kn-62 and Neuroblastoma

Pannexin1 (Panx1), a protein related to the gap junction proteins of invertebrates, forms nonjunctional channels that open upon depolarization and in response to mechanical stretch and purinergic receptor stimulation. Importantly, ATP can be released through Panx1 channels, providing a possible role for these channels in non-vesicular signal transmission. In this study we expressed exogenous human and mouse Panx1 in the gap junction deficient Neuro2A neuroblastoma cell line and explored the contribution of Panx1 channels to cell-cell communication as sites of ATP release. Electrophysiological (patch clamp) recordings from Panx1 transfected Neuro2A cells revealed membrane conductance that increased beyond 0 mV when applying voltage ramps from -60 to +100 mV; threshold was correlated with extracellular K+, so that at 10 mM K+, channels began to open at -30 mV. Evaluation of cell-cell communication using dual whole cell recordings from cell pairs revealed that activation of Panx1 current in one cell of the pair induced an inward current in the second cell after a latency of 10-20 s. This paracrine response was amplified by an ATPase inhibitor (ARL67156, 100 μM) and was blocked by the ATP-degrading enzyme apyrase (6.7 U/ml), by the P2 receptor antagonist suramin (50 μM) and by the Panx1 channel blocker carbenoxolone. These results provide additional evidence that ATP release through Panx1 channels can mediate nonsynaptic bidirectional intercellular communication. Furthermore, current potentiation by elevated K+ provides a mechanism for enhancement of ATP release under pathological conditions.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Apyrase; Carbenoxolone; Cell Communication; Cell Line, Tumor; Connexins; Gap Junctions; Humans; Membrane Potentials; Mice; Nerve Tissue Proteins; Neuroblastoma; Patch-Clamp Techniques; Purinergic P2X Receptor Antagonists; Signal Transduction; Suramin

Choline-O-acetyltransferase (ChAT) is the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine in cholinergic neurons. Here we show that in mouse cholinergic NS-20Y neuroblastoma cells cultured in the presence of either okadaic acid (serine/threonine phosphatases 1 and 2A inhibitor) or KN-62 (CaM kinase inhibitor) ChAT activity and mRNA either increased or decreased as a function of the drug concentration, respectively. After 24 h exposure, okadaic acid exerted a dramatic effect on cell morphology; cells became round and had no more neurites. On the contrary, KN-62 induced a slight morphological differentiation of the cells. The present results suggest that phosphatases 1 and 2A and CaM kinase could mediate regulation of ChAT gene expression.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Choline O-Acetyltransferase; Enzyme Induction; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Mice; Neoplasm Proteins; Neuroblastoma; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Processing, Post-Translational; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Lithium, carbamazepine and sodium valproate are mood stabilizers used in the treatment of bipolar disorder, and although their mechanisms of action remain unknown, signal transduction systems and the associated modulation of gene expression may constitute significant actions. We examined if acute or chronic treatments with these agents modulated the activation of the AP-1 transcription factor or the increased intracellular calcium levels in human neuroblastoma SH-SY5Y cells caused by stimulation with carbachol. AP-1 activation stimulated by carbachol was reduced by pretreatment for 1 h, 24 h or 7 days with 1 mM lithium by 15%, 37%, and 60%, respectively, and with 0.05 mM carbamazepine by 3%, 21%, and 46%, respectively, but not by pretreatment with 0.5 mM sodium valproate. AP-1 DNA binding activity stimulated by carbachol or by phorbol ester-induced activation of protein kinase C was inhibited by the protein kinase C inhibitor Ro31-8220, but phorbol ester-stimulated AP-1 activation was unaltered by 7-day pretreatments with lithium or carbamazepine. Activation of AP-1 by carbachol was dependent on calcium, as it was inhibited by treatment with the extracellular calcium chelator EGTA, the intracellular calcium chelator BAPTA-AM, and the calcium/calmodulin kinase II inhibitor KN62. Pretreatment for 7 days with lithium or carbamazepine had no significant effect on carbachol-stimulated increases in intracellular calcium levels, but reduced the stimulation of AP-1 by the calcium ionophore ionomycin by 30% to 40%. Thus, chronic treatment with the antibipolar agents lithium and carbamazepine attenuates carbachol-stimulated AP-1 DNA binding activity, and these agents preferentially inhibit signaling cascades activated by the calcium rather than the protein kinase C arm of the phosphoinositide signaling pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Antipsychotic Agents; Bipolar Disorder; Calcium; Calcium Signaling; Carbachol; Carbamazepine; Chelating Agents; DNA; Dose-Response Relationship, Drug; Edetic Acid; Egtazic Acid; Gene Expression Regulation; Humans; Indoles; Ionomycin; Ionophores; Lithium; Muscarinic Agonists; Nerve Tissue Proteins; Neuroblastoma; Neurons; Phosphatidylinositols; Protein Binding; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Tumor Cells, Cultured; Valproic Acid

