title: Multiple polyphosphoinositide pathways regulate apoptotic signalling in a dorsal root ganglion derived cell line.
abstract: The polyphosphoinositides play important roles in transