Eiki Satoh

Integrin-linked Kinase Controls Neurite Outgrowth in N1E-115 Neuroblastoma Cells

Mouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against β1 integrin inhibits neurite outgrowth. Thus, β1 integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of β1 integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whereas that in the DN-ILK-transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinase kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK-expressed cells. The time course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus, activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells.

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Abstract: Ouabain (5 X 10−8‐5 X 10−4 M) was confirmed to cause a dose‐dependent increase in [3H]acetylcholine (pHJACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose‐dependent decrease in membrane potential. In a low‐Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10−6 M) had no effect on the ouabain‐induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10−6‐5 X 10−4 M) inhibited the ouabain‐induced increase in both [3H]ACh release and [Ca2+]i in a dose‐dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mMMg2+ blocked ouabain‐induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.

Comparison of the antioxidant activity of roasted tea with green, oolong, and black teas

Although the antioxidant properties of green, oolong, and black teas have been well studied, antioxidant activity has not been examined in roasted tea. Therefore, in the current studies, we investigated the antioxidant activity of roasted tea in comparison with those of green, oolong, and black teas. Using water extracts of the various teas, we examined the total phenolic content as well as the antioxidant activities, including the reducing power, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, and the inhibition of hemolysis caused by 2,2’-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-induced lipid oxidation in erythrocyte membranes. The roasted tea contained lower levels of total phenolics than green, oolong, or black tea (green tea>oolong tea>black tea>roasted tea). The relative reducing power and DPPH scavenging activity decreased in the following order: green tea>roasted tea>oolong tea>black tea. Also, green tea was more effective against AAPH-induced erythrocyte hemolysis than other teas (green tea>roasted tea=oolong tea=black tea). These results suggest that roasted tea is beneficial to health, in humans, because of its high antioxidant activity.

Mechanism of cadmium-induced blockade of neuromuscular transmission☆

Abstract The effect of cadmium ion (Cd 2+ ) on neuromuscular transmission was examined in phrenic nerve-diaphragm preparations (PND) of mice and rats.The indirectly induced muscle twitch (IT) was inhibited by Cd 2+ (0.1–1.0×10 −4 M) in a dose-dependent manner, but the directly induced twitch (DT) was rarely affected. The inhibition was reversible and reproducible. An increase in external Ca 2+ concentration antagonized and an increase in Mg 2+ concentration potentiated the inhibitory effect of Cd 2+ . The inhibitory effect of Cd 2+ (2.8×10 −5 M) was antagonized by cysteine (5.6×10 −5 M) applied before or after the Cd 2+ treatment. Hg 2+ (mercuric chloride, 2.8×10 −5 M) or p-chloromercuribenzoic acid (PCMB, 2.5×10 −5 M) also inhibited IT but in a different manner. The effects of Hg 2+ or PCMB were not antagonized by cysteine applied after the treatment. Pretreatment with Hg 2+ (10 −6 M) or PCMB (5×10 −6 M) did not potentiate the inhibitory effect of Cd 2+ . Resting membrane potential and frequency of miniature end-plate potential (m.e.p.p.) were not affected by Cd 2+ , but m.e.p.p. amplitude was slightly reduced. The end-plate potential was blocked by Cd 2+ (10 −4 M), and this effect was partially antagonized by an increase in Ca 2+ concentration. In bioassay experiments, the evoked output of acetylcholine from rat PND was reversibly reduced by Cd 2+ . The inhibitory effect of Cd 2+ on the output of [ 14 C]acetylcholine evoked from rat PND was antagonized by an increase in Ca 2+ concentration and potentiated by an increase in Mg 2+ . From these results, it is suggested that Cd 2+ reversibly inhibits neuromuscular transmission by reducing transmitter release from motor nerve terminals resulting from the inhibition of Ca 2+ influx at the membrane of axon terminals. We did not obtain conclusive evidence that Cd 2+ produces neuromuscular blockade by acting on thiol groups of membrane constituents.

Effects of Monensin and Veratridine on Acetylcholine Release and Cytosolic Free Ca2+ Levels in Cerebrocortical Synaptosomes of Rats

Monensin (10−8‐10−4M) caused a dose‐dependent increase in the release of [3H] acetylcholine ([3H] ACh) from purified rat cerebrocortical synaptosomes, with an EC50 of ∼1.6 × 10−6M. Extracellular Na+, but not Ca2+, was required for a monensin‐induced increase in the release of [3H] ACh. Monensin also increased the cytosolic free Ca2+ concentration ([Ca2+]i) and uptake of 22Na+ in a dose‐dependent manner. Monensin continued to cause a dose‐dependent increase in [Ca2+]i in the absence of extracellular Ca2+, although an ∼50% reduction was noted at concentrations of >10−5M. The EC50 for the monensin‐induced increase in [Ca2+]i was similar to that noted in the release of [3H]ACh. Veratridine exhibited effects similar to those of monensin, but a large portion of the increase in [Ca2+]i and [3H] ACh release was dependent on extracellular Ca2+. Measurements of rhodamine 6G fluorescence indicated that monensin and veratridine caused synaptosomal hyperpolarization and depolarization, respectively. Tetrodotoxin (10−6M) completely blocked all the effects of veratridine but had no effect on the activity of monensin. These results suggest that monensin increases the release of ACh at least in part by increasing [Ca2+]i, resulting from the increase in the Na+ influx through tetrodotoxin‐insensitive mechanisms in rat cerebrocortical synaptosomes.

