Toshimasa Yoshioka

Inhibitory effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)-induced microvascular permeability change in mouse skin.

Anti‐inflammatory effects of cyclic AMP elevating agents were examined in a mouse model of lipopolysaccharide (LPS)‐induced microvascular permeability change. Vascular permeability on the back skin was measured by the local accumulation of Pontamine sky blue (PSB) after subcutaneous injection of LPS (400u2003μg site−1) from Salmonella typhimurium. Dye leakage in the skin was significantly increased 2u2003h after injection of LPS. This LPS‐induced dye leakage was suppressed by phosphodiesterase inhibitors, including pentoxifylline (160u2003mgu2003kg−1), milrinone (5u2003–u200310u2003mgu2003kg−1), rolipram (0.5u2003–u200310u2003mgu2003kg−1) and zaprinast (5u2003–u200310u2003mgu2003kg−1). The dye leakage was also inhibited by β‐adrenoceptor agonists, including isoproterenol (0.5u2003–u20035u2003mgu2003kg−1) and salbutamol (0.05u2003–u20035u2003mgu2003kg−1), an adenylate cyclase activator, forskolin (5u2003mgu2003kg−1), and a cell permeable cyclic AMP analogue, 8‐bromo‐cyclic AMP (8‐Br‐cAMP, 10u2003mgu2003kg−1). LPS caused a transient increase in serum TNF‐α level peaking at 1u2003h after the injection. This increase in serum TNF‐α was completely blocked by a pretreatment with pentoxifylline (160u2003mgu2003kg−1), milrinone (5u2003mgu2003kg−1), rolipram (1u2003mgu2003kg−1), zaprinast (10u2003mgu2003kg−1), salbutamol (0.5u2003mgu2003kg−1), forskolin (1u2003mgu2003kg−1) and 8‐Br‐cAMP (10u2003mgu2003kg−1). LPS caused an increase in serum IL‐1α level peaking at 3u2003h after injection. This increase in serum IL‐1α was not significantly suppressed by the cyclic AMP elevating agents. Our study suggests that cyclic AMP elevating agents attenuate LPS‐induced microvascular permeability change by suppressing TNF‐α up regulation.

Role of nitric oxide, prostaglandins and tyrosine kinase in vascular endothelial growth factor-induced increase in vascular permeability in mouse skin

Abstract We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5–2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by NG-nitro-l-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5–20 mg/kg, i.v.), but not by an inactive enantiomer, NG-nitro-d-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1–100 μg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE2 (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 μg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 μg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE2, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin.

Nitric oxide mediates down regulation of lipoprotein lipase activity induced by tumor necrosis factor-α in brown adipocytes

Abstract We previously reported that tumor necrosis factor- α (TNF- α )/cachectin suppresses lipoprotein lipase activity and its gene expression in brown adipocytes differentiated in culture. Recent evidence suggests that the effect of TNF- α over various cells is related to the enhanced production of nitric oxide (NO). The present study examined whether the suppressive effect of TNF- α on lipoprotein lipase activity is mediated by production of NO in the brown adipocytes. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that TNF- α caused a concentration- and time-dependent expression of inducible NO synthase in brown adipocytes. Increasing concentrations of TNF- α (0.5–50 ng/ml) for 24 h resulted in a concentration-dependent decrease in lipoprotein lipase activity with reciprocal increase in nitrite production in the medium. The suppressive effect of TNF- α on lipoprotein lipase activity was significantly prevented by NO synthase inhibitors, N G -nitro- l -arginine methyl ester ( l -NAME) and aminoguanidine, but not by d -NAME, an inactive isomer. Furthermore, 8-bromoguanosine 3′,5′-cyclic monophosphate, cell permeant cGMP, suppressed lipoprotein lipase activity and 1 H -[1,2,4] oxadiazolo[4,3- a ]quinoxalin-1-one, a selective inhibitor for soluble guanylate cyclase, restored the TNF- α -suppressed lipoprotein lipase activity. These results suggest that TNF- α stimulates brown adipocytes to express inducible NO synthase, followed by production of NO, which in turn mediates the suppressive effect of TNF- α on lipoprotein lipase activity. The effect of NO is mediated, at least partly, through production of cGMP.

Evaluation of iNOS‐dependent and independent mechanisms of the microvascular permeability change induced by lipopolysaccharide

Subcutaneous injection of lipopolysaccharide (LPS) increases plasma leakage in mouse skin. Pretreatment with LPS conditions mice tolerant to the LPS‐induced plasma leakage. Nitric oxide (NO) has been suggested to be involved in these LPS effects. A specific role of inducible NO synthase (iNOS) was investigated in the LPS‐induced plasma leakage using iNOS deficient mice. Plasma leakage in mouse skin was measured by the local accumulation of Pontamine sky blue at the site of subcutaneous injection of LPS (Sal. typhimurium). LPS (100–400u2003μgu2003site−1) produced a dose‐related increase in dye leakage in both iNOS deficient and wild‐type mice with about 40% less dye leakage in iNOS deficient mice. Indomethacin (5u2003mgu2003kg−1), N‐[‐2‐cyclohexyloxy]‐4‐nitrophenyl methanesulphonamide (NS‐398) (1u2003mgu2003kg−1), diphenhydramine (10u2003mgu2003kg−1) and anti‐TNF‐α antibody (dilution 1u2003:u2003400, 10u2003mlu2003kg−1) inhibited the LPS‐induced dye leakage in both iNOS deficient and wild‐type mice, whereas NG‐nitro‐L‐arginine methyl ester (L‐NAME) (10u2003mgu2003kg−1) or aminoguanidine (10u2003mgu2003kg−1) inhibited that in wild‐type but not in iNOS deficient mice. Pretreatment with LPS (0.15u2003mgu2003kg−1 i.p.) 4u2003h before decreased the LPS‐induced dye leakage in wild‐type but not in iNOS deficient mice. LPS pretreatment increased serum corticosterone levels in both mice, while it increased the serum nitrate/nitrite levels in wild‐type but not in iNOS deficient mice. These studies indicate that an increase in vascular permeability induced by LPS is mediated by NO produced by iNOS, eicosanoids, histamine and TNF‐α. The tolerance against LPS‐induced vascular permeability change may be mediated by iNOS induction but not by an increased release of endogenous corticosteroids.

Cationic amino acid transporter-2 mRNA induction by tumor necrosis factor-α in vascular endothelial cells

Nitric oxide (NO) synthesis may be coupled to the activity of the cellular L-arginine transporter, namely the cationic amino acid transporter. The present study examined tumor necrosis factor (TNF)-alpha-induced alterations in the gene expression of the cationic amino acid transporter (CAT) and NO production in human umbilical vein endothelial cells. In quiescent endothelial cells, CAT-1 mRNA expression, determined by reverse transcription-polymerase chain reaction, was dominant to that of CAT-2. TNF-alpha (10 ng/ml for 1-24 h) induced a time-dependent increase in CAT-2 but not CAT-1 expression. Moreover, TNF-alpha (1-30 ng/ml) treatment for 6 h induced a concentration-dependent increase in CAT-2 mRNA expression. The upregulation of CAT-2 expression by TNF-alpha was associated with enhanced nitrite accumulation in the culture medium (70% increase compared with vehicle-treated cells at 24 h). Thus, induction of the cationic amino acid transporter may constitute one mechanism for the TNF-alpha-induced NO production in human umbilical vein endothelial cells.

Protein kinase C mediates tumor necrosis factor-α-induced inhibition of obese gene expression and leptin secretion in brown adipocytes

Abstract. Previously we showed that tumor necrosis factor-α (TNF-α) inhibits lipoprotein lipase (LPL) activity and its gene expression, an early marker of adipocyte differentiation, in cultured brown adipocytes. To know whether TNF-α also affects late events in brown adipocyte maturation, we examined the effect of TNF-α on obese gene expression and leptin secretion in mouse brown adipocytes differentiated in culture. TNF-α caused a concentration-dependent decrease in leptin accumulation in culture medium and leptin mRNA amount in brown adipocytes which constitutively express the ob gene. Time-course study showed that TNF-α significantly suppressed leptin secretion during incubation for 16, 24 and 48xa0h. Since some effect of TNF-α is mediated by activation of protein kinase C (PKC), the role of PKC in TNF-α-induced downregulation of ob gene expression and leptin secretion was studied. The suppressive effect of TNF-α on both ob gene expression and leptin secretion was blocked by PKC inhibitors such as bisindolylmaleimide I (BIM) and l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7). Incubation of brown adipocytes with TNF-α (20xa0ng/ml, 15xa0min) caused a rapid shift of PKC activity from the cytosolic to the membrane fraction, suggesting an activation of PKC by TNF-α in brown adipocytes. This effect of TNF-α was blocked by a selective PKC inhibitor, BIM. These results suggest that TNF-α promotes dedifferentiation of the brown adipocytes as evidenced by a downregulation in ob gene expression and leptin secretion via PKC-dependent mechanisms.

A lipid A analog ONO-4007 induces tolerance to plasma leakage in mice

Abstract.Objective: The effects of pretreatment with ONO-4007, a lipid A analog, on cutaneous plasma leakage induced by ONO-4007, lipopolysaccharide (LPS) and inflammatory mediators were investigated.¶Material: Male ddY strain mice.¶Treatment: Mice were pretreated with ONO-4007 (up to 6 mg/kg i.p.), 0-24 h prior to plasma leakage study.¶Methods: Plasma extravasation was determined by dye leakage.¶Results: Systemic ONO-4007 (6 mg/kg i.p.) pretreatment for 2 to 12 h inhibited plasma extravasation in the mouse skin elicited by ONO-4007 and LPS. The inhibition was dose-dependent. Plasma leakage induced by platelet-activating factor (PAF), histamine and 5-hydroxytryptamine (5-HT) was also inhibited by ONO-4007 pretreatment. Plasma corticosterone levels increased 2 and 4 h after systemic ONO-4007 (6 mg/kg) administration and returned to the control level 24 h later. Adrenalectomy and metyrapone but not propranolol reversed the inhibition by ONO-4007 pretreatment of LPS-induced plasma leakage.¶Conclusions: A single injection of ONO-4007 in mice induced transient tolerance to plasma leakage elicited by LPS, ONO-4007 and inflammatory mediators. Endogenous corticosterone, at least in part, plays a role in the development of tolerance.