A cyclic AMP-regulated negative feedforward system for neuritogenesis revealed in a neuroblastomaxglioma hybrid cell line.

We examined the role of second messengers during the neuritogenesis that accompanies neuronal differentiation in a neuroblastomaxglioma hybrid cell line (NG108-15). NG108-15 cells extended neurites after treatment with dibutyryl cyclic AMP. This dibutyryl cyclic AMP treatment evoked the synthesis of voltage-dependent Ca(2+) channel proteins in the cells. The number of neurites was decreased by Ca(2+) influx under condition of high K(+). Interestingly, the increase of neurites stimulated by dibutyryl cyclic AMP and the decrease of neurites caused by high K(+) were both reversible. This is the first study to demonstrate that cyclic AMP regulates a negative feedforward system for neuritogenesis, which links with Ca(2+) signaling. Such a dual role of cyclic AMP may play an important part in precise neurite targeting.

Three-dimensional characterization of interior structures of exocytotic apertures of nerve cells using atomic force microscopy.

We examined the interior structure of exocytotic apertures in synaptic vesicles of neuroblastoma x glioma hybrid cells using atomic force microscopy. The atomic force microscopy detected apertures of 50-100nm in diameter at various depths within the varicosities of these cells. We were also able to image a regular radial pattern on the wall and lump-like structures at the bottom of these apertures. In contrast, scanning electron microscopy could only detect the apertures but not the fine details of their interior. The cells examined here exhibited the same electrophysiological properties and expression of synaptophysin and syntaxin 1 as presynaptic terminals, as studied by various electrophysiological and imaging techniques. Our results indicate that atomic force microscopy allows three-dimensional viewing of the fine structures located inside exocytotic apertures in nerve cells.

A novel function of synapsin II in neurotransmitter release.

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-II-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells.

Ca2+ signaling regulated by an ATP-dependent autocrine mechanism in astrocytes.

Although the mechanisms of Ca2+ wave propagation in astrocytes induced by mechanical stimulation have been well studied, it is still not known how the [Ca2+]i increases in the stimulated cells. Here, we have analyzed the mechanisms of [Ca2+]i increase in single, isolated astrocytes. Our results showed that there was an autocrine mechanism of Ca2+ regulation mediated by ATP in mechanically stimulated astrocytes. This autocrine mechanism induced the activation of phospholipase C via a G-protein, resulting in Ca2+ release from intracellular Ca2+ stores. A second pathway mediating a [Ca2+]i increase was via a Ca2+ influx from the extracellular space, which, interestingly, suppressed an intracellular Ca2+ oscillation. These two different Ca2+ cascades are involved in signal transduction and may function separately during intercellular communication.

Acquisition of neuronal proteins during differentiation of NG108-15 cells.

The differentiated type of neuroblastomaxglioma hybrid cell line, NG108-15, has widely been used in in vitro studies instead of primary-cultured neurons. Here we examined whether NG108-15 cells can be used as a model for studying the neuronal differentiation process. We compared the expression of neuronal proteins (neurofilament 200 (NF200), phosphorylated-NF200 (p-NF200), microtubule associated protein 2, synaptophysin, syntaxin 1, choline acetyltransferase, and acetylcholinesterase (AChE)) and a glial protein (vimentin) between undifferentiated and differentiated NG108-15 cells by immunocytochemistry and immunoblot analysis. The expression of all neuronal proteins, with the exception of NF200 and p-NF200, was positive in differentiated cells, but almost negative in undifferentiated cells. On the other hand, cytoskeletal intermediate filaments (NF200 and p-NF200) for neurons and that (vimentin) for glia were present in both undifferentiated and differentiated cells. Furthermore, a high expression of AChE mRNA was confirmed in differentiated cells by reverse transcription-PCR analysis. Our results showed that even though the expression of cytoskeletal filaments does not change during differentiation of NG108-15 cells, these cells during differentiation can serve as an appropriate tool for investigating and understanding the mechanisms involved in neuronal development and differentiation.

Bimodal effects of acetylcholine on synchronized calcium oscillation in rat cultured cortical neurons.

To understand the plastic changes in neural network dynamics, we analyzed the modulating effects of cholinergic agonists on synchronized Ca(2+) oscillation in the rat primary cultured cortical neurons. At 5-10 days after establishment of the culture, neurons exhibited synchronized Ca(2+) oscillation. This Ca(2+) oscillation was derived from glutamatergic and gamma-aminobutyric acid (GABA)ergic synaptic inputs from neighboring neurons. Application of acetylcholine and nicotine increased, decreased, or did not change the frequency of synchronized Ca(2+) oscillation, depending on the colonies in the culture. The presence of cholinergic neurons in the culture was confirmed by immunocytochemical staining. Our study provided evidence for the first time that cholinergic neurons exert excitatory influences on GABAergic neurons as well as glutamtergic neurons, resulting in bimodal effects on the frequency of synchronized Ca(2+) oscillation in the cortical neural network.

Effects of capsaicin and ethanol on hepatic drug-metabolizing enzymes in rat.

Capsaicin (CAP; 50 mg/kg body wt., p.o.) and/or ethanol (EtOH; 10% (v/v) in the drinking water) were given for a period of 30 days to male rats, and changes in hepatic drug-metabolizing enzymes were investigated. The EtOH alone tended to increase the microsomal parameters such as cytochrome P-450 content and enzyme activities of aniline hydroxylase, aminopyrine demethylase and UDP-glucuronyl transferase, while it did not affect the cytosolic enzyme activities of sulfotransferase and glutathione S-transferase. The administration of CAP without EtOH tended to decrease all of the parameters studied. In contrast, the ingestion of CAP with EtOH tended to increase even the elevated microsomal enzyme activities by EtOH alone. These data suggest that chronic alcohol ingestion could modify the potential for detoxification of xenobiotics in persons who regularly consume a large amount of hot peppers.

