observed 2 other patients with 14577 T/C substitution from another group of 252 unrelated diabetic patients, whereas no individual from a group of 529 control subjects had 14577 T/C substitution. Furthermore, these 6 substitutions were in linkage disequilibrium. Mitochondrial respiratory chain complex I activity and O2 consumption rates of the proband cybrid cells, which were obtained by the fusion of mtDNA-deleted (rho0) HeLa cells and mtDNA from the proband, showed 64.5 and 61.5% reductions, respectively, compared with control cybrid cells. The present study strongly indicates that the new mtDNA mutation at 14577 T/C is probably a major pathogenic mutation for type 2 diabetes in this family.

Aged↗

PubMed1999

Extracellular adenosine increases Na+/I- symporter gene expression in rat thyroid FRTL-5 cells.

We studied the effect of extracellular adenosine on iodide (I-) transport in FRTL-5 thyroid cells. I- accumulation increases after a 48 h exposure to adenosine in a concentration-dependent manner, reaching a maximum of 7.9-fold basal levels at 72 h after the addition of 300 microM adenosine. Neither I- efflux nor intracellular cyclic adenosine monophosphate accumulation is affected by the exposure to adenosine. The stimulation of I- transport by adenosine is partly as a result of an increase in Na+/I- symporter (NIS) mRNA and protein levels. Northern blot analysis revealed that adenosine increases NIS mRNA levels at 24 h, reaching a maximum at 36 h. Western blot analysis demonstrated that adenosine increases NIS protein levels at 36 h, reaching a maximum at 72 h, in parallel with the kinetics of adenosine-induced I- transport. Adenosine increased the promoter activity of a full-length NIS promoter-luciferase chimera, suggesting that the effect of adenosine on NIS mRNA levels is transcriptional. The stimulatory effect of adenosine on NIS mRNA levels, is mimicked by N6-(L-2-phenylisopropyl) adenosine (PIA), an A1 adenosine receptor agonist, and inhibited by 1,3-dipropyl-8-cyclopentylxanthine, an A1 adenosine receptor antagonist, suggesting that the effect is mediated via the A1 adenosine receptor stimulation in FRTL-5 cells. Incubating cells with islet-activating protein inhibited the adenosine-induced NIS mRNA levels. In sum, extracellular adenosine increases NIS gene expression and stimulates I- transport via the A1 adenosine receptor-Gi/Go protein signal transduction pathway.

Adenosine↗

PubMed1999

An aggressive familial amyloidotic polyneuropathy caused by a new variant transthyretin Lys 54.

Histologic examination of sural nerve of a 32-year-old man with an aggressive polyneuropathy associated with autonomic failure demonstrated amyloid deposition, and familial amyloidotic polyneuropathy (FAP) was diagnosed. Immunohistochemical staining showed transthyretin (TTR) staining of the amyloid deposits in nerve. Sequencing revealed G to A transition in the codon 54 causing TTR Lys 54. This is a new variant TTR associated with aggressive FAP.

Adult↗

PubMed1999

CBP-dependent and independent enhancing activity of steroid receptor coactivator-1 in thyroid hormone receptor-mediated transactivation.

Full-length of steroid receptor coactivator-1 (F-SRC-1) has been shown to interact with thyroid hormone receptors (TRs) in a ligand-dependent manner and to stimulate receptor-dependent transcription. To identify functional domains of F-SRC-1, several internal deletion mutants of F-SRC-1 were constructed. Although in vitro pull down assay with TR showed interaction of all of these mutants with TR, lack of mid legion (amino acids 398-1172) lost enhancing activity of TR-mediated transcription in a transient transfection assay. However, F-SRC-1 mutant lacking CBP-interacting domain still preserved enhancing activity. Surprisingly, F-SRC-1 mutants also increased basal level of viral promoter activity depending upon their deleted region. Yeast activation function assay revealed that these F-SRC-1 mutants had intrinsic activation function when bound to DNA. Analyses of small fragments of F-SRC-1 identified three separable activation domains. In vitro binding assay showed that TBP and TFIIB bound to C-terminal half of F-SRC-1. These results suggest that F-SRC-1 can function via both CBP-dependent and independent manners using various sets of activation domains and that direct interactions between F-SRC-1 and TBP or TFIIB may not be important for CBP-independent transcription.

Animals↗

PubMed1999

The procedure of polymerase chain reaction-restriction fragment-single strand conformation polymorphism analysis by Hha I/Hinc II to detect mitochondrial DNA mutations.

We reported 56 mitochondrial DNA (mtDNA) mutations in Japanese compared with the Cambridge Sequence by polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) analysis. Here we report the principle and the detailed procedures of PCR-RF-SSCP analysis. Restriction map of the 15,673 bp PCR product of mtDNA was designed by MacMolly Tetra, and Hha I and Hinc II were selected. Restriction fragment length polymorphism (RFLP) analysis of 15,673 bp PCR products by Hha I/Hinc II revealed one common pattern and 10 polymorphic patterns compared with the prediction based on the Cambridge Sequence. When the samples were electrophoresed by SSCP analysis, DNA fragments of 912 bp, 772 bp, 761 bp, 634 bp, 431 bp, 327 bp, 226 bp and 171 bp showed 6, 4, 5, 4, 5, 7, 2 and 3 polymorphic patterns, respectively. The determinations of nucleotide sequence of these 46 polymorphic patterns revealed 56 mutations compared with the Cambridge Sequence and 7 of these mutations were common in Japanese. Among these, 20 mutations have not been reported before.

DNA, Mitochondrial↗

PubMed1999

Z-4 allele upstream of the aldose reductase gene is associated with proliferative retinopathy in Japanese patients with NIDDM, and elevated luciferase gene transcription in vitro.

We determined by PCR the number of (A-C)n repeats in the 2. 1 kb upstream of the aldose reductase (AR2) gene in healthy control subjects and in patients with NIDDM in Japanese. Sixty-one patients were recruited based on the severity of retinopathy and subdivided into two groups with proliferative retinopathy and without retinopathy. Japanese exhibited 10 different alleles in this region. The most prevalent allele was designated Z ((A-C)24 repeats) allele. The Z-4 allele was significantly associated with patients with proliferative retinopathy, whereas the Z+2 allele was significantly associated with patients without retinopathy. Erythrocyte AR2 protein levels were significantly elevated in patients exhibiting the Z-4 allele compared to those exhibiting other alleles. When Z-4 allele was ligated in transfection experiments to luciferase vector containing the promoter region of the AR2 gene, the construct showed significantly higher transcription of the reporter gene compared to constructs without (A-C) repeat or with Z-2, Z or Z+2 alleles. Our results suggest that the Z-4 allele in the 2. 1 kb upstream of the AR2 gene may enhance gene transcription and may be a genetic risk factor, which determines the predisposition to retinopathy in Japanese patients with NIDDM.

Aldehyde Reductase↗

PubMed1999

Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells.

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.

Cilostazol↗

PubMed1999

Exophthalmos, pretibial myxedema, osteoarthropathy syndrome associated with papillary fibroelastoma in the left ventricle.

EMO syndrome, a rare complication of Graves' disease, exhibits exophthalmos, pretibial myxedema, and osteoarthropathy. The presence of functional thyrotropin receptors (TSHR) in adipocytes and osteoblasts, both of which we have recently observed, may be related to these extrathyroidal manifestations of Graves' disease. In addition, the expression of TSHR in the heart has recently been reported. We describe here a patient with Graves' disease exhibiting EMO syndrome with a papillary fibroelastoma in the left ventricle. Pathological examinations showed that the fibroelastoma contained Alcian blue-stained mucinous materials that were also observed in the subcutaneous tissue of pretibial myxedema.

Fibroma↗