Ca2+ entry in CHO cells, after Ca2+ stores depletion, is mediated by arachidonic acid.
Transfected Chinese hamster ovary cells expressing the rat neurotensin receptor (CHO-NTR cells) were used to study the 'Ca2+ stores depletion-Ca2+ entry' coupling which follows stimulation with neurotensin and liberation of inositol 1,4,5-trisphosphate. This coupling could be dissociated in time: the stores were emptied by stimulation with neurotensin in the absence of extracellular Ca2+; thereafter, readmission of extracellular Ca2+ produced a transient entry of Ca2+ that was progressively restored in the endoplasmic reticulum. We showed previously that the rise of [Ca2+]i during Ca2+ stores depletion controls the subsequent entry of Ca2+ and that unknown protein kinases and phosphatases may also be involved in this coupling. Here we show that: 1. W-7 (25 microM), KN-62 (10 microM) and a myristoylated autocamtide-2 related inhibitory peptide (20 microM), three inhibitors of the calcium-calmodulin-dependent protein kinase II (CaM kinase II) inhibit the entry of Ca2+ induced by emptying the stores of Ca2+ with neurotensin and thapsigargin. 2. Ca2+ stores depletion-Ca2+ entry coupling is also greatly diminished by 10 microM ONO-RS-082, an inhibitor of the phospholipase A2 (PLA2). 3. Arachidonic acid (5-100 microM) produces an entry of Ca2+; the same result is obtained by use of 5, 8, 11, 14-eicosatetraynoic acid, a non-metabolizable analog of arachidonic acid. 4. NTR-CHO cells are labeled with [3H] arachidonic acid for 24 h (progressively incorporated in membrane phospholipids). Upon neurotensin (1 nM) and thapsigargin (1 microM) stimulation, these cells produce a release of arachidonic acid which lasts for as long as the stores are empty and stops when they are reloaded with Ca2+. This production of arachidonic acid is significantly diminished by suppressing the [Ca2+]i transient during stores depletion (with cell permeant EGTA), by the PLA2 inhibitor ONO-RS-082 (10 microM) and by the CaM kinase II inhibitor KN-62 (10 microM). 5. The rise of [Ca2+]i by itself (induced by flash photolysis of nitrophenyl-EGTA), i.e. without depletion of the stores, is not sufficient to trigger an entry of Ca2+. 6. The reloading process of Ca2+ into the endoplasmic reticulum is inhibited by 10 microM chelerythrine, 100 nM GF 109203X, two inhibitors of protein kinases C (PKC) or by their downregulation by a prolonged treatment of the cells with 1 microM phorbol-12, 13-dibutyrate. We therefore suggest the involvement of CaM kinase II and PLA2 in the 'Ca2+ stores depletion-Ca2+ entry' coupling in these transfected CHO cells.
Gailly
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Department of Physiology and Pharmacology, Catholic University of Louvain, Brussels, Belgium. gailly@fymu.ucl.ac.be
D000818:Animals; D016718:Arachidonic Acid / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006224:Cricetinae; D009496:Neurotensin / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D018028:Receptors, Neurotensin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism
D018028:Receptors, Neurotensin; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D009496:Neurotensin; D019284:Thapsigargin; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D000252:Calcium-Transporting ATPases; D002118:Calcium