2 DISTINCT ATP SIGNALING MECHANISMS IN DIFFERENTIATED NEUROBLASTOMA XGLIOMA HYBRID NG108-15 CELLS

The ATP signaling mechanism in neuroblastoma x glioma hybrid NG108-15 cells differentiated by exposure to dibutyryl-cAMP was characterized. In cells loaded with fura-2, ATP rapidly raised the cytosolic Ca2+ con centration ([Ca2+]i); the magnitude of the rise was inversely proporti onal to the extracellular Na+ concentration. Large increases in cytoso lic Na+ concentration, measured with the fluorescent Na+ indicator sod ium-binding benzofuran isophthalate, were dose-dependently elicited by ATP. ATP also evoked the entry of ethidium bromide into cells, and th is process was inhibited by Mg2+. Inositol-1,4,5-trisphosphate (IP3) g eneration induced by ATP was totally blocked by removal of extracellul ar Ca2+, but residual IP3 generation still remained in nondifferentiat ed cells. In addition, ATP produced a concentration-, time-, and Mg2+- dependent biphasic uptake of Ca-45(2+). A range of nucleotides and ATP analogues, including CTP, UTP, and GTP, induced only 9-29% of the ATP response. However, adenosine 5'-thiotriphosphate evoked 79% of ATP-in duced Ca-45(2+) uptake. Ca-45(2+) uptake elicited by ATP could be pote ntly blocked by purinoceptor antagonists, but other tested reagents le ss effectively blocked the action of ATP. When bradykinin was used as an agonist, the [Ca2+]i rise was transient and was insensitive to the extracellular Na+ concentration. Na+ influx, entry of ethidium bromide , and Ca-45(2+) uptake were unaffected by bradykinin. Furthermore, bra dykinin-evoked IP3 generation was insensitive to extracellular Ca2+. N either ATP nor bradykinin had any effect on cAMP levels within cells. These data suggest that ATP induces a [Ca2+]i rise in differentiated N G108-15 cells via two distinct Ca2+ influx mechanisms, i.e., a recepto r-operated cation channel and pores formed by ATP4 . These mechanisms are distinct from those elicited by bradykinin.