Palytoxin-induced increase in cytosolic-free Ca2+ in mouse spleen cells

The effect of palytoxin (C(129)H(223)N(3)O(54)) on Ca(2+) homeostasis in immune cells has not been studied. Therefore, we investigated the effect of palytoxin on the cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) in mouse spleen cells using a fluorescence Ca(2+) indicator, fura-2. Palytoxin (0.1-100 nM) increased [Ca(2+)](i) in a concentration-dependent manner. The palytoxin-induced increase in [Ca(2+)](i) was abolished by the omission of extracellular Ca(2+) or 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100 microM), and was greatly inhibited by Ni(2+) (2 mM). Ouabain (0.5-1 mM) partially inhibited the palytoxin-induced response. There was no effect of decreased extracellular Na(+) (6.2 mM), tetrodotoxin (1 microM), verapamil (10 microM), nifedipine (10 microM), omega-agatoxin IVA (200 nM), omega-conotoxin GVIA (1 microM), omega-conotoxin MVIIC (500 nM), or La(3+) (100 microM). These results suggest that palytoxin increases [Ca(2+)](i) in mouse spleen cells by stimulating Ca(2+) entry through an SKF-96365-, Ni(2+)-sensitive pathway.

Black tea extract, thearubigin fraction, counteract the effects of botulinum neurotoxins in mice

Botulinum neurotoxin type A (BoNT/A, 1.5 nM) completely inhibited indirectly evoked twitches in in vitro mouse phrenic nerve‐diaphragm preparations within 40 – 45 min. Black tea extract, thearubigin fraction (TRB), mixed with BoNT/A blocked the inhibitory effect of the toxin. The protective effect of TRB extended to botulinum neurotoxins types B and E (BoNT/B and BoNT/E) and tetanus toxin, but not to tetrodotoxin. TRB was also effective against oral toxicity of BoNT/A, B and E. Thus, TRB may be of potential benefit in protecting the paralytic actions of botulinum neurotoxins (BoNTs), but its use is limited by mixing with the toxin.

Acute Restraint Stress Enhances Calcium Mobilization and Glutamate Exocytosis in Cerebrocortical Synaptosomes from Mice

Acute stress is known to enhance the memory of events that are potentially threatening to the organisms. Glutamate, the most abundant excitatory neurotransmitter in the mammalian central nervous system, plays a critical role in learning and memory formation and calcium (Ca2+) plays an essential role in transmitter release from nerve terminals (synaptosomes). In the present study, we investigated the effects of acute restraint stress on cytosolic free Ca2+ concentration ([Ca2+]i) and glutamate release in cerebrocortical synaptosomes from mice. Acute restraint stress caused a significant increase in resting [Ca2+]i and significantly enhanced the ability of the depolarizing agents K+ and 4-aminopyridine (4-AP) to increase [Ca2+]i. It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K+- and 4-AP-induced Ca2+-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca2+ channel blocker) and ω-conotoxin GVIA (an N-type Ca2+ channel blocker) completely suppressed the enhancements of [Ca2+]i and Ca2+-dependent glutamate release in acute restraint-stressed mice. These results indicate that acute restraint stress enhances K+- or 4-AP-induced glutamate release by increasing [Ca2+]i via stimulation of Ca2+ entry through P- and N-type Ca2+ channels.

Acute restraint stress enhances calcium mobilization and proliferative response in splenic lymphocytes from mice

Calcium (Ca2+ ) plays an essential role in lymphocyte activation and maturation. Acute and chronic stress has been shown to modulate the lymphocyte immune response; but the relationship between cytosolic free Ca2+ concentration ([Ca2+ ]i) and the immune response in lymphocytes following exposure to stress has not been examined. In the present study, we investigated the effects of acute restraint stress on [Ca2+ ]i and the proliferation of splenic lymphocytes from mice. We observed that 2 h of restraint significantly increased plasma corticosterone levels in mice. On examining [Ca2+ ]i and the proliferation ex vivo of splenic lymphocytes isolated from restraint-stressed mice using fura-2 and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, respectively, we found that acute restraint stress caused a significant increase in resting [Ca2+ ]i and significantly enhanced the ability of concanavalin A (Con A; a T-cell-selective mitogen) to increase [Ca2+ ]i but not that of lipopolysaccharide (LPS; a B-cell-selective mitogen). In addition, acute restraint stress significantly enhanced Con A-stimulated but not LPS-stimulated lymphocyte proliferation. Overall, there was a positive correlation between [Ca2+ ]i and T-cell proliferation following acute restraint stress. The enhancements of [Ca2+ ]i and T-cell proliferation were completely suppressed by verapamil (a Ca2+ channel blocker). These results suggest that acute restraint stress enhances Con A-stimulated T-cell proliferation by increasing [Ca2+ ]i via stimulation of Ca2+ entry.

Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; KT−4‐10−6 M) caused a dose‐dependent increase in the release of [3H] acetylcholine ([3H] ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose‐response curves for the PTX‐induced increases in [3H] ACh release and in [Ca2+]iwere depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H] ACh induced by concentrations of PTX < 10−10M was more dependent on the simultaneous presence of both Ca2+ and Na+than the release induced by higher concentrations of PTX. The PTX‐induced increase both in [3H] ACh release and in {Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10−6M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin‐insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H] ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na+‐Ca2+ exchange mechanism.