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Cytosolic phospholipase A2 is coupled to muscarinic receptors in the human astrocytoma cell line 1321N1: Characterization of the transducing mechanism

AuthorsBayón, Yolanda ; Hernández, Marita ; Alonso, Andrés ; Núñez, Lucía ; García-Sancho, Javier ; Sánchez Crespo, Mariano; Nieto, María Luisa
Issue Date1997
PublisherPortland Press
CitationBiochemical Journal 323(1): 281-287 (1997)
AbstractThe cholinergic agonist carbachol induced the release of arachidonic acid in the 1321N1 astrocytoma cell line, and this was blocked by atropine, suggesting the involvement of muscarinic receptors. To assess the mechanisms of signalling involved in the response to carbachol, a set of compounds characterized by eliciting responses through different mechanisms was tested. A combination of 4β-phorbol 12β-myristate 13α-acetate and thapsigargin, an inhibitor of endomembrane Ca 2+-ATPase that induces a prolonged elevation of cytosolic Ca 2+ concentration, induced an optimal response, suggesting at first glance that both protein kinase C (PKC) and Ca 2+ mobilization were involved in the response. This was consistent with the observation that carbachol elicited Ca 2+ mobilization and PKC-dependent phosphorylation of cytosolic phospholipase A 2 (cPLA 2;phosphatide sn-2-acylhydrolase, EC as measured by a decrease in electrophoretic mobility. Nevertheless, the release of arachidonate induced by carbachol was unaltered in media containing decreased concentrations of Ca 2+ or in the presence of neomycin, a potent inhibitor of phospholipase C which blocks phosphoinositide turnover and Ca 2+ mobilization. Guanosine 5'-[γ-thio]triphosphate added to the cell-free homogenate induced both [ 3H]arachidonate release and cPLA 2 translocation to the cell membrane fraction in the absence of Ca 2+, thus suggesting the existence of an alternative mechanism of cPLA 2 translocation dependent on G-proteins and independent of Ca 2+ mobilization. From the combination of experiments utilizing biochemical and immunological tools the involvement of cPLA 2 was ascertained. In summary, these data indicate the existence in the astrocytoma cell line 1321N1 of a pathway involving the cPLA 2 which couples the release of arachidonate to the occupancy of receptors for a neurotransmitter, requires PKC activity and G-proteins and might operate in the absence of Ca 2+ mobilization.
Identifiersissn: 0264-6021
e-issn: 1470-8728
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