Katsushige Mizuno

Putative membrane-bound estrogen receptors possibly stimulate mitogen-activated protein kinase in the rat hippocampus

We investigated whether 17beta-estradiol affects the activity of extracellular signal-regulated kinase (ERK) in the central nervous system in vivo. 17beta-Estradiol was administered intracerebroventricularly, and then ERK activity in the rat hippocampus was measured. We found that ERK activity in the rat hippocampus had increased to approximately threefold its basal level at 5 min. This rapid effect was mimicked by the membrane-impermeable estradiol, bovine serum albumin-conjugated 17beta-estradiol, and was not inhibited by tamoxifen and 7alpha,9-(4,4,5,5, 5-pentafluoropentylsulphinyl)nonylestra-1,3,5(10)-triene-3, 17beta-diol (ICI 182780), classical nuclear estrogen receptor antagonists. These data suggest that the rapid activation of ERK by estradiol in vivo is mediated through a putative membrane estrogen receptor in the rat hippocampus.

Neuroprotection by estrogen via extracellular signal-regulated kinase against quinolinic acid-induced cell death in the rat hippocampus

Extracellular signal‐regulated kinase (ERK) belongs to the family of mitogen‐activated protein kinases (MAPKs), which are serine‐threonine kinases activated by phosphorylation in response to a variety of mitogenic signals. We previously reported that 17β‐estradiol rapidly activates ERK in the rat hippocampus. However, the physiological role of this rapid activation of ERK by estrogen in vivo has not yet been elucidated. This study investigated whether ERK may participate in mediating the neuroprotective effects of estrogen against quinolinic acid (QA) toxicity in the rat hippocampus in vivo. Injection of QA into the hippocampi of male rats produced a loss of Nissl‐stained neurons in the CA1 after 24 h. Prior administration of 17β‐estradiol (50 pmol/animal) to the ventricles prevented the QA‐induced decrease in Nissl‐stained neurons. Pretreatment with U0126, an inhibitor of MAPK/ERK kinase, inhibited the rapid activation of ERK by 17β‐estradiol in the rat hippocampus. Moreover, the neuroprotective effects of 17β‐estradiol against QA toxicity were blocked by the pretreatment with U0126. U0126 alone did not produce a loss of neurons. These results indicate that ERK mediates estrogen neuroprotection after QA toxicity in the rat hippocampus.

Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway.

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood. We have previously reported that thrombin activates p38 mitogen‐activated protein kinase (p38 MAPK) by a tyrosine kinase‐dependent mechanism, and that p38 MAPK has a role in thrombin‐induced mitogenic response in rat VSMC. In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin‐induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose‐dependent manner, whereas it had no effect on the response to platelet‐derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA‐AM and GF109203X, which suggest a requirement for intracellular Ca2+ increase and protein kinase C. We next examined the effect of AG1478 on thrombin‐induced DNA synthesis. AG1478 inhibited thrombin‐induced DNA synthesis in a dose‐dependent manner. In contrast, PDGF‐induced DNA synthesis was not affected by AG1478. In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin‐induced proliferation of VSMC.

Targeted DNA transfection into the mouse central nervous system using laser- induced stress waves

We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.

The stimulation of β3-adrenoceptor causes phosphorylation of extracellular signal-regulated kinases 1 and 2 through a Gs- but not Gi-dependent pathway in 3T3-L1 adipocytes

Abstract The treatment of 3T3-L1 adipocytes with three β 3 -adrenoceptor agonists, (±)-( R *, R *)-(4-[2-([2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]phenoxy)acetic acid (BRL37344), 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1, 3-dihydro-2H-benzimidazol-2-one (CGP12177) and [(7S)7-{(2R)2-(3-chlorophenyl)-2-hydroxyethyl-amino}-5,6,7,8-tetrahydronapht-2-yl]ethyl oxyacetate, hydrochloride (SR58611) induces phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). The phosphorylations were not affected by pretreatment of the adipocytes with pertussis toxin, whereas the same treatment completely abolished lisophosphatidic acid-induced phosphorylation of ERK1/2, suggesting the role of pertussis toxin-insensitive G protein in the ERK1/2 phosphorylation by stimulation with the β 3 -adrenoceptor agonists. The phosphorylation of ERK1/2 was mimicked by treating the adipocytes with cholera toxin, a direct activator of stimulatory G (G s ) protein. In addition, the ERK1/2 phosphorylations by the β 3 -adrenoceptor agonists were completely diminished by long-term treatment of the adipocytes with cholera toxin (100 ng/ml, 24 h), whereas that obtained with lisophosphatidic acid stimulation was not. Our findings strongly suggest that the three β 3 -adrenoceptor agonists induce ERK1/2 phosphorylation in 3T3-L1 adipocytes through a G s protein-dependent cascade.

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.

Stimulation of β3‐adrenoceptors causes phosphorylation of p38 mitogen‐activated protein kinase via a stimulatory G protein‐dependent pathway in 3T3‐L1 adipocytes

This study deals with phosphorylation and activation of p38 mitogen‐activated protein kinase (MAPK) via β3‐adrenoceptor (AR) and the signal transduction pathway in 3T3‐L1 adipocytes. β3‐AR agonist BRL37344A (10 nM) caused phosphorylation and activation of p38 MAPK in 3T3‐L1 adipocytes but not in fibroblasts. BRL37344A and also the other β3‐AR agonists, CGP12177A and SR58611A, caused p38 MAPK phosphorylation in dose‐dependent manners. The p38 MAPK phosphorylations by BRL37344A (10 nM), CGP12177A (100 nM), and SR58611A (10 nM) were not antagonized by β1‐ and β2‐ARs antagonist 1‐propranolol (100 nM) but blocked by β3‐AR antagonist SR59230A (10 μM), suggesting the phosphorylation was caused via β3‐AR. The phosphorylations of p38 MAPK were completely abolished by treatment with cholera toxin (CTX) but not pertussis toxin (100 ng ml−1, 24 h). Activation of Gs by CTX (100 ng ml−1) and adenylyl cyclase by forskolin mimicked p38 MAPK phosphorylation. p38 MAPK phosphorylation by BRL37344A was reduced to almost 50% by cyclic AMP‐dependent protein kinase (PKA) inhibitors such as H89 (10 μM) and PKI (10 μM). A src‐family tyrosine kinases inhibitor PP2 (1 μM) also halved the p38 MAPK phosphorylation. Combined use of H89 (10 μM) and PP2 (10 μM) did not bring about further inhibition. These results suggest that β3‐AR caused phosphorylation of p38 MAPK via Gs protein and partly through a pathway involving PKA and src‐family kinase(s), although the contribution of the unidentified pathway remains to be clarified.

Methamphetamine increases the hippocampal α2A-adrenergic receptor and Gαo in mice

Abstract This study investigates the involvement of α2-adrenergic receptors (AR) in mouse brain induced by a low dose of methamphetamine (METH, 2 mg/kg). Immunohistochemical studies show that α2A-AR increased in the dentate gyrus area of the hippocampus 24 h after five repeated administrations of METH. The hippocampal α2A-AR proteins rose 3.2-fold when compared to the saline-administered mice. The other adrenergic receptor, α1D-AR, were not changed by the treatment. Moreover, αo-subunits of GTP-binding proteins (Gαo), one of the downstream molecules of α2A-AR, was also increased by the treatment. These suggest that the repeated administration of low-doses of METH causes quantitative changes of the signaling of α2A-AR in the mouse hippocampus.