inosine-triphosphate and Brain-Neoplasms

1. In this study we have investigated neuropeptide Y (NPY) and somatostatin (SRIF) receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line SH-SY5Y. 2. The Ca(2+)-sensitive dye fura 2 was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither NPY (30-100 nM) nor SRIF (100 nM) elevated [Ca2+]i when applied alone. However, when either NPY (300 pM-1 microM) or SRIF (300 pM-1 microM) was applied in the presence of the cholinoceptor agonist carbachol (1 microM or 100 microM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone. 3. The elevation of [Ca2+]i by NPY was independent of the concentration of carbachol. In the presence of 1 microM or 100 microM carbachol NPY elevated [Ca2+]i with a pEC50 of 7.80 and 7.86 respectively. 4. In the presence of 1 microM carbachol the NPY Y2 selective agonist peptide YY(3-36) (PYY(3-36)) elevated [Ca2+]i with a pEC50 of 7.94, the NPY Y1 selective agonist [Leu31, Pro34]-NPY also elevated [Ca2+]i when applied in the presence of carbachol, but only at concentrations > 300 nM. The rank order of potency, PYY(3-36) > or = NPY > > [Leu31, Pro34]-NPY indicates that an NPY Y2-like receptor is involved in the elevation of [Ca2+]i. 5. In the presence of 1 microM carbachol, SRIF elevated [Ca2+]i with a pEC50 of 8.24. The sst2 receptor-preferring analogue BIM-23027 (c[N-Me-Ala-Tyr-D-Trp-Lys-Abu-Phe]) elevated [Ca2+]i with a pEC50 of 8.63, and the sst5-receptor preferring analogue L-362855 (c[Aha-Phe-Trp-D-Trp-Lys-Thr-Phe]) elevated [Ca2+]i with a pEC50 of approximately 6.1. Application of the sst3 receptor-preferring analogue BIM-23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2, 1 microM) to SH-SY5Y cells in the presence of carbachol neither elevated [Ca2+]i nor affected the elevations of [Ca2+]i caused by a subsequent coapplication of SRIF. The rank order of potency, BIM-23026 > or = SRIF > > L-362855 > > > BIM-23026 suggests that an sst2-like receptor is involved in the elevation of [Ca2+]i. 6. Block of carbachol activation of muscarinic receptors with atropine (1 microM) abolished the elevation of [Ca2+]i by the SRIF and NPY. 7. Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the NPY or SRIF response. The Ca2+ channel activator maitotoxin (2 ng ml-1) also elevated [Ca2+]i but subsequent application of either NPY or SRIF in the presence of maitotoxin caused no further changes in [Ca2+]i. 8. The elevations of [Ca2+

Topics: Brain Neoplasms; Calcium; Calcium Channels; Carbachol; Electrophysiology; Humans; Inosine Triphosphate; Muscarinic Agonists; Neuroblastoma; Neuropeptide Y; Pertussis Toxin; Receptors, Gastrointestinal Hormone; Receptors, Opioid, delta; Receptors, Somatostatin; Somatostatin; Tumor Cells, Cultured; Virulence Factors, Bordetella

We have proposed recently that a pertussistoxin-insensitive Ca2+ influx stimulated by Y2-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca2+ concentration ([Ca2+]i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca2+ and independent of internal Ca2+ stores. We describe here the actions of NPY in these same cells, using the activity of Ca(2+)-activated K+ channels as an indicator of [Ca2+]i. The elementary slope conductance of these channels was 110 +/- 3 pS (with an asymmetrical K+ gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca2+, increased the channel open probability. ATP applied in the absence of external Ca2+ caused rises both in channel open probability and [Ca2+]i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca2+ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5'-triphosphate (GTP) by (guanosine 5'-O-(2-thiodiphosphate) (GDP[ beta S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y2-type receptors to Ca2+ influx in CHP-234 cells.

Topics: Adenosine Triphosphate; Brain Neoplasms; Calcium; Electrophysiology; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Inosine Triphosphate; Inositol 1,4,5-Trisphosphate; Ionomycin; Ionophores; Neuroblastoma; Patch-Clamp Techniques; Potassium Channels; Receptors, Neuropeptide Y; Thionucleotides; Tumor Cells, Cultured