The role of Ca2+/calmodulin-dependent protein kinase (CaM kinase; EC 2.7.1.123) in the generation of Ca2+ signals by muscarinic acetylcholine receptors (mAChR) was studied. Changes in intracellular Ca2+ concentrations ([Ca2+]i) induced by mAChR activation were monitored in SK-N-SH human neuroblastoma cells using the dye Fura-2. SK-N-SH cells express M3 mAChR, as well as CaM kinase types II and IV, which are specifically inhibited by the CaM kinase antagonist KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne). Carbamylcholine (100 microM) elicited an initial transient peak in [Ca2+]i due to mobilization of Ca2+ from internal stores, followed by a sustained elevation in [Ca2+]i that depended on the influx of extracellular Ca2+ and which was inhibited by EGTA and Ni2+. These mAChR-induced Ca2+ signals were diminished to an equal extent by preincubating the cells with 0.01 to 100 microM KN-62. KN-62 inhibited mAChR-induced Ca2+ influx and mobilization from internal stores by about 25-30%, producing a half-maximal effect at approximately 1 microM. In contrast, KN-62 (25 microM) almost completely abolished carbamylcholine-stimulated entry of divalent cations through Mn2+-permeant channels, as revealed by Mn2+ quenching of Fura-2 fluorescence. KN-62 also almost completely abolished Ca2+ influx induced by depolarization of the cells with 25 mM K+ (IC50 = 3 microM). These results suggest that CaM kinases regulate both the mobilization of intracellular Ca2+ and the stimulation of Ca2+ influx that are induced by mAChR activation, and indicate that the mAChR-induced influx of Ca2+ occurs through Ca2+ channels other than, or in addition to, the voltage-gated calcium channels or Mn2+-permeant channels which are inhibited by KN-62.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Enzyme Inhibitors; Humans; Neuroblastoma; Piperidines; Receptor, Muscarinic M3; Receptors, Muscarinic; Signal Transduction; 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

Intercellular adhesion molecule-1 (ICAM-1) is an important cell surface adhesion receptor of the immune system. Its cell surface expression on a wide variety of cells, including cancer cells, is regulated by various proinflammatory cytokines. In the present study, we investigated the role of calcium (Ca2+) and calmodulin (CaM) in the retinoic acid and gamma-interferon (IFN-gamma) signaling in the human neuroblastoma cell line SK-N-SH for up-regulating ICAM-1 expression. A 24-h incubation in the presence of Ca(2+)-mobilizing agents (A23187 and thapsigargin) resulted in the induction of ICAM-1 expression. Both Ca(2+)-mobilizing agents stimulated ICAM-1 expression additively to IFN-gamma but not to retinoic acid, suggesting that IFN-gamma does not use Ca2+ to stimulate ICAM-1, whereas retinoic acid might use it in part. As a second messenger, Ca2+ can be coupled with calmodulin. Using calmodulin inhibitors (W7 and calmidazolium), we found that retinoic acid-stimulated, A23187-stimulated, and thapsigargin-stimulated but not FIN-gamma-stimulated ICAM-1 were inhibited. Calmodulin signaling elicited by retinoic acid was an early event occurring within the first h of retinoic acid treatment, providing evidence that they may both be coupled to regulate gene expression. Using a novel CaM kinase II inhibitor, KN-62, we demonstrated that retinoic acid stimulated ICAM-1 expression in a CaM kinase II-dependent fashion. The mechanisms whereby CaM kinase II mediates retinoic acid activity on ICAM-1 expression remain to be elucidated.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Adhesion Molecules; Enzyme Activation; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Interferon-gamma; Isoquinolines; Neuroblastoma; Piperazines; Protein Kinase C; Sulfonamides; Terpenes; Thapsigargin; Tretinoin; Tumor Cells, Cultured; Up-Regulation