Sachie Asada

Essential Role of p38 Mitogen-activated Protein Kinase in Contact Hypersensitivity

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in the pathogenesis of inflammation, using a mouse contact hypersensitivity (CHS) model induced by 2,4-dinitro-1-fluorobenzene (DNFB). Ear swelling was induced by challenge with DNFB, accompanied by infiltration of mononuclear cells, neutrophils, and eosinophils and a marked increase in mRNA levels of cytokines such as interleukin (IL)-2, interferon (IFN)-γ, IL-4, IL-5, IL-1β, IL-18, and tumor necrosis factor-α in the challenged ear skin. Both ear swelling and the number of infiltrated cells in DNFB-challenged ear skin were significantly inhibited by treatment with SB202190, a p38 inhibitor. Furthermore, the DNFB-induced expression of all cytokines except IL-4 was significantly inhibited by treatment with SB202190. Ribonuclease protection assay revealed that the mRNA levels of chemokines such as IP-10 and MCP-1 in ear skin were markedly increased at 24 h after challenge with DNFB. The induction of these chemokines was significantly inhibited by treatment with SB202190. In p38α +/− mice, both ear swelling and infiltration of cells induced by DNFB were reduced compared with those in wild-type mice. However, induction of cytokines by DNFB was also observed in p38α +/− mice, although the induction of IFN-γ, IL-5, and IL-18 was typically reduced compared with that in wild-type mice. Challenge with DNFB slightly induced IP-10 and MCP-1 mRNA in p38α +/− mice, with weaker signals than those in SB202190-treated wild-type mice. These results suggest that p38 plays a key role in CHS and is an important target for the treatment of CHS.

Bis-N-nitroso-caged nitric oxides: photochemistry and biological performance test by rat aorta vasorelaxation

Three new caged nitric oxides (NOs)-BNN3, BNN5Na, and BNN5M were tested for biological use. BNNs have a strong ultraviolet (UV) absorption band (lambda(max): 300 nm, epsilon: 13.5 mM(-1) cm (-1)) extended to 420 nm and produce NO upon irradiation with 300-360 nm light in quantum yields about 2. A photoexcited BNN molecule yields two NOs with time constants of less than 10 ns for phase 1 and less than 20 micros for phase 2 at 37 degrees C, suggesting usefulness of BNNs for measuring in vivo and in vitro fast NO reactions. Upon irradiating with UV light, caged nitric oxides-loaded rat aortic strips maintained in a state of active tonic contraction effectively relaxed ( < 3 microM BNN5M loading solution concentration). BNN3 is incorporated in the lipid membrane. BNN5Na, insoluble in organic solvents but water soluble, localizes in the water phase. BNN5M, is muscle-cell-permeable and hydrolysed to BNN5Na to remain in cytosol. BNNs were thermally stable and demonstrated no observable toxicity.

Microtubule dynamics regulates the level of endothelin-B receptor in rat cultured astrocytes

Abstract: We investigated the effect of cytoskeleton modulators on endothelin‐B (ETB) receptor expression in rat primary cultured astrocytes. Northern blot analysis and a binding study revealed that colchicine and nocodazole, microtubule‐disrupting agents, decreased the levels of both ETB receptor mRNA and the number of ET‐1 binding sites in quiescent astrocytes. Down‐regulation of both ETB receptor mRNA and the number of binding sites for ET‐1 was also observed in quiescent astrocytes treated with taxol, a microtubule‐stabilizing agent. In contrast, neither β‐lumicolchicine, an inactive isomer of colchicine, nor cytochalasin D, a microfilament‐disrupting agent, influenced ETB receptor expression. The level of ETB receptors in astrocytes was affected by the cell state, namely, proliferative, quiescent, or differentiated state. The order of ETB receptor expression according to the cell state was proliferative state < quiescent state ≪ differentiated state induced by dibutyryl cyclic AMP. Also, in proliferative astrocytes and differentiated astrocytes, colchicine significantly down‐regulated both ETB receptor mRNA and the number of binding sites for ET‐1. However, thymidine assay revealed that colchicine did not change quiescent astrocytes and differentiated astrocytes to a proliferative state. Furthermore, the increase in glutamine synthetase activity in differentiated astrocytes was not affected by colchicine. These results suggest that microtubule dynamics possibly regulates ETB receptor expression in astrocytes without affecting the cell state.

Endothelin-1-induced downregulation of ETB receptor mRNA: participation of cAMP.

Summary: In this study we examined the participation of cAMP formation in endothelin-1 (ET-l)-induced down-regulation of ETB receptor mRNA in ROS 17/2 rat osteosarcoma cells. Dibutyryl cAMP induced downregulation of ETB receptor mRNA in a time-dependent manner. ET-1 induced production of inositol phosphates and an increase of cAMP level in ROS 17/2 cells. A stimulatory effect on cAMP level was also observed when A23187 plus PMA was added to the cells. The increase in cAMP level induced either by ET-1 or by A23187 plus PMA was inhibited by indomethacin. The downregulation of ETB receptor mRNA induced by ET-1 was significantly inhibited by indomethacin. These results suggest that the ET-1-induced downregulation of ETB receptor mRNA in ROS 17/2 cells may be partly mediated through the increase in cAMP level secondary to the activation of the phosphoinositide hydrolysis/Ca2+ transduction cascade.

Cytodifferentiation enhances Erk activation induced by endothelin-1 in primary cultured astrocytes.

We have previously demonstrated that endothelin-1 (ET-1)-induced extracellular signal-regulated kinase (Erk) activity via the ETB receptor (EDNRB) is mediated through two independent pathways, a protein kinase C-dependent pathway and a pertussis toxin (PTX)-sensitive pathway, in astrocytes. In this study, we showed that the molar potency of ET-1 to induce Erk activation was two orders of magnitude higher in dibutyryl cAMP (DBcAMP)-treated astrocytes than in quiescent astrocytes. This DBcAMP-enhanced molar potency of ET-1 in Erk activation was selectively inhibited by pretreatment of astrocytes with PTX. The expression level of EDNRB in astrocytes was markedly upregulated by DBcAMP-induced cytodifferentiation. However, this up-regulation was simply attributed to the high expression of lowaffinity sites. The molar potency of ET-1 to induce both stimulation of inositol trisphosphate production and activation of protein kinase C in DBcAMP-treated astrocytes was similar to that in quiescent astrocytes. On the contrary, the molar potency of ET-1 to induce accumulation of Ras-GTP was two orders of magnitude higher in DBcAMP-treated astrocytes than in quiescent astrocytes, which was consistent with the case of ET-1-induced Erk activation. Moreover, the ET-1-induced Ras activation was PTX sensitive. These results suggest that cytodifferentiation selectively enhances the PTXsensitive Ras/Erk pathway induced by ET-1 in astrocytes, and that cytodifferentiation-induced EDNRB up-regulation might not contribute to this selective potentiation of ET-1 signaling.