title: Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells.
abstract: C62B rat glioma cells respond to muscarinic cholinergic stimulation with transient inositol phosphate formation and phospholipase A2-dependent arachidonic acid liberation. Since phospholipase A2 is a Ca2+-sensitive enzyme, we have examined the role of the agonist-stimulated Ca2+ response in production of the arachidonate signal. The fluorescent indicator fura-2 was used to monitor changes in cytoplasmic Ca2+ levels ([Ca2+]i) of C62B cells following acetylcholine treatment. In the presence of extracellular Ca2+, acetylcholine induces a biphasic [Ca2+]i response consisting of an initial transient peak that precedes arachidonate liberation and a sustained elevation that outlasts the phospholipase A2 response. The initial [Ca2+]i peak is not altered by the absence of external Ca2+ and therefore reflects intracellular Ca2+ mobilization. The sustained elevation phase is dependent on the influx of external Ca2+; it is lost in Ca2+-free medium and restored on the addition of Ca2+. Pretreating cells with phorbol dibutyrate substantially inhibits acetylcholine-stimulated inositol phosphate formation and the peak [Ca2+]i response without affecting the sustained elevation in [Ca2+]i. This suggests that the release of internal Ca2+ stores by inositol 1,4,5-trisphosphate can be blocked without interfering with Ca2+ influx. Pretreatment with phorbol also fails to affect acetylcholine-stimulated arachidonate liberation, demonstrating that phospholipase A2 activation does not require normal intracellular Ca2+ release. Stimulated arachidonate accumulation is totally inhibited in Ca2+-free medium and restored by the subsequent addition of Ca2+. Pretreatment with verapamil, a voltage-dependent Ca2+ channel inhibitor, also blocks both the sustained [Ca2+]i elevation and arachidonate liberation without altering peak intracellular Ca2+ release. We conclude that the influx of extracellular Ca2+ is tightly coupled to phospholipase A2 activation, whereas large changes in [Ca2+]i due to mobilization of internal Ca2+ stores are neither sufficient nor necessary for acetylcholine-stimulated phospholipase A2 activation.
authors.affiliation: Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill 27599-7250.
authors.forename: R C
authors.initials: RC
authors.lastname: Brooks
authors.forename: K D
authors.initials: KD
authors.lastname: McCarthy
authors.forename: E G
authors.initials: EG
authors.lastname: Lapetina
authors.forename: P
authors.initials: P
authors.lastname: Morell
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D016257:Fura-2; D005910:Glioma; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology
chemical_list: D001572:Benzofurans; D005456:Fluorescent Dyes; D011976:Receptors, Muscarinic; D015240:Phorbol 12,13-Dibutyrate; D014700:Verapamil; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D000109:Acetylcholine; D002118:Calcium; D016257:Fura-2