Adenosine as an active component of Antrodia cinnamomea that prevents rat PC12 cells from serum deprivation-induced apoptosis through the activation of adenosine A(2A) receptors

Antrodia cinnamomea (formerly named Antrodia camphorata) is a rare medicinal fungus. We previously reported that it exhibits antioxidative, vasorelaxative, anti-inflammatory, and anti-angiogenic effects. When serum deprivation-induced apoptosis in neuronal-like PC12 cells was used as a stress model, the extract of A. cinnamomea displayed effectiveness in preventing serum-deprived apoptosis. Since our previous data show that the extract of A. cinnamomea contains adenosine (ADO), we attempt to investigate if the active component is ADO and to identify its targeting site in this study. After pre-incubation with ADO deaminase, neither ADO nor the extract of A. cinnamomea exerted any protection, demonstrating that the active component of A. cinnamomea is ADO. Furthermore, an ADO A(2A) receptor (A(2A)-R) antagonist was used and was able to block the protective effects of ADO and the extract of A. cinnamomea, demonstrating that the ADO targeting site in this model is A(2A)-R. Taken together, the protective effect of A. cinnamomea is owed to its active component, ADO, which acts through activation of A(2A)-R to prevent serum deprivation-induced PC12 cell apoptosis.     

Tetrasialoganglioside GQ1b Reactive Monoclonal Antibodies: Their Characterization and Application for Quantification of GQ1b in Some Cell Lines of Neuronal and Adrenal Origin(S)

Seven monoclonal antibodies (MAbs) directed to tetrasialoganglioside (GQ1b) were established, purified GQ1b being used for immunization and hybridoma screening. All of the MAbs reacted strongly with GQ1b, although they also reacted with other gangliosides, with different specificities and reactivities. Some MAbs (1H10, 2C7, and 3F4) reacted with GD3, GT1a, GQ1b, and GP1c. MAb 1H4 showed broad specificity. It reacted with GD3, GD1b, GD2, GT1a, GT1b, GO1b, GQ1c, and GP1c. MAbs 7F5, 4E7, and 4F10 recognized GT1a, GQ1b, and GP1c. MAb 4F10 was more specific for GQ1b than the other MAbs. Using MAb 4F10, we determined, by means of an immunoassay, the quantities of endogenous GQ1b in some neuronal and adrenal cell lines, GOTO (human neuroblastoma), Neuro2a (mouse neuroblastoma), and PC12 (rat pheochromocytoma). PC12 and Neuro2a cells contained at least 5.1 X 10(6) and 3.9 X 10(5) molecules/cell of GQ1b, respectively. In contrast, no GQ1b was detected in GOTO cells, which are known for their specific neuritogenic response to this particular ganglioside when exogenously added.     

Does Phospholipid Methylation Play a Role in the Primary Mechanism of Action of Nerve Growth Factor?

Nerve growth factor (NGF)-untreated (naive) and neurite-bearing NGF-treated ("primed") PC12 rat pheochromocytoma cells were used as model system to study the role of phospholipid methylation in the NGF mechanism of action. The neurite-bearing cultures were deprived of NGF for 3 h before experimentation. Under both experimental conditions, the cells were labelled with [methyl-3H]methionine and then challenged with NGF for time periods ranging from 5 s to 30 min. Methylated phospholipids were extracted and then resolved and identified by TLC as phosphatidyl mono-, di-, and trimethyl ethanolamine. Quantification of the amount of radioactivity incorporated into each of the phospholipids indicated that NGF does not significantly alter phospholipid methylation either in naive or in neurite-bearing cells. Furthermore, using a methyltransferase inhibitor, it was found that neurite outgrowth still occurs when phospholipid methylation is almost completely blocked. These results indicate that phospholipid methylation does not play a primary role in the mechanism of action of NGF.     

Nerve Growth Factor Affects Cyclic AMP Metabolism, but Not by Directly Stimulating Adenylate Cyclase Activity

Published experiments both support and contradict the hypothesis that nerve growth factor (NGF) can regulate adenylate cyclase activity. Using a sensitive assay that measures the conversion of [2-3H]adenine to [3H]cyclic AMP, we have shown that NGF alone cannot measurably stimulate cyclic AMP production, whereas the adenosine analog phenylisopropyladenosine (PIA) stimulates adenylate cyclase 20-fold over basal activity. NGF potentiates the capacity of both PIA and cholera toxin to stimulate cyclic AMP accumulation at all concentrations tested. This potentiation occurs at the earliest measurable times and does not require RNA synthesis. Therefore, we conclude that cyclase activation alone does not account for the effect of NGF on cyclic AMP accumulation and we discuss possible mechanisms.     

Ceramide Selectively Decreases Tau Levels in Differentiated PC12 Cells Through Modulation of Calpain I

Abstract: Ceramide has been recently proposed to be a signal mediator in several important physiological processes including apoptosis, cellular growth, and differentiation. Because the microtubule-associated protein tau plays an important role in the establishment and maintenance of neuronal morphology, the effects of ceramide on tau were examined. Treatment of differentiated PC12 cells with the cell-permeable ceramide derivative N-acetylsphingosine (C2) resulted in a significant reduction in tau levels. Significant decreases in tau levels were also observed when the cells were treated with another ceramide derivative, N-hexanoylsphingosine (C6). In addition, C2 treatment increased the levels of a calpain-derived spectrin breakdown product but did not alter the levels of two cytoskeletal proteins, α-actin and α-tubulin. Because both tau and spectrin are proteolyzed in vitro by the calcium-activated cysteine protease calpain, the effects of ceramide analogues on the activity of this protease were examined. Treatment of PC12 cells with C2 enhanced calcium-stimulated proteolytic activity significantly, as revealed by monitoring the hydrolysis of the membrane-permeable calpain-selective fluorescence probe N-succinyl-l -leucyl-l -leucyl-l -valyl-l -tyrosine-7-amido-4-methylcoumarin. This activity increase was not due to a direct effect of C2 on calpains, because C2 did not alter the activities of purified calpain I or II. In addition, C2 treatment of PC12 cells resulted in a significant increase in the levels of calpain I and, to a lesser extent, the levels of calpastatin (an endogenous calpain inhibitor protein), whereas the levels of calpain II were not changed. Moreover, treatment of the cells with the synthetic calpain-specific inhibitor N-carbobenzoxy-l -leucyl-l -leucyl-l -tyrosine diazomethyl ketone blocked the C2-induced decreases in tau levels. These results indicate that tau levels are regulated in response to a physiological factor and, thus, have implications for ceramide-mediated changes in normal and pathological neuronal processes.     

