Tsugunobu Andoh

The roles of thioredoxin in protection against oxidative stress-induced apoptosis in SH-SY5Y cells.

Using models of serum deprivation and 1-methyl-4-phenylpyridinium (MPP+), we investigated the mechanism by which thioredoxin (Trx) exerts its antiapoptotic protection in human neuroblastoma cells (SH-SY5Y) and preconditioning-induced neuroprotection. We showed that SH-SY5Y cells are highly sensitive to oxidative stress and responsive to both extracellularly administered and preconditioning-induced Trx. Serum deprivation and MPP+ produced an elevation in the hydroxyl radicals, malondialdehyde and 4-hydroxy-2,3-nonenal (HNE), causing the cells to undergo mitochondria-mediated apoptosis. Trx in the submicromolar range blocked the observed apoptosis via a multiphasic protection mechanism that includes the suppression of cytochromec release (most likely via the induction of Bcl-2), the inhibition of procaspase-9 and procaspase-3 activation, and the elevated level of Mn-SOD. The reduced form of Trx suppresses the serum-free-induced hydroxyl radicals, lipid peroxidation, and apoptosis, indicating that H2O2 is removed by Trx peroxidase. The participation of Trx in preconditioning-induced neuroprotection is supported by the observation that inhibition of Trx synthesis with antisense oligonucleotides or of Trx reductase drastically reduced the hormesis effect. This effect of Trx-mediated hormesis against oxidative stress-induced apoptosis is striking. It induced a 30-fold shift in LD50 in the MPP+-induced neurotoxicity.

Differential Activation of Brain-derived Neurotrophic Factor Gene Promoters I and III by Ca2+ Signals Evoked vial-type Voltage-dependent andN-Methyl-d-aspartate Receptor Ca2+Channels

Although the brain-derived neurotrophic factor (BDNF) gene is activated by the intracellular Ca2+signals evoked via Ca2+ influx into neurons, little is known about how the activation of alternative BDNF gene promoters is controlled by the Ca2+ signals evoked viaN-methyl-d-aspartate receptors (NMDA-R) and L-type voltage-dependent Ca2+ channels (L-VDCC). There is a critical range in the membrane depolarization caused by high K+ concentrations (25–50 mm KCl) for effective BDNF mRNA expression and transcriptional activation of BDNF gene promoters I and III (BDNF-PI and -PIII, respectively) in rat cortical culture. The increase in BDNF mRNA expression induced at high K+ was repressed not only by nicardipine, an antagonist for L-VDCC, but also by dl-amino-5-phosphonovalerate, an antagonist for NMDA-R, which was supported by the effects of antagonists on the Ca2+ influx. Although the promoter activations at 25 and 50 mm KCl were different, BDNF-PIII was activated by either the Ca2+ influx through NMDA-R or L-VDCC, whereas BDNF-PI was predominantly by the Ca2+influx through L-VDCC. Direct stimulation of NMDA-R supported the activation of BDNF-PIII but not that of BDNF-PI. Thus, the alternative BDNF gene promoters responded differently to the intracellular Ca2+ signals evoked via NMDA-R and L-VDCC.

Intradermal leukotriene B4, but not prostaglandin E2, induces itch-associated responses in mice

The itch-associated responses induced by intradermal injection of leukotriene B4 and prostaglandin E2 were studied in mice. Leukotriene B4(0.001-1 nmol/site) elicited scratching of the injected site; the dose-response curve was bell-shaped with a peak effect at 0.03 nmol/site. The effect of leukotriene B4 (0.03 nmol/site) started within 3 min, peaked in the second 10-min period, had almost subsided by 30 min, and was inhibited by the simultaneous injection of the leukotriene B4 receptor antagonist ONO-4057, 5-[2-(2carboxyethyl)-3-(6-( p-methoxyphenyl)-5E-hexenyl) oxyphenyoxy] valeric acid. Prostaglandin E2 (0.003-300 nmol/site) did not significantly elicit scratching. The results raise the possibility that leukotriene B4 is an endogenous itch mediator in the skin.

Allodynia and hyperalgesia induced by herpes simplex virus type-1 infection in mice

&NA; Human subjects infected with herpes or varicella‐zoster viruses complain of pain, such as allodynia, in or near the region with vesicles. However, the mechanisms of the pain are unclear. We show for the first time that infection with herpes simplex virus type‐1 (HSV‐1) induces allodynia and hyperalgesia in mice. When HSV‐1 was inoculated on the hind paw of the mouse, eruption appeared on the back on day 5 post‐inoculation, and zosteriform skin lesions were developed on the inoculated side. Allodynia and hyperalgesia became apparent in the hind paw on the inoculated side on day 5 and persisted until at least day 8. HSV‐1 DNA was detected in the dorsal root ganglia from days 2 to 8 post‐inoculation, with a peak effect on day 5. The application of heat‐inactivated HSV‐1 induced no allodynia, hyperalgesia and skin lesion. When started from days 0 or 2, repeated treatment with acyclovir, anti‐HSV‐1 agent, inhibited the appearance of allodynia, hyperalgesia, eruption and the viral proliferation in the dorsal root ganglia. In contrast, when started from days 5 or 6, acyclovir treatment slightly inhibited the development of skin lesions and the viral proliferation, but not allodynia and hyperalgesia. These results suggest that the propagation of HSV‐1 in the dorsal root ganglia produces allodynia and hyperalgesia as a result of functional abnormality of the sensory neurons in mice. This may be a useful model for studying the mechanisms of herpetic pain.

