Norimichi Nakahata

Thromboxane A2 : Physiology/pathophysiology, cellular signal transduction and pharmacology

Thromboxane A(2) (TXA(2)), an unstable arachidonic acid metabolite, elicits diverse physiological/pathophysiological actions, including platelet aggregation and smooth muscle contraction. TXA(2) has been shown to be involved in allergies, modulation of acquired immunity, atherogenesis, neovascularization, and metastasis of cancer cells. The TXA(2) receptor (TP) communicates mainly with G(q) and G(13), resulting in phospholipase C activation and RhoGEF activation, respectively. In addition, TP couples with G(11), G(12), G(13), G(14), G(15), G(16), G(i), G(s) and G(h). TP is widely distributed in the body, and is expressed at high levels in thymus and spleen. The second extracellular loop of TP is an important ligand-binding site, and Asp(193) is a key amino acid. There are two alternatively spliced isoforms of TP, TPalpha and TPbeta, which differ only in their C-terminals. TPalpha and TPbeta communicate with different G proteins, and undergo hetero-dimerization, resulting in changes in intracellular traffic and receptor protein conformations. TP cross-talks with receptor tyrosine kinases, such as EGF receptor, to induce cell proliferation and differentiation. TP is glycosylated in the N-terminal region for recruitment to plasma membranes. Furthermore, TP conformation is changed by coupling to G proteins, showing several states of agonist binding. Finally, several drugs modify TP-mediated events; these include cyclooxygenase inhibitors, TXA(2) synthase inhibitors and TP antagonists. Some flavonoids of natural origin also have TP receptor antagonistic activity. Recent advances in TP research have clarified TXA(2)-mediated events in detail, and further study will supply more beneficial information about TXA(2) pathophysiology.

Inhibition of cyclooxygenase and prostaglandin E2 synthesis by γ-mangostin, a xanthone derivative in mangosteen, in C6 rat glioma cells

The fruit hull of mangosteen, Garcinia mangostana L., has been used for many years as a medicine for treatment of skin infection, wounds, and diarrhea in Southeast Asia. In the present study, we examined the effect of gamma-mangostin, a tetraoxygenated diprenylated xanthone contained in mangosteen, on arachidonic acid (AA) cascade in C6 rat glioma cells. gamma-Mangostin had a potent inhibitory activity of prostaglandin E2 (PGE2) release induced by A23187, a Ca2+ ionophore. The inhibition was concentration-dependent, with the IC50 value of about 5 microM. gamma-Mangostin had no inhibitory effect on A23187-induced phosphorylation of p42/p44 extracellular signal regulated kinase/mitogen-activated protein kinase or on the liberation of [14C]-AA from the cells labeled with [14C]-AA. However, gamma-mangostin concentration-dependently inhibited the conversion of AA to PGE2 in microsomal preparations, showing its possible inhibition of cyclooxygenase (COX). In enzyme assay in vitro, gamma-mangostin inhibited the activities of both constitutive COX (COX-1) and inducible COX (COX-2) in a concentration-dependent manner, with the IC50 values of about 0.8 and 2 microM, respectively. Lineweaver-Burk plot analysis indicated that gamma-mangostin competitively inhibited the activities of both COX-1 and -2. This study is a first demonstration that gamma-mangostin, a xanthone derivative, directly inhibits COX activity.

The dorsomedial hypothalamic nucleus is not necessary for food-anticipatory circadian rhythms of behavior, temperature or clock gene expression in mice

Circadian rhythms in mammals are regulated by a light‐entrainable circadian pacemaker in the hypothalamic suprachiasmatic nucleus and food‐entrainable oscillators located elsewhere in the brain and body. The dorsomedial hypothalamic nucleus (DMH) has been proposed to be the site of oscillators driving food‐anticipatory circadian rhythms, but this is controversial. To further evaluate this hypothesis, we measured clock gene, temperature and activity rhythms in intact and DMH‐ablated mice. A single 4‐h midday feeding after an overnight fast induced mPer1 and mPer2 mRNA expression in the DMH, arcuate nucleus, nucleus of the solitary tract and area postrema, and reset daily rhythms of mPer1, mPer2 and mBMAL1 in the DMH, arcuate and neocortex. These rhythms persisted during 2 days of food deprivation after 12 days of scheduled daytime feeding. Acute induction of DMH mPer1 and mPer2 was N‐methyl‐d‐aspartate (NMDA) receptor‐dependent, whereas rhythmic expression after 6 days of restricted feeding was not. Thermal DMH lesions did not affect acute induction or rhythmic expression of clock genes in other brain regions in response to scheduled daytime feeding. DMH lesions attenuated mean daily activity levels and nocturnality but did not affect food‐anticipatory rhythms of activity and body temperature in either light–dark or constant darkness. These results confirm that the DMH and other brain regions express circadian clock gene rhythms sensitive to daytime feeding schedules, but do not support the hypothesis that DMH oscillations drive food‐anticipatory behavioral or temperature rhythms.

