icatibant and Neuroblastoma

Confocal fluorescence microscopy was used to study the bradykinin-induced calcium signals in the neuroblastoma x glioma cell line NG 108-15. We found that bradykinin induced a rise in free calcium, not only in the cytoplasm but also in the nucleus. The nuclear and cytosolic calcium concentrations were not significantly different and rose to about 1.2 microM. The signal was mediated by the B2-receptor subtype as confirmed using the specific antagonist Hoe 140. Both the onset and the intensity of the calcium signals were concentration-dependent. The rise of nuclear calcium level was independent of extracellular calcium and suppressed by thapsigargin which is known to deplete inositol 1,4,5-trisphosphate-sensitive calcium stores. Bradykinin-induced calcium increase desensitizes rapidly. This desensitization was shown not to involve activation of protein kinase C.

Topics: Animals; Bradykinin; Calcium; Cell Nucleus; Fura-2; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Microscopy, Confocal; Neuroblastoma; Protein Kinase C; Rats; Signal Transduction; Tumor Cells, Cultured

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Topics: Animals; Atropine; Bradykinin; Bradykinin Receptor Antagonists; Capillary Action; Carcinoma, Hepatocellular; Cell Line; Electrophoresis; Glioma; Humans; Ligands; Liver Neoplasms; Mice; Muscarinic Antagonists; Neuroblastoma; PC12 Cells; Rats; Receptors, Bradykinin; Receptors, Muscarinic; Tumor Cells, Cultured