h-89 and Neuroblastoma

Prenatal ethanol exposure can cause extensive apoptotic neurodegeneration throughout the developing central nervous system (CNS), which results in cognitive deficits and memory decline. However, the underlying mechanisms need further study. Single-minded 2 (Sim2), a transcriptional repressor, is reportedly involved in diseases that impair learning and memory, such as Down syndrome (DS) and Alzheimer's disease. It is still unknown whether Sim2 is involved in regulating ethanol-mediated neuronal injury that might ultimately lead to neuronal dysfunction and subsequent learning and memory deficits. To study the effects of ethanol on Sim2 expression and neuronal injury, we used animal models and cell culture experiments. Our results indicated that in SH-SY5Y cells, ethanol exposure increased Sim2 expression and levels of cleaved caspase 3, which is a marker for cells undergoing apoptosis. Silencing Sim2 expression attenuated caspase 3 activation and cellular apoptosis. We also found that protein kinase A (PKA) activation induced Sim2 expression, as did ethanol. Inhibiting the PKA signaling pathway with H-89 decreased Sim2 expression and cleavage of caspase 3 that was induced by ethanol in vivo and in vitro. We further found that PKA regulated Sim2 expression at the transcriptional level. These results demonstrate that ethanol leads to increased Sim2 expression via the PKA pathway, ultimately resulting in apoptotic cell death.

Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Caspase 3; Cell Line, Tumor; Cells, Cultured; Cerebral Cortex; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Ethanol; Humans; Isoquinolines; Mice; Neoplasm Proteins; Neuroblastoma; Neurons; Primary Cell Culture; Rats; RNA Interference; RNA, Small Interfering; Sulfonamides; Transcription, Genetic; Transfection

Calcium/calmodulin-dependent serine kinase (CASK), a causative gene in X-linked mental retardation, acts as a multi-domain scaffold protein and interacts with more than 20 cellular proteins in different subcellular regions of neurons. It is of interest, therefore, to explore whether post-translational modification regulates CASK's protein-protein interactions. Here, we provide evidence that CASK is phosphorylated by protein kinase A (PKA), identifying residue S562 in the PSD-95-Dlg-ZO-1 domain and residue T724 in the guanylate kinase domain as PKA sites by an in vitro PKA kinase reaction and site-directed mutagenesis. Although the role of S562 phosphorylation is not clear, T724 phosphorylation up-regulates the interaction between CASK and T-box transcription factor T-brain-1 (Tbr-1). NMDAR2b, a downstream target of the CASK-Tbr-1 complex, was then used to explore the significance of CASK phosphorylation by PKA. In cultured cortical neurons, the PKA pathway stimulates both the protein expression and the promoter activity of NMDAR2b. Deletion of the Tbr-1-binding sites greatly reduces the 3'-5'-cyclic AMP responsiveness of the NMDAR2b promoter, and the CASK T724A mutation does not promote the 3'-5'-cyclic AMP responsiveness of NMDAR2b. In conclusion, our data provide evidence that PKA phosphorylates CASK, regulates the nuclear function of CASK, and consequently modulates NMDAR2b expression.

Topics: Animals; Brain; Cell Line; Cricetinae; Cricetulus; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disks Large Homolog 4 Protein; Enzyme Inhibitors; Gene Expression Regulation; Guanylate Kinases; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Isoquinolines; Membrane Proteins; Mutagenesis, Site-Directed; Mutation; Nerve Tissue Proteins; Neuroblastoma; Neurons; Phosphoproteins; Phosphorylation; Rats; Receptors, N-Methyl-D-Aspartate; Serine; Signal Transduction; Sulfonamides; Syndecan-2; T-Box Domain Proteins; Tetrodotoxin; Threonine; Transfection; Up-Regulation; Zonula Occludens-1 Protein

