Signal Transduction Pathways Coupled to a P_2U Receptor in Neuroblastoma × Glioma (NG108‐15) Cells

Abstract

Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P2 purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P2U receptor in a cultured neuroblastoma x glioma hybrid cell line (NG108-15 cells). The addition of > or = 1 microM ATP to NG108-15 cells caused a transient increase in [Ca2+]i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations > or = 500 microM also elicited a sustained increase in [Ca2+]i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca2+]i elicited by ATP occurred concomitantly with the hydrolysis of [32P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca2+]i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP4-) in the medium and not with the concentration of magnesium-ATP (MgATP2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca2+]i. ADP, ITP, TTP, GTP, ATP gamma S, 2-methylthio ATP, beta, gamma-imidoATP or 3'-O-(4-benzoyl)benzoylATP, but not CTP, AMP, beta, gamma-methylene ATP, or adenosine, also caused an increase in [Ca2+]i. In cells labeled with [32P]P(i) or [14C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [14C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P2 nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A2 and D.

Streptozotocin, an inducer of NAD+ decrease, attenuates M‐potassium current inhibition by ATP, bradykinin, angiotensin II, endothelin 1 and acetylcholine in NG108‐15 cells

“…5 mM nicotinamide alone had a small but negligible effect (Table 1). signal pathways from these five receptors, which are originally reported to couple to phospholipase C [4,6,8,12,14,15,17,20,21], to M channels merge into one, because M-current inhibition was susceptible to streptozotocin treatment and was restored by an addition of nicotinamide. Our previous measurement shows that in streptozotocin-treated NGPM1 27 cells, cellular NAD ÷ content is reduced to about 40-50% of the control level Table 1 Agonist-induced M-current inhibition in NGPM1 27 cells under various conditions [l 1] and that this level is recovered by simultaneous addition of nicotinamide, a precursor of NAD ÷.…”

P2Y Receptor Linked to Phospholipase C: Stimulation of Neuro 2A Cells by UTP and ATP and Possible Regulation by Protein Kinase C Subtype ε

“…The P2Y7 receptor was cloned from human erythroleukemic cells (Akbar et al, 1996). In neuronal cells, the P2Y2 receptor has been reported in NG1O8-15, NCB-20, and PC12 cells (Garritsen et al, 1992;Murrin and Boarder, 1992;Lin et al, 1993). In the present study, we have examined whether P2Y receptors occur in Neuro 2A mouse neuroblastoma cells, and if they are involved in the control of phospholipase C (PLC) -coupled signaltransduction pathways.…”

Desensitization of P2U receptor in neuronal cell line. Different control by the agonists ATP and UTP, as demonstrated by single-cell Ca2+ responses

“…Moreover, the potency of UTP proved the receptor to be of the P #U type. The purinoceptor present in the neuronal cell line is coupled to activation of phospholipase C and Ca# + influx -the nature of this influx pathway [8] and even its unequivocal existence is still unclear -and has been characterized molecularly by cloning the corresponding cDNA [9]. The protein has structural similarities and homologies with other members of the family of G-proteincoupled receptors that have seven putative transmembrane domains.…”