Yasuo Oda

The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2-activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2-mediated secretion from the salivary glands. Intravenous calcitonin gene-related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2-mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2-mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.

3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection.

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10(-16) mol (injected amount) with helium-cadmium laser induced detection.

Comparative studies on the analysis of glycosylation heterogeneity of sialic acid-containing glycoproteins using capillary electrophoresis.

Comparative studies concerning glycoform analysis of sialoglycoproteins by capillary electrophoresis were performed using a few separation modes hitherto reported. Glycoprotein samples examined in the present study were successfully separated to their respective glycoforms using surface-modified capillaries commercially available for capillary gas chromatography in the running buffer near their isoelectric points. The analysis times were less than 50 min and reproducibilities in migration times were excellent (less than 2.0% RSD for both run-to-run and day-to-day analyses). We present a method for the glycoform analysis of alpha1-acid glycoprotein in sera by simple pre-treatment as an application. The present technique will become one of the general methods for the evaluation of glycosylation heterogeneity of commercially available glycoprotein drugs.

High-performance capillary electrophoresis of hyaluronic acid: determination of its amount and molecular mass

The amount and the molecular mass of hyaluronic acid (HA) were determined by high-performance capillary electrophoresis. HA was observed at around 15 min by using an untreated fused-silica capillary (75 microns I.D.) of 58 cm length (effective length, 50 cm) at 20 kV in 50 mM phosphate buffer (pH 4.0). Calibration curves showed good linearity from 0.01 mg/ml to 3.3 mg/ml for all HA samples examined. The lower limit of detection by monitoring the absorbance at 185 nm was 1.0 microgram/ml at the signal-to-noise ratio of 5. HA samples were examined in a buffer containing pullulan (PU) as an additive for the matrix formation material. The HA samples showed marked peak-broadening when analyzed in the buffer solution containing PU with a specified molecular mass. The peak broadening was based on the dispersion of the molecular mass of the HA sample analyzed.

Effect of saikosaponin on the immune responses in mice

The in vivo effects of saikosaponin, isolated from Bupleurum radix, on the immune responses are still poorly understood. We have already shown that saikosaponin-d increases phagocytic activities of murine peritoneal macrophages such as spreading activity, phagocytosis, lysosomal enzyme activity and intracellular killing activity of living yeast. This work extends these observations by showing that treatment with saikosaponin also increased the antibody response in plaque-forming cell numbers after in vivo immunization with sheep red blood cells (SRBC) and an augmentation of spleen cell proliferation responses to stimulation with T- or B-cell mitogens both before and after immunization. Furthermore, after SRBC immunization, the macrophages from mice treated with saikosaponin-d revealed significant increases in spreading activity and lysosomal enzyme activity. The chemiluminescences of the macrophages from mice treated with saikosaponin-d stimulated by opsonized zymosan and PMA were enhanced and interleukin-1 production by the cells was increased in a dose-dependent manner. These results demonstrate that saikosaponin-d may stimulate in vivo immunological lymphocyte functions, partly by activating some macrophage functions.

Glucagonlike peptide-1 (7–36)amide suppresses glucagon secretion and decreases cyclic AMP concentration in cultured in-R1-G9 cells

We previously reported that GLP-1(7-36)amide had glucagonostatic action as well as insulinotropic action in the perfused rat pancreas. In this study, we examined the effect of GLP-1(7-36)amide on glucagon secretion and cAMP concentration in glucagon-secreting cell line, In-R1-G9. GLP-1(7-36)amide (1nM) significantly suppressed glucagon secretion and decreased cAMP concentration in the cells. GLP-1(1-37) did not affect glucagon secretion. It is suggested that inhibitory effect of GLP-1(7-36)amide on glucagon secretion is at least partly mediated by adenylate cyclase system.

Effects of propranolol and atenolol on immobilization stress-induced hypertension and down-regulation of central β-adrenoceptors in rats

Effects of chronic treatment with propranolol or atenolol on stress-induced changes in blood pressure, body weight, and cerebral beta-adrenoceptors in rats were examined and compared with the effects of chronic treatment with prazosin. Immobilization stress (2 h daily for 2 weeks) induced a moderate elevation of blood pressure, loss of body weight gain, and downregulation of cerebral beta-adrenoceptors, but produced no changes in the cerebral alpha 1-adrenoceptors. Chronic administration of propranolol (5 or 50 mg.kg-1), atenolol (5 or 50 mg.kg-1) or prazosin (2 or 20 mg.kg-1) inhibited stress-induced hypertension but did not affect loss of body weight gain. Propranolol increased the density of cerebral beta-adrenoceptors by 77% and reduced the downregulation induced by stress. Atenolol also increased the density of cerebral beta-adrenoceptors by 34% and abolished the stress-induced downregulation in cerebral beta-adrenoceptor density. In contrast, prazosin had no effect on the cerebral beta-adrenoceptors in nonstressed or stressed rats. These results suggest that the antihypertensive action of propranolol and atenolol may be partly associated with the inhibition of stress-activated central beta-adrenoceptor transmission.

Anomalous migration of hyaluronic acid oligomers in capillary electrophoresis: correlation to susceptibility to hyaluronidase.

During high‐resolution capillary electrophoresis analysis in an electrolyte solution containing a neutral polymer, small oligomers of regularly arranged acidic polysaccharides such as hyaluronic acid and N‐acetylneuraminic acid polymers showed reversal of the migration order. This anomalous migration was well correlated with their reported biological activity. In the present study, we analyzed hyaluronidase action on the purified hyaluronic acid oligomers using capillary electrophoresis and found that hydrolytic and transglycosylation actions by hyaluronidase were dependent on the molecular sizes of hyaluronic acid oligomers, and well correlated to their migration profiles. Furthermore, fluorescent polarization technique was employed for understanding the relationship between molecular size of hyaluronic acid oligomers and their electromigrations.

Effects of neurotransmitter candidates on 45Ca uptake by cortical slices of rat brain: Stimulatory effect of l-glutamic acid

The effects of neurotransmitter candidates and the characteristics of the stimulatory effect of L-glutamic acid (L-Glu) on 45Ca uptake by rat brain slices were investigated. 45Ca uptake was significantly stimulated by acetylcholine, serotonin and especially L-Glu, but not by other neurotransmitter candidates. L-Glu caused dose-dependent stimulation of 45Ca uptake (L-Glu-stimulated 45Ca uptake), its effect being half-maximal at 1 microM. The related compounds D-glutamic acid, D,L-alpha-aminoadipic acid and N-methyl-D,L-glutamic acid (final conc. of 10 microM) also stimulated 45Ca uptake, but less than 10 microM L-Glu. D,L-alpha-Methylglutamic acid and L-glutamic acid diethylether (final conc. of 10 microM), which are specific inhibitors of L-Glu, inhibited L-Glu-stimulated 45Ca uptake. Mg,Ca-ATPase activity was hardly affected by a concentration of 10 microM L-Glu that caused maximal stimulation of 45Ca uptake. These findings suggest that L-Glu-stimulated 45Ca uptake by brain cortical slices is linked to L-Glu receptor.

Lactone formation of N-acetylneuraminic acid oligomers and polymers as examined by capillary electrophoresis.

We examined lactone‐formation reaction of oligomers and polymers of N‐acetylneuraminic acid in diluted hydrochloric acid solution and found that the time course of the lactonization reaction was easily traced by capillary electrophoresis. The reaction proceeded more rapidly with increasing the molecular mass of oligomers because the conformation of inner units became rigid and more favorable for the formation of lactone linkage. Present results obtained using capillary electrophoresis will be useful in understanding of physical and chemical properties of oligo/polysialic acids.