17β-Estradiol activates ICI 182,780-sensitive estrogen receptors and cyclic GMP-dependent thioredoxin expression for neuroprotection

Clinical studies suggest that estrogen may improve cognition in Alzheimers patients. Basic experiments demonstrate that 17β‐estradiol protects against neurodegeneration in both cell and animal models. In the present study, a human SH‐SY5Y cell model was used to investigate molecular mechanisms underlying the receptor‐mediated neuroprotection of physiological concentrations of 17β‐estradiol. 17β‐Estradiol (<10 nM) concomitantly increased neuronal nitric oxide synthase (NOS1) expression and cell viability. 17β‐Estradiol‐induced neuroprotection was blocked by the receptor antagonist ICI 182,780, also prevented by inhibitors of NOS1 (7‐nitroindazole), guanylyl cyclase (LY 83,583), and cGMP‐dependent protein kinase (PKG) (Rp‐8‐pCPT‐cGMPs). In addition to the expression of NOS1 and MnSOD, 17β‐estradiol increased the expression of the redox protein thioredoxin (Trx), which was blocked by the inhibition of either cGMP formation or PKG activity. The expression of heme oxygenase 2 and brain‐derived neurotrophic factor was not altered. Estrogen receptor‐enhanced cell viability against oxidative stress may be linked to Trx expression because the Trx reductase inhibitor, 5,5′‐dithio‐bis(2‐nitrobenzoic acid) significantly reduced the cytoprotective effect of 17β‐estradiol. Furthermore, Trx (1 μM) inhibited lipid peroxidation, proapoptotic caspase‐3, and cell death during oxidative stress caused by serum deprivation. We conclude that cGMP‐dependent expression of Trx–the redox protein with potent antioxidative and antiapoptotic properties–may play a pivotal role in estrogen‐induced neuroprotection.

Nociceptin gene expression in rat dorsal root ganglia induced by peripheral inflammation

NOCICEPTIN has been suggested to be involved in nociceptive processing in the spinal dorsal horn. To investigate whether the biosynthesis of nociceptin is altered in animals with nociceptive hypersensitivity, we examined effects of peripheral inflammation on the expression of prepronociceptin mRNA, especially in the dorsal root ganglion of the rat. Although little or no expression of prepronociceptin mRNA was observed in the dorsal root ganglia of naive animals, carrageenan-elicited inflammation markedly induced its expression, peaking within 30 min, a time when nociceptive hypersensitivity was not yet apparent. The results suggest that peripheral inflammation induced nociceptin expression in primary sensory neurons, which may be associated with the production of nociceptive hypersensitivity.

Preconditioning‐Mediated Neuroprotection

Abstract: Preconditioning adaptation induced by transient ischemia can increase brain tolerance to oxidative stress, but the underlying neuroprotective mechanisms are not fully understood. Recently, we developed a human brain‐derived cell model to investigate preconditioning mechanism in SH‐SY5Y neuroblastoma cells. 1 Our results demonstrate that a non‐lethal serum deprivation‐stress for 2 h (preconditioning stress) enhanced the tolerance to a subsequent lethal oxidative stress (24 h serum deprivation) and also to 1‐methyl‐4‐phenyl‐pyridinium (MPP+). 2 Two‐hour non‐lethal preconditioning stress increased the expression of neuronal nitric oxide (NOS1/nNOS) mRNA, Fos, Ref‐1, NOS protein, and then nitric oxide (•NO) production. As well as MnSOD expression, the •NO‐cGMP‐PKG pathway mediated the preconditioning‐induced upregulation of antiapoptotic protein Bcl‐2 and the downregulation of adaptor protein p66shc. We also propose that cGMP‐mediated preconditioning‐induced adaptation against oxidative stress may be due to the synthesis of a new protein, such as thioredoxin (Trx) since the protective effect can be blocked by Trx reductase inhibitor. 3 The antioxidative potency of Trx was approximately 100 and 1,000 times greater than GSNO and GSH, respectively. These results suggest that •NO‐cGMP‐PKG signaling pathway plays an important role in the preconditioning‐induced neuroprotection, and perhaps cardioprotection, against oxidative stress.