Isolation and Characterization of Chromogranins A, B, and C from Bovine Chromaffin Granules and a Rat Pheochromocytoma

Bovine chromaffin granules from adrenal medulla contain three acidic secretory proteins: chromogranins A, B, and C. For isolation of these proteins, methods based mainly on high performance liquid chromatography were developed. After removal of contaminating glycoproteins by lectin affinity chromatography, chromogranins were separated by high performance anion-exchange, gel-filtration, and reverse phase liquid chromatography. As a final purification step sodium dodecyl sulfate-gel electrophoresis was performed. Amino acid analysis of isolated bovine chromogranins revealed a similar composition of all three proteins, with glutamic acid being the most prominent amino acid. The methods developed for bovine proteins also proved suitable for isolating rat chromogranins A and B from a transplantable pheochromocytoma. Chromogranin C was not present in sufficient amounts to be isolated from this tissue. The chromogranins purified by these methods were used to raise specific antibodies in rabbits. The use of purified chromogranins together with specific antisera may be valuable in understanding the still undiscovered function of these proteins.     

Release of Chromaffin Granule Glycoproteins and Proteoglycans from Potassium-Stimulated PC12 Pheochromocytoma Cells

Abstract: Cultured PC12 pheochromocytoma cells were labeled with [³H]gIucosamine, and the glycoproteins and proteoglycans released following potassium-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs-Ringers-HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM ). The released complex carbohydrates include chromogranins, dopamine β-hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(β l ± 3 )N-ace tylgalactosamine, as well as several mono- and disialyl O -glycosidically-linked oligosaccharides, and the tetra-saccharide AcNeu(α2 ± 3)Gal(β l ± 3)[AcNeu(α2 ± (6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23–68%), heparan sulfate (16–23%), and glycoprotein oligosaccharides (16–54%), which are of the triand tetraantennary and O -glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans.     

膀胱副神经节瘤的诊治分析(附1 例病例报告并文献复习)

目的:总结膀胱副神经节瘤的诊治方法及体会,提高膀胱副神经节瘤的诊断和治疗水平。方法:报告1例膀胱嗜铬细胞瘤的临床资料并结合文献复习分析总结膀胱副神经节瘤的诊断治疗方法。结果:患者行腹腔镜下膀胱部分切除术,手术成功,术后顺利出院,随访至今无转移。结论:膀胱副神经节瘤的诊断主要依靠临床表现和实验室检查,血尿、高血压和排尿时典型发作三联征为膀胱副神经节瘤的主要症状,对于瘤体较小的患者要警惕CT检查漏诊的可能性,可行活组织检查进行确诊。腹腔镜下切除是目前治疗膀胱副神经节瘤最有效、创伤最小的方法,但术后要严格随访。     

Role of STIM1 in Regulation of Store-Operated Ca<Superscript>2+</Superscript> Influx in Pheochromocytoma Cells

Changes in the local environment such as pH (acidosis/alkalosis), temperature (hypothermia/hyperthermia), and agonist (glutamate) can adversely affect neuronal function, and are important factors in clinical situations such as anesthesia and intensive care. Regulation of intracellular Ca²⁺ ([Ca²⁺]_i) is key to neuronal function. Stromal interaction molecule (STIM1) has been recently recognized to trigger store-operated Ca²⁺ entry (SOCE), an important component of [Ca²⁺]_i regulation. Using differentiated, fura-2 loaded rat pheochromocytoma (PC12) cells transfected with small interference RNA for STIM1 (or vehicle), we examined the role of STIM1 in SOCE sensitivity to temperature, pH, and glutamate. SOCE was triggered following endoplasmic reticulum depletion. Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40°C), or to glutamate. In non-transfected cells, SOCE was inhibited by acidosis or hypothermia, but increased with alkalosis and hyperthermia. Increasing glutamate concentrations progressively stimulated SOCE. STIM1 siRNA decreased SOCE at normal temperature and pH, and substantially decreased sensitivity to acidosis and hypothermia, eliminating the concentration-dependence to glutamate. Sensitivity of SOCE to these environmental parameters was less altered by decreased extracellular Ca²⁺ alone (with STIM1 intact). We conclude that STIM1 mediates exquisite susceptibility of SOCE to pH, temperature, and glutamate: factors that can adversely affect neuronal function under pathological conditions.     

Aberrant positive metaiodobenzylguanidine (MIBG) scan in a patient with gastrointestinal stromal tumor

¹²³I-metaiodobenzylguanidine (MIBG) scintigraphy is an effective means of diagnosing chromaffin-cell tumors such as pheochromocytoma. We report a very rare case of a 45-year-old patient with large intra-abdominal mass and elevated urinary normetanephrines. ¹²³I-MIBG scintigraphy showed mild MIBG uptake. Although initial findings suggested a pheochromocytoma, histologic examination along with immunohistochemical staining revealed features suggestive of gastrointestinal stromal tumor (GIST).