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 mu M), KN-62 (10 mu M) and a myristoylated autocamtide-2 related inhibitory peptide (20 mu M), 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 mu M ONO-RS-082, an inhibitor of the phospholipase A(2) (PLA(2)). 3. Arachidonic acid (5-100 mu M) 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 [H-3] arachidonic acid for 24 h (progressively incorporated in membrane phospholipids). Upon neurotensin (1 nM) and thapsigargin (1 mu M) 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+], transient during stores depletion (with cell permeant EGTA), by the PLA, inhibitor ONO-RS-082 (10 CIM) and by the CaM kinase II inhibitor KN-62 (10 CIM) 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 mu M 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 CIM phorbol-12,13-dibutyrate. We therefore suggest the involvement of CaM kinase II and PLA(2) in the 'Ca2+ stores depletion-Ca2+ entry' coupling in these transfected CHO cells.