Nitric oxide enhances substance P-induced itch-associated responses in mice

Substance P (SP) elicits itch and itch‐associated responses in humans and mice, respectively. In mice, NK1 tachykinin receptors are involved in SP‐induced itch‐associated responses, scratching, and mast cells do not play a critical role. The present study was conducted to elucidate the role of nitric oxide (NO) on SP‐induced scratching in mice. An intradermal injection of SP (100 nmol site−1) elicited scratching in mice, and it was suppressed by an intravenous injection of the NO synthase (NOS) inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME), but not by its inactive enantiomer D‐NAME. Intradermal injections of L‐NAME (100 nmol site−1), another NOS inhibitor 7‐nitroindazole (10 nmol site−1) and the NO scavenger haemoglobin (0.01–10 nmol site−1) also inhibited SP‐induced scratching. L‐NAME (100 nmol site−1) did not affect scratching induced by an intradermal injection of 5‐hydroxytryptamine (100 nmol site−1). Intradermal injections of L‐arginine (300 nmol site−1) and the NO donor (±)‐(E)‐4‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexenamide (NOR3; 100 nmol site−1) increased scratching induced by SP. Intradermal injections of L‐arginine (1–1000 nmol site−1) or NOR3 (1–100 nmol site−1) alone were without effects on scratching. Intradermal injections of SP (10–100 nmol site−1) increased the intradermal concentration of NO in a dose‐dependent manner in mice. An increase in NO levels induced by SP was inhibited by L‐NAME and the NK1 tachykinin receptor antagonist L‐668,169, but not by the NK2 tachykinin receptor antagonist L‐659,877. SP (1–10 μM) elicited NO production in cultured human keratinocytes and the SP‐induced NO production was inhibited by L‐NAME and L‐668,169. We conclude that intradermal SP increases NO in the skin, possibly through the action on NK1 tachykinin receptors on the epidermal keratinocytes and that NO enhances SP‐induced itch‐associated responses.

Induction of thioredoxin and mitochondrial survival proteins mediates preconditioning-induced cardioprotection and neuroprotection.

Abstract: Delayed cardio‐ and neuroprotection are observed following a preconditioning procedure evoked by a brief and nontoxic oxidative stress due to deprivation of oxygen, glucose, serum, trophic factors, and/or antioxidative enzymes. Preconditioning protection can be observed in vivo and is under clinical trials for preservation of cell viability following organ transplants of liver. Previous studies indicated that ischemic preconditioning increases the expression of heat‐shock proteins (HSPs) and nitric oxide synthase (NOS). Our pilot studies indicate that the treatment of neuronal NOS inhibitor (7‐nitroindazole) and 6Br‐cGMP blocks and mimics, respectively, preconditioning protection in human neuroblastoma SH‐SY5Y cells. This minireview focuses on nitric oxide‐mediated cellular adaptation and the related cGMP/PKG signaling pathway in a compensatory mechanism underlying preconditioning‐induced hormesis. Both preconditioning and 6Br‐cGMP increase the induction of human thioredoxin (Trx) mRNA and protein for cytoprotection, which is largely prevented by transfection of cells with Trx antisense but not sense oligonucleotides. Cytosolic Trx1 and mitochondrial Trx2 suppress free radical formation, lipid peroxidation, oxidative stress, and mitochondria‐dependent apoptosis; knock out/down of either Trx1 or Trx2 is detrimental to cell survival. Other recent findings indicate that a transgenic increase of Trx in mice increases tolerance against oxidative nigral injury caused by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Trx1 can be translocated into nucleus and phosphoactivated CREB for a delayed induction of mitochondrial anti‐apoptotic Bcl‐2 and antioxidative MnSOD that is known to increase vitality and survival of cells in the brain and the heart. In conclusion, preconditioning adaptation or a brief oxidative stress induces a delayed nitric oxide‐mediated compensatory mechanism for cell survival and vitality in the central nervous system and the cardiovascular system. Preconditioning‐induced adaptive tolerance may be signaling through a cGMP‐dependent induction of cytosolic redox protein Trx1 and subsequently mitochondrial proteins such as Bcl‐2, MnSOD, and perhaps Trx2 or HSP70.

Potential ability of morphine to inhibit the adhesion, invasion and metastasis of metastatic colon 26-L5 carcinoma cells

Morphine is frequently used for cancer patients terminal medical care to relieve cancer pain. In the present study, we examined the inhibitory effect of morphine on experimental lung metastasis and invasion of colon 26-L5 cells. Morphine was found to significantly reduce the number of tumor colonies and the weight of the tumor-containing lung. Morphine inhibited the adhesion and migration of colon 26-L5 cells to extracellular matrix components and invasion into reconstituted basement membrane Matrigel, without affecting the cell proliferation in vitro. Notably, naloxone, an antagonist of morphine, abrogated morphine-induced inhibition of tumor cell adhesion, but did not affect the inhibitory effect on the production of matrix metalloproteinases (MMPs) from tumor cells. These results suggest that morphine inhibited the adhesive and invasive properties of tumor cells by different inhibitory mechanisms that involved the mediation of an opioid receptor.