Rap1 potentiates endothelial cell junctions by spatially controlling myosin II activity and actin organization

Rap1 potentiates endothelial cell junctions by spatially controlling non-muscle myosin II activity through activation of the Cdc42–MRCK pathway and suppression of the Rho–ROCK pathway.

Thromboxane A2 activates phospholipase C in astrocytoma cells via pertussis toxin-insensitive G-protein

The properties of thromboxane A2 (TXA2) receptors were examined in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of TXA2, stimulated the accumulation of inositol phosphates (IPs) with an EC50 of about 50 nM. The STA2-induced accumulation of IPs was inhibited concentration dependently by ONO3708, a TXA2 receptor antagonist, with an inhibition constant (Ki) of about 10 nM. Inositol trisphosphate (IP3) was accumulated more rapidly than inositol bisphosphate (IP2) in response to STA2. HPLC analysis indicated that inositol 1,4,5-trisphosphate accumulated in the presence of STA2. STA2 alone had no effect on the accumulation of IPs in membrane preparations but it potentiated the accumulation induced by GTP gamma S. [3H]SQ29548, a TXA2 receptor antagonist, bound specifically to TXA2 receptors, expressing a single binding site with a dissociation constant (Kd) of 10.9 nM. The competition curve for STA2 inhibition of [3H]SQ29548 binding was shifted to the right and was steeper in the presence of GTP gamma S. Pertussis toxin (IAP) elicited ADP-ribosylation of 41KD protein but had no effect on the sensitivity to GTP of the STA2 inhibition of SQ29548 binding or of STA2-induced accumulation of IPs. It is concluded from these results that the stimulation of TXA2 receptors results in activation of phospholipase C via a GTP binding protein and that the protein is not a substrate for IAP.

Scabronine A, a novel diterpenoid having potent inductive activity of the nerve growth factor synthesis, isolated from the mushroom, Sarcodon scabrosus

Abstract A novel diterpenoid, scabronine A, having strong inductive activity of the nerve growth factor synthesis was isolated from the fruit body of the mushroom, Sarcodon scabrosus (Fr.) Karst. The stereostructure of scabronine A ( 1 ) was elucidated on the basis of the spectroscopic analysis of natural scabronine A and its derivatives.

ERK5 activity is required for nerve growth factor-induced neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells

Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt2cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 μm, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt2cAMP, but BIX02188 and BIX02189, at 30 μm, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt2cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt2cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process.

INVOLVEMENT OF CYCLOOXYGENASE-2 IN LIPOPOLYSACCHARIDE-INDUCED IMPAIRMENT OF THE NEWBORN CELL SURVIVAL IN THE ADULT MOUSE DENTATE GYRUS

There is growing evidence indicating that neurogenesis in adulthood is influenced by certain types of the central diseases such as neuroinflammation, however, its mechanism is not fully understood. This study was, therefore, designed to examine the effects of lipopolysaccharide (LPS), a bacterial endotoxin known to cause the neuroinflammation, on the neurogenesis in the dentate gyrus of adult mice using the bromodeoxyuridine (BrdU) -pulse chase method. LPS failed to affect the number of BrdU-labeled cells in the dentate gyrus 2 h after BrdU injection, indicating no effects of LPS on the proliferation of the neural stem cells (NSCs). On the other hand, we found that LPS dose-dependently (0.1, 0.5, 1 mg/kg) decreased the number of BrdU-labeled cells 7 and 21 days after BrdU injection. We also observed that LPS increased cell death in the dentate gyrus using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, suggesting that LPS impaired the survival of newborn cells derived from the NSCs. The double-immunostaining for BrdU and specific cell type markers revealed that LPS did not alter the commitment of the NSCs to the neurons and astrocytes. The systemic injection of indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and NS398, a selective COX-2 inhibitor, but not SC560, a selective COX-1 inhibitor, did not only ameliorate LPS-induced suppression of the newborn cell survival, they fully protected against the LPS effect. Furthermore, the central injection of NS398 also ameliorated LPS-induced suppression of the newborn cell survival in the dentate gyrus. The treatment with LPS increased the expression of COX-2 protein 7 h and 7 days after the injection in the dentate gyrus. These results suggest that LPS impairs the survival of newly generated cells derived from the NSCs in the dentate gyrus without affecting the differentiation fate, and these effects of LPS were mediated presumably by COX-2 expression in the dentate gyrus.