In our previous observations, adenosine triphosphate (ATP) was found to evoke immediate elevations in intracellular free calcium concentration ([Ca2+]i) in HT4 neuroblastoma cells of mice. We tried to see if a brief pretreatment of glucocorticoids could inhibit the Ca2+ response and reveal the underlying signaling mechanism.. Measurement of [Ca2+]i was carried out using the dual-wavelength fluorescence method with Fura-2 as the indicator.. Pre-incubation of HT4 cells for 5 min with corticosterone (B) or bovine serum albumin conjugated corticosterone (B-BSA) inhibited the peak [Ca2+]i increments in a concentration-dependent manner. Cortisol and dexamethasone had a similar action, while deoxycorticosterone and cholesterol were ineffective. Both extracellular Ca2+ influx and internal Ca2+ release contributed to ATP-induced [Ca2+]i elevation. The brief treatment with only B attenuated Ca2+ influx. Furthermore, the [Ca2+]i elevation induced by the P2X receptor agonist adenosine 5'-(beta, gamma-methylene) triphosphate (beta, gamma-meATP) was also suppressed. The rapid inhibitory effect of B can be reproduced by forskolin 1 mmol/L and blocked by H89 20 mmol/L. Neither nuclear glucocorticoid receptor antagonist mifepristone nor protein kinase C inhibitors influenced the rapid action of B.. Our results suggest that glucocorticoids modulate P2X receptor-medicated Ca2+ influx through a membrane-initiated, non-genomic and PKA-dependent pathway in HT4 cells.

Topics: Adenosine Triphosphate; Animals; Calcium; Cell Line, Tumor; Corticosterone; Cyclic AMP-Dependent Protein Kinases; Dexamethasone; Dose-Response Relationship, Drug; Hydrocortisone; Isoquinolines; Mice; Neuroblastoma; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2X; Serum Albumin; Signal Transduction; Sulfonamides

To investigate mechanisms of neurite outgrowth, murine Neuro-2a neuroblastoma cells were exposed to ganglioside GM1 in the presence or absence of specific protein kinase inhibitors. Isoquinolinesulfonamide (H-89), an inhibitor of cyclic AMP dependent protein kinase A (PKA), and bisindolylmaleimide I (BIM), which inhibits protein kinase C, each stimulated neurite outgrowth in a dose-dependent manner in the absence of exogenous GM1. Minimally effective (threshold) concentrations of H-89 or BIM potentiated outgrowth when they were used in combination with GM1. To search for a shared component in the mechanisms of GM1, H-89 and BIM, phosphorylation of ERK1/2 was examined. Inhibition of the activation of extracellular signal regulated kinases (ERK1/2) by U0126, prevented neuritogenesis of Neuro-2a by all the three agents. Pretreatment of serum-depleted Neuro-2a cultures with GM1 or BIM enhanced ERK1/2 phosphorylation when the serum level was restored to 10%. In contrast, H-89 did not alter the serum-mediated response. In cells exposed to GM1 or BIM without additional serum, a transitory decrease in ERK phosphorylation occurred. These data suggest that GM1 influences two neuritogenic pathways, one modulated by PKC and the other regulated by PKA. Therefore, GM1 may have the potential to stimulate alternate pathways resulting in outgrowth.

Topics: Animals; Enzyme Inhibitors; G(M1) Ganglioside; Indoles; Isoquinolines; Maleimides; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neurites; Neuroblastoma; Phosphorylation; Sulfonamides; Tumor Cells, Cultured

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.

Topics: Cilostazol; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dexamethasone; Humans; Inositol; Isoquinolines; Neuroblastoma; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phosphodiesterase Inhibitors; Sodium-Potassium-Exchanging ATPase; Sorbitol; Sulfonamides; Tetrazoles; Tumor Cells, Cultured