Survey of formaldehyde, acetaldehyde and oligomers in polyethylene terephthalate food-packaging materials.

Polyethylene terephthalate (PET) is frequently used as a packaging material for beverage bottles, fruit and vegetable trays, and egg crates in Japan. Levels of formaldehyde (FA), acetaldehyde (AA) and PET oligomers in various PET food packaging were determined. PET samples were initially dissolved in trifluoroacetic acid with 2,4-dinitrophenylhydrazine to derivatize formaldehyde and acetaldehyde to their dinitrophenylhydrazones. The stable derivatives along with the oligomers were analysed using HPLC with ultraviolet light detection at 360 and 254 nm, respectively. The PET pellets contained 3.5-12.4 microg g-1 AA and 4.0-7.2 mg g-1 oligomers, while FA was below the determination limit. FA, AA and oligomer levels in Japanese bottles were 0.6-3.0 microg g-1, 8.4-25.7 microg g-1 and 5.0-8.7 mg g-1, ND-1.6 microg g-1, 5.0-13.1 microg g-1 and 4.9-8.2 mg g-1 in French and Italian bottles, and ND-1.2 microg g-1, 9.1-18.7 microg g-1 and 5.6-8.0 mg g-1 in US and Canadian bottles, respectively. Compared with European bottles, Japanese bottles contain higher FA and AA levels. In sheet-moulding products, their contents were determined as ND-1.1 microg g-1, 11.5-43.1 microg g-1 and 4.6-9.2 mg g-1, respectively. The results show that sheet-moulding products contain lower FA and higher AA in comparison with bottles. FA and AA are considered to be generated from PET during the heating process for moulding the pellets to bottles or sheet-moulding articles and de-aeration during the sheet-moulding process is effective in removing FA. In contrast, the level of the oligomers remains unchanged during the moulding process from pellets to bottles or sheet products.

Beta-cyclodextrin-linked chitosan beads: preparation and application to removal of bisphenol A from water.

Water-insoluble and highly porous chitosan beads having an ability to form inclusion complexes have been synthesized by treatment of a solution of chitosan in aqueous acetic acid with aqueous NaOH followed by crosslinking and reductive alkylation with 2-O-formylmethyl-beta-cyclodextrin. Preliminary experiments for its application to removal of endocrine disrupting chemicals were carried out using a column of the resulting beads and bisphenol A as the model substrate.

Rectal absorption enhancement of gentamicin in rabbits from hollow type suppositories by sodium salicylate or sodium caprylate.

The absorption of gentamicin (GM) from the rectum of rabbits after coadministration of GM (60 mg) and sodium salicylate (SA) or sodium caprylate (CA) as absorption-enhancing agents was investigated. Two types of hollow type suppositories were used--a conventional type (Type I) and a release-restricted type (Type II). Without SA or CA, GM was not absorbed. However, GM absorption was marked when 90 mg of solid SA or CA was added (the bioavailability of GM was 58% with SA, and 59% with CA). The enhancing effect of SA or CA (30 mg) in solid or aqueous solution form on GM absorption was evaluated using the Type I suppository. In the case of SA, the highest plasma GM level (Cmax 15.3 +/- 1.7 micrograms/ml, AUC0-4 27.3 +/- 3.9 h.micrograms/ml) was obtained following coadministration of powdered GM and SA; the plasma GM level (Cmax 1.5 +/- 0.6 micrograms/ml, AUC0-4 3.0 +/- 1.3 h.micrograms/ml) following the administration of a solution of GM and SA was significantly decreased as compared with the results using the powdered form. In the case of CA, the plasma GM level (Cmax 14.8 +/- 4.5 micrograms/ml, AUC0-4 25.4 +/- 8.7 h.micrograms/ml) following administration of the solution form was not significantly decreased in comparison with the results obtained using the powdered form. A marked increase in the enhancing effect of SA on the rectal GM absorption was found following use of Type II suppositories when GM and SA were coadministered in solution form. However, the GM absorption after coadministration of GM and CA using Type II suppositories was not significantly different from the absorption resulting from use of Type I suppositories. Our results suggest that the form and concentration of drug should not be ignored in evaluating the enhancing effects of SA or CA on the rectal absorption of poorly absorbed drugs such as GM.

Effects of rice bran hemicellulose on 1,2-dimethylhydrazine-induced intestinal carcinogenesis in Fischer 344 rats.

The effect of rice bran hemicellulose (RBH) on 1,2-dimethylhydrazine- (DMH) induced intestinal carcinogenesis was studied in male Fischer 344 rats. Rats were fed a basal control diet or a diet containing 2% or 4% RBH at five weeks of age. At 6 weeks of age, all animals were given an intraperitoneal injection of DMH (20 mg/kg body wt) at weekly intervals for 20 weeks and autopsied 7 weeks after the last injection. The incidence of DMH-induced colon tumors was significantly lower in rats fed the 4% RBH diet than in rats fed the basal control diet (p < 0.05). The number of colon tumors per rat was also significantly lower in rats fed the 4% RBH diet than in rats fed the basal control diet (p < 0.05). The present study suggested that the water-soluble RBH played a preventive role in DMH-induced large bowel carcinogenesis in Fischer 344 rats.