Natural Products: Baicalin and Baicalein, Constituents of an Important Medicinal Plant, Inhibit Intracellular Ca2+ Elevation by Reducing Phospholipase C Activity in C6 Rat Glioma Cells

Glial cells have a role in maintaining the function of neural cells. This study was undertaken to clarify the effects of baicalin and baicalein, flavonoids isolated from an important medicinal plant Scutellariae Radix (the root of Scutellaria baicalensis Georgi), on glial cell function using C6 rat glioma cells.

Thromboxane A2-mediated shape change: independent of Gq-phospholipase C--Ca2+ pathway in rabbit platelets.

1 Thromboxane A2 (TXA2) receptor‐mediated signal transduction was investigated in washed rabbit platelets to clarify the mechanisms of induction of shape change and aggregation. 2 The TXA2 agonist, U46619 (1 nM to 10 μ) caused shape change and aggregation in a concentration‐dependent manner. A forty‐times higher concentration of U46619 was needed for aggregation (EC50 of 0.58 μ) than shape change (EC50 of 0.013 μ). The aggregation occurred only when external 1 mM Ca2+ was present, but the shape change could occur in the absence of Ca2+. 3 SQ29548 at 30 nM and GR32191B at 0.3 μ (TXA2 receptor antagonists) competitively inhibited U46619‐induced shape change and aggregation with similar potency, showing that both aggregation and shape change induced by U46619 were TXA2 receptor‐mediated events. However, ONO NT‐126 at 1 nM, another TXA2 receptor antagonist, inhibited U46619‐induced aggregation much more potently than the shape change, suggesting the possible existence of TXA2 receptor subtypes. 4 ONO NT‐126 (2 nM to 3 μ) by itself caused a shape change without aggregation in a concentration‐dependent manner, independent of external Ca2+. Therefore, ONO NT‐126 is a partial agonist at the TXA2 receptor in rabbit platelets. 5 U46619 (10 nM to 10 μ) increased internal Ca2+ concentration ([Ca2+]i) and activated phosphoinositide (PI) hydrolysis in a concentration‐dependent manner with a similar concentration‐dependency. 6 U46619 (3 nM to 10 μ) also activated GTPase concentration‐dependently in the membranes derived from platelets. U46619‐induced activation of GTPase was partly inhibited by treatment of membranes with QL, an antibody against Gq/11. 7 The EC50 values of U46619 in Ca2+ mobilization (0.15 μ), PI hydrolysis (0.20 μ) and increase in GTPase activity (0.12 μ) were similar, but different from the EC50 value in shape change (0.013 μ), suggesting that activation of TXA2 receptors might cause shape change via an unknown mechanism. 8 U46619‐induced shape change was unaffected by W‐7 (30 μ), a calmodulin antagonist or ML‐7 (30 μ), a myosin light‐chain kinase inhibitor, indicating that an increase in [Ca2+]i might not be involved in the shape change. In fact, U46619 (10 nM) could cause shape change without affecting [Ca2+]i level, determined by simultaneous recordings. 9 [3H]‐SQ29548 and [3H]‐U46619 bound to platelets at a single site with a Kd value of 14.88 nM and Bmax of 106.1 fmol/108 platelets and Kd value of 129.8 nM and Bmax of 170.4 fmol/108 platelets, respectively. The inhibitory constant Ki value for U46619 as an inhibitor of 3H‐ligand binding was similar to the EC50 value of U46619 in GTPase activity, phosphoinositide hydrolysis and Ca2+ mobilization, but significantly different (P<0.001 by Students t test) from the effect on shape change. 10 Neither U46619 nor ONO NT‐126 affected the adenosine 3′,5′‐cyclic monophosphate (cyclic AMP) level in the presence or absence of external Ca2+ and/or isobutyl methylxanthine. 11 The results indicate that TXA2 receptor stimulation causes phospholipase C activation and increase in [Ca2+]i via a G protein of the Gq/11 family leading to aggregation in the presence of external Ca2+, and that shape change induced by TXA2 receptor stimulation might occur without involvement of the Gq‐phospholipase C‐Ca2+ pathway.