We investigated the relation between cyclic AMP (cAMP) and nitric oxide (NO) production, as well as the effect of NO on Na , K+-ATPase activity in the human neuroblastoma cell line SH-SY5Y. Two cAMP agonists, dibutyryl cAMP (DBC) and beraprost sodium (BPS), increased cAMP accumulation and NO production in a time and dose dependent manner at 50 mmol/l glucose. On the other hand, cellular sorbitol and myo-inositol contents and protein kinase C activity were not altered by DBC or BPS. A specific protein kinase A inhibitor, H-89, suppressed increases in nitrite/nitrate and cyclic GMP (cGMP) and protein kinase A activity stimulated by DBC or BPS. This finding suggests that cAMP stimulates NO production by activating protein kinase A via a pathway different from the sorbitol-myo-inositol-protein kinase C pathway. We observed that an NO donor, sodium nitroprusside, and an NO agonist, L-arginine, enhanced ouabain sensitive Na+, K+-ATPase activity at 50 mmol/l glucose. We also found that a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), inhibited Na+, K+-ATPase activity at 5 mmol/l glucose, and partially suppressed the enzyme activity stimulated by DBC or BPS. The results of this study suggest that cAMP regulates protein kinase A activity, NO production and ouabain sensitive Na+, K+-ATPase activity in a cascade fashion. The results also suggest that protein kinase A at least partially regulates Na+, K+-ATPase activity without mediation by NO in SH-SY5Y cells. We speculate that cAMP and NO are two important regulatory factors in the pathogenesis of diabetic neuropathy.

Topics: Bucladesine; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Enzyme Inhibitors; Epoprostenol; Humans; Isoquinolines; Kinetics; Neuroblastoma; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrites; Ouabain; Sodium-Potassium-Exchanging ATPase; Sulfonamides; Tumor Cells, Cultured

The human neuroblastoma cell line SK-N-SH has been used as a model system to study the interactions of the human estrogen receptor (hER) with neurotransmitters. We have successfully transfected these cells using an adenoviral delivery system and have reconstituted ligand-dependent responses to estradiol and ligand-independent responses to a series of dopamine D1 receptor agonists. The full agonist for the D1 receptor, SKF 82958, shows a robust activation of hER, comparable to that induced by estradiol. This activation is blocked by the protein kinase A inhibitor H-89, is mimicked by forskolin, and is therefore thought to be mediated in part through the cAMP/protein kinase A pathway. We have examined deletion mutants of hER for activation by SKF 82958 and find that both its transactivation domains, AF-1 and AF-2, must cooperate to impart the full response to the agonist. Significantly, an agonist of the muscarinic acetylcholine receptor, carbachol, though not active by itself, synergistically activates hER in conjunction with suboptimal doses of SKF 82958. This is the first reported instance of two neurotransmitters synergizing to activate a member of the nuclear receptor superfamily, and might predict a role for multiple neural inputs modulating the effects of these receptors in the central nervous system.

Topics: Adenoviridae; Benzazepines; Carbachol; Cyclic AMP-Dependent Protein Kinases; Dopamine; Enzyme Inhibitors; Estradiol; Genetic Vectors; Humans; Isoquinolines; Ligands; Muscarinic Agonists; Neuroblastoma; Neurons; Neurotransmitter Agents; Quinpirole; Receptors, Dopamine D1; Receptors, Dopamine D2; Receptors, Estrogen; Sequence Deletion; Sulfonamides; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Since the alpha and beta isoforms of CaM kinase II are known to be expressed almost exclusively in the brain, we compared the effect of overexpression of the beta isoform of CaM kinase II with that of the alpha isoform. The subcellular distribution of the alpha isoform was different from that of the beta isoform, although the catalytic properties of the alpha and beta isoforms expressed in transfected cells were similar to those of brain CaM kinase II. The alpha isoform was found in the soluble fraction more than in the particulate fraction, whereas most of the beta isoform bound to subcellular structures. In the cell overexpressing alpha and beta isoforms of CaM kinase II, neurite extension was promoted when compared with the morphology of neo transfectants. Neurite outgrowth of cells overexpressing CaM kinase II was further stimulated by the treatment of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a selective but not absolutely specific inhibitor of protein kinase C. The morphological change was rapid and observed within 1 h followed by H-7 treatment. Morphological changes, such as the number of cells with neurites and length of neurites were greater in the beta cells than in the alpha cells. Chelerythrine, a specific inhibitor of protein kinase C, also stimulated the neurite outgrowth of these cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing CaM kinase II stimulated with H-7. These results suggest that CaM kinase H and protein kinase C play an important role in the control of cell change, and that the subcellular distribution of CaM kinase II is important for regulating cellular functions efficiently.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acid Sequence; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cloning, Molecular; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Immunoblotting; Isoenzymes; Isoquinolines; Mice; Molecular Sequence Data; Neurites; Neuroblastoma; Phosphorylation; Substrate Specificity; Sulfonamides; Transfection; Tumor Cells, Cultured

We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 mM DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.

Topics: Animals; Apoptosis; Bucladesine; Cell Differentiation; Cell Division; Cell Line; Central Nervous System Neoplasms; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; DNA, Neoplasm; Enzyme Inhibitors; Isoquinolines; Kinetics; Neurites; Neuroblastoma; Neurons; Rats; Sulfonamides; Time Factors; Tumor Cells, Cultured

1. In SH-SY5Y cells, mu-opioids cause a rapidly desensitizing activation of phospholipase C (PLC), that appears secondary to Ca2+ influx via L-type voltage-sensitive Ca2+ channels (VSCCs). The aim of the present study was to characterize the mechanisms of desensitization of the mu-opioid-induced inositol (1,4,5) triphosphate (Ins(1,4,5)P3) response, by use of a stereospecific radioreceptor mass assay. 2. (R+)-Bay K 8644 (1 nM-10 microM) dose-dependently inhibited fentanyl-induced Ins(1,4,5)P3 formation, with an IC50 of 28.5 nM, confirming our earlier observations that mu-opioids open L-type VSCCs, thus allowing Ca2+ influx to activate PLC. 3. Ro 31-8220 (0.1 nM-10 microM), a protein kinase C inhibitor, dose-dependently enhanced fentanyl-induced Ins(1,4,5)P3 formation (EC50 = 20.0 nM), whilst acute phorbol 12,13-dibutrate (1 microM) abolished the response. 4. H-89 (1 nM-10 microM), a protein kinase A inhibitor, also dose-dependently enhanced fentanyl-induced Ins(1,4,5)P3 formation (EC50 = 93 nM), whilst dibutryl cyclic AMP (0.5 mM) abolished the response. 5. Blockade of Ca(2+)-activated K+ currents with 4-aminopyridine (2 mM) or iberiotoxin (10 nM) had no effect on fentanyl-induced Ins(1,4,5)P3 formation but further increased the Ro 31-8220-enhanced response. 6. All three mechanisms had additive, or even supra-additive, effects, but only at later (120-300 s) time points. In addition, fentanyl-induced Ins(1,4,5)P3 formation, even if enhanced by H-89, Ro 31-8220 and/or 4-aminopyridine, was inhibited by nifedipine (1 nM-10 microM). 7. In conclusion, desensitization of the mu-opioid-induced activation of PLC is multifactorial, involving protein kinases C and A and Ca(2+)-activated K+ efflux, but the L-type VSCC is of critical importance and may be a possible common site of action.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Analgesics, Opioid; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Fentanyl; Humans; Indoles; Inositol 1,4,5-Trisphosphate; Isoquinolines; Narcotics; Neuroblastoma; Nifedipine; Potassium Channels; Protein Kinase C; Receptors, Opioid, mu; Sensitivity and Specificity; Sulfonamides; Tumor Cells, Cultured; Type C Phospholipases

In retinoic acid (RA)-differentiated SH-SY5Y cell cultures, colchicine could induce cell death, accompanied by the typical ladder pattern of DNA fragmentation found in apoptotic cells. This effect could be attenuated by the protein synthesis inhibitor cycloheximide. The protein kinase inhibitor H-89 efficiently reduced colchicine-induced cell death. These results suggest that the mechanism for colchicine-induced cell death may act, at least in part, through the activation of apoptosis in differentiating SH-SY5Y cells.

Topics: Apoptosis; Cell Differentiation; Cell Line; Colchicine; Dose-Response Relationship, Drug; Humans; Isoquinolines; Kinetics; L-Lactate Dehydrogenase; Neuroblastoma; Neurotoxins; Protein Kinase Inhibitors; Sulfonamides; Tretinoin; Tumor Cells, Cultured