Receptor-stimulated phospholipase A2 activation is coupled to influx of external calcium and not to mobilization of intracellular calcium in C62B glioma cells.
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.
Brooks
R C
RC
Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill 27599-7250.
McCarthy
K D
KD
Lapetina
E G
EG
Morell
P
P
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
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