Nobuyuki Banba

Dexamethasone suppresses iNOS gene expression by inhibiting NF-κB in vascular smooth muscle cells

Bacterial lipopolysaccharide (LPS) and other immunostimulants induce an isoform of nitric oxide synthase (iNOS) gene expression in vascular smooth muscle cells (VSMC). This process is dependent on nuclear factor-kappa B (NF-kappaB) activation and is suppressed by glucocorticoids. The aim of this study was to investigate the molecular mechanisms of inhibition of iNOS expression by the synthetic glucocorticoid, dexamethasone (DEX), in rat VSMC. Treatment of VSMC with LPS plus interferon-gamma (LPS/IFN) caused activation of NF-kappaB and the iNOS promoter. LPS/IFN induced iNOS mRNA and NO synthesis. DEX markedly depressed LPS/IFN-stimulated iNOS mRNA expression and NO production. DEX also suppressed LPS/IFN-stimulated activity of a 1.7-kb iNOS promoter, indicating that the inhibition of iNOS expression by DEX occurs at the level of transcription. NF-kappaB activation by LPS/IFN was repressed by DEX. The inhibition of NF-kappaB by DEX exhibited dose-dependent kinetics, which corresponded to DEX suppression of iNOS promoter activation, iNOS mRNA expression, and NO production. However, activation of activator protein-1 (AP-1), which is also contained in the iNOS promoter, was not enhanced by LPS/IFN or inhibited by DEX. Thus, glucocorticoids appear to block iNOS expression, at least in part, through inhibition of NF-kappaB activation, which results in decreased NO production.

Immunoreactive endothelin concentrations in maternal and fetal blood

Immunoreactive-endothelin (ir-ET) concentrations were determined in peripheral maternal blood and in umbilical cord blood just after delivery. The concentrations in both the umbilical artery (2.83 +/- 1.36 pmol/l plasma, Mean +/- SD) and vein (3.37 +/- 1.53 pmol/l) were significantly higher than those found in maternal venous blood (1.43 +/- 1.02 pmol/l). On the other hand, ir-ET levels in maternal blood were not significantly different when compared with those found in non-pregnant women (1.50 +/- 0.83 pmol/l). No significant difference of ir-ET levels between the umbilical artery and vein was observed. A highly significant correlation (r = 0.60, p less than 0.01) of ir-ET levels between the umbilical artery and vein was observed. Also, a significant correlation (r = 0.48, p less than 0.01) between umbilical vein and maternal vein ir-ET levels with a weaker correlation (r = 0.36, p less than 0.05) between umbilical artery and maternal vein ir-ET levels was demonstrated. The present study indicates that ir-ET may be actively secreted in fetal circulation and the plasma levels in maternal and fetal circulation may have a possible relation.

Expression of monocyte chemoattractant protein-1 mRNA and protein in cultured human thyrocytes

Monocytes as well as lymphocytes infiltrate in the stroma of thyroid tissue in autoimmune and destructive thyroiditis. Monocyte chemoattractant protein‐1 (MCP‐1) is a cytokine that attracts T‐lymphocytes as well as monocytes. Using human thyrocytes in primary cultures, we show that expression of MCP‐1 mRNA and protein is remarkably stimulated by both interleukin‐1 (IL‐1) and tumor necrosis factor‐α (TNF‐α), and also that interferon‐γ (IFN‐γ) by itself is a weak stimulant but has a synergistic activity with either IL‐1 or TNF‐α. The finding indicates that MCP‐1 can be produced by thyrocytes themselves, suggesting a possible role of thyrocytes on accumulation of monocytes and T‐lymphocytes to the tissue from the blood in autoimmune and destructive thyroiditis.

Interleukin-6 and transforming growth factor-β regulate the expression of monocyte chemoattractant protein-1 and colony-stimulating factors in human thyroid follicular cells

Monocytes and T-lymphocytes, both of which play a pivotal role in immune/inflammatory responses, can be attracted from the circulation into tissues by monocyte chemoattractant protein-1 (MCP-1), and monocytes can be further activated by colony-stimulating factors (CSFs), granulocyte/macrophage CSF (GM-CSF) or macrophage CSF (M-CSF). We examined whether either interleukin-6 (IL-6) or transforming growth factor-beta (TGF-beta), both of which are produced by thyroid follicular cells (TFC), can regulate the production of MCP-1 or CSF(s) in human TFC. IL-6, being effective only in the presence of soluble IL-6 receptor (sIL-6R), stimulated the expression of both MCP-1 and M-CSF, but was inhibitory on GM-CSF expression. On the other hand, TGF-beta stimulated the expression of both MCP-I and GM-CSF, but suppressed M-CSF expression. These results suggest a possible role of IL-6 or TGF-beta on the initiation and/or modulation of thyroid immune/inflammatory responses via MCP-1 production and differential production of GM-CSF or M-CSF by TFC.

Effects of pituitary adenylate cyclase activating polypeptide (PACAP) on the cardiovascular system

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on the cardiovascular system were examined. When PACAP-38 (270 or 420 pmol/kg body weight) was administered intravenously to the anesthetized dogs, both mean arterial pressure and left ventricular systolic pressure increased within 2 min after a temporal depression. Pulmonary arterial systolic pressure increased promptly. These hemodynamic values and heart rates (HR) 5 min after injection were significantly higher than the corresponding values in physiological saline injected dogs, and some effects were still sustained over 15 min. Cardiac output and stroke volume also increased and the values at 5 min were significantly higher than those in controls. The high dose of PACAP-38 (420 pmol/kg) evoked greater responses than those induced by the low dose (270 pmol/kg). Plasma adrenaline, but neither noradrenaline nor dopamine concentration significantly increased 15 min after injection of 420 pmol/kg PACAP-38. Moreover, PACAP-38 clearly stimulated cyclic AMP production in rat cardiac myocytes with EC50 of 1.5 x 10(-9) M and plasma cAMP levels significantly and dose-dependently increased in dogs 5 min after administration. These results first demonstrated that PACAP has inotropic and chronotropic actions on the heart possibly by a direct stimulation of adenylate cyclase in cardiac myocytes and also that the cardiovascular functions may be possibly modified by an evoked adrenaline secretion in vivo.

Effects of calcium channel antagonists on the induction of nitric oxide synthase in cultured cells by immunostimulants

We investigated whether calcium channel antagonists would alter the induction of nitric oxide (NO) synthesis by bacterial lipopolysaccharide (LPS) alone or in combination with interferon-gamma (IFN gamma) in cultured J774 macrophages, rat vascular smooth muscle cells, rat renal mesangial cells, and rat cardiac myocytes. The induction of NO synthesis was determined by measuring nitrite, the stable end-product. The dihydropyridine calcium channel antagonists, nifedipine, manidipine, nitrendipine, benidipine, barnidipine, perdipine, and nilvadipine all reduced the LPS-induced nitrite production in a dose-dependent manner, each with a differing half-maximal inhibitory concentration, in cultured J774 macrophages. Nifedipine also inhibited nitrite production in vascular smooth muscle cells, mesangial cells, and cardiac myocytes. The half-maximal inhibitory concentrations of nifedipine were ranked as follows: smooth muscle cells < mesangial cells < cardiac myocytes. Diltiazem, at nontoxic concentrations, had no effect on the nitrite formation in the three cell types. Verapamil markedly increased the formation of nitrite in cardiac myocytes in response to LPS and IFN gamma, but not in vascular smooth muscle or mesangial cells. Exposure of cardiac myocytes to LPS and IFN gamma caused the expression of NO synthase mRNA that was significantly increased by verapamil. Thus, certain calcium channel antagonists modulate NO synthesis by altering the induction of NO synthase.

Nitric oxide inhibits cell growth in cultured human thyrocytes

Effect of NO induced by interleukin-1 (IL-1) or IL-1/interferon- gamma (IL-1/IFN-gamma) was investigated on cell growth using primary cultures of human thyrocytes. Cytokine-induced NO production was associated not only with an increase in cyclic GMP (cGMP) formation but also with an inhibition of cell growth determined by bromo-deoxyuridine (Br-dU) incorporation into DNA. When NO synthesis was blocked by NG-monomethyl-L-arginine (L-MMA), cGMP formation was prevented in parallel with NO production and inversely a restoration of cell growth was evident. S-nitroso-N-acetyl-penicillamine, a NO donor, but not a cell permeable cGMP analog, 8-bromo-cGMP, inhibited cell growth in a dose-dependent manner. The present findings strongly indicate that endogenous NO produced by the cytokine treatment as well as exogenous NO, has a cGMP-independent inhibitory action on human thyrocyte growth.

Effect of NG-nitro-l-arginine on shock induced by endotoxin and by platelet activating factor in dogs

When NG-nitro-L-arginine, a nitric oxide synthase inhibitor, administration was started 5 min prior to shock induction in anesthetized dogs, a partial restoration was observed in endotoxin-induced shock and a complete recovery in platelet activating factor (PAF)-induced shock. When NG-nitro-L-arginine infusion was started 5 min after shock induction, no significant recovery was observed in endotoxin-induced shock and a complete recovery in PAF-induced shock. These data indicate that enhanced production of nitric oxide by vascular endothelial cells may contribute to endotoxin- or PAF-induced shock and also that some mediators including inducible nitric oxide synthase and/or cellular damage might be involved in endotoxin-induced shock.

Human urinary epidermal growth factor: effects of age, sex and pregnancy

Urinary concentrations of immunoreactive human epidermal growth factor (hEGF) were determined by specific homologous radioimmunoassay in 169 healthy men (aged 20-69 years), 275 healthy women (20-8 years). healthy women (20-68 years) and 413 pregnant women (20-39 years). Relative hEGF concentrations in urine (micrograms/g creatinine) decreased significantly in both sexes between 24 and 64 years of age. The relative concentrations of hEGF in urine were significantly higher in women than in men at ages 20-69 years. The mean values of relative urinary hEGF concentrations in pregnant women in their twenties and thirties (30.0 +/- 0.7 micrograms/g creatinine and 29.6 +/- 1.2 micrograms/g creatinine) were significantly higher than those in age-matched nonpregnant women (27.3 +/- 1.8 micrograms/g creatinine and 22.8 +/- 0.7 micrograms/g creatinine). Among the trimesters, it was highest in the 2nd trimester of women in the twenties and thirties (33.4 +/- 1.3 micrograms/g creatinine and 31.7 +/- 1.9 micrograms/g creatinine). The significance of the increased urinary excretion of hEGF (micrograms/g creatinine) in pregnancy is not known. Further studies are required to find a source of hEGF in urine and a possible relation between increased hEGF excretion and fetoplacental growth and development.

The herbal medicine sho-saiko-to induces nitric oxide synthase in rat hepatocytes.

We have examined the effects of the herbal medicine sho-saiko-to (SST) on nitric oxide (NO) biosynthesis in rat hepatocytes by measuring the stable end-product nitrite and the mRNA of inducible NO synthase (iNOS). Interferon-gamma (IFN) by itself failed to induce NO synthesis (IFN: 1-1,000 u/ml). SST also did not elicit NO synthesis at concentrations up to 300 micrograms/ml when administered alone, but dose-dependently induced NO production in the presence of IFN. Whereas SST or IFN induced barely detectable levels of iNOS mRNA when administered alone, a combination of SST and IFN markedly induced iNOS mRNA in the cells. SST also modestly increased NO synthesis caused by interleukin-1 or bacterial lipopolysaccharide as a single agent, or in combination with IFN. On the other hand, SST had no effects on the NO synthesis produced by iNOS which were already induced. Thus, we found that SST stimulates cultured hepatocytes to produce NO by inducing iNOS gene expression under appropriate conditions. The capability of SST to induce NO biosynthesis might be related to the therapeutic efficacy of SST on the liver diseases.