Yasuyuki Sugishita

Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes.

OBJECTIVE Inducible nitric oxide synthase (iNOS) has been implicated to contribute to myocardial dysfunction in various settings, but considerable species differences have been noted in the levels of iNOS expression and its function in several tissues. The aim of this study was to elucidate evolutional changes in myocardial iNOS expression and function. METHODS An iNOS cDNA clone was isolated by RT-PCR from the 10-day old cultured chick embryonic ventricular myocytes stimulated with 10 micrograms/ml of lipopolysaccharide. Expression of the iNOS mRNA was analyzed with Northern blot analysis and RNase protection assay. The iNOS activity was estimated from conversion rates of L-arginine to L-citrulline and intracellular cGMP contents were measured with radioimmunoassay. Furthermore, both [Ca2+]i (fluorescent dye indo-1) and cell contraction (video motion detector) were simultaneously recorded. RESULTS Aside from the primer sequences, the insert (1026 bp) of the cDNA clone showed 66.4% identity at the deduced amino acid level to the human iNOS cDNAs. Northern blot analysis revealed that chicken iNOS mRNA of approximately 4.5 kb was induced by lipopolysaccharide within 6 h in the cultured myocytes. RNase protection assay also showed that lipopolysaccharide provoked 14.6 +/- 5.1-fold increases (n = 6, p < 0.05) in the iNOS mRNA signals within 6 h. The iNOS activity (+300%, P < 0.05) as well as the intracellular cGMP contents (+75%, P < 0.01) were significantly augmented in the lipopolysaccharide-stimulated cells. Both the cell contraction and [Ca2+]i were significantly reduced after the administration of a large amount (10 mM) of L-arginine in the myocytes pretreated with both lipopolysaccharide and NG-monomethyl-L-arginine (100 microM). CONCLUSION As like as the nucleotide and amino acid sequences, the myocardial effects of the iNOS may also be evolutionary conserved.

Ets-1 is involved in transcriptional regulation of the chick inducible nitric oxide synthase gene in embryonic ventricular myoctyes

In order to elucidate roles of Ets family of transcription factors in transcriptional activation of inducible nitric oxide synthase (iNOS) genes, we analyzed the chick iNOS gene expression in cultured chick embryonic ventricular myocytes (CEVM). Deletional analysis and site-directed mutagenesis demonstrated that both the Ets/PEA3 site (–221 to –216 bp) and the κB site (–101 to –93 bp) of the 5′-flanking region of the chick iNOS gene were involved in the maximal activation of the lipopolysaccharide (LPS)-induced expression of the reporter (luciferase) gene, although the proximal κB site played the more essential role. Electrophoretic mobility shift assay revealed that LPS augmented the nuclear protein bindings to the Ets/PEA3 as well as κB motifs. Ets-1, one of the Ets proteins, was suggested to be bound to the Ets/PEA3 oligonucleotide. By Northern blot analysis, LPS was shown to induce iNOS mRNA in CEVM, along with a preceding increase in the levels of c-ets-1 mRNA. Ets-1 may be involved in the iNOS gene transcription in CEVM, presumably through interacting with the NF-κB.

Pulmonary Embolism Due to Popliteal Venous Aneurysm

A 33-year-old man was admitted to our hospital for shortness of breath on exertion. His symptoms started suddenly a week before admission when he was driving a car and worsened daily. He was amateur football player and had no history of hypertension, dyslipidemia, diabetes mellitus, smoking, or leg injury. On admission, an arterial pulse oxygen saturation monitor showed that his arterial blood oxygen saturation was 94% with room air. His blood pressure was 114/82 mm Hg and his pulse rate was 92/min with regular rhythm. His height was 162 cm and body weight was 62 kg. No other outstanding physical abnormalities were observed. Laboratory data showed slightly an elevated C-reactive protein level of 0.87 mg/dL with a normal white blood cell count of 6100 cells/mL. Arterial blood sampling revealed a normal CO2 level of 41 mm Hg and pH of 7.42 with low oxygen tension (52 mm Hg). An ECG showed a small S wave in lead I and a small Q wave and inverted T wave in …

Expression of Vascular Endothelial Growth Factor and Hypoxia-Inducible Factor 1α in the Myocardium

Expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α), a transcription factor involved in the VEGF induction, was examined in the myocardium. In the 10-day-old chick embryonic hearts and ventricular myocytes, the mRNA species encoding VEGF and HIF-1α were abundantly expressed even at the basal conditions. The steady-state levels of the VEGF mRNA were regulated by various protein kinases. In the cultured neonatal rat ventricular myocytes, VEGF expression was upregulated by lipopolysaccharide. These data suggest that VEGF and HIF-1α expressed in cardiac myocytes may play significant roles in cardiovascular development. Furthermore, the cardiac myocyte-derived VEGF may also contribute to the pathogenesis of systemic inflammatory response syndrome, including myocardial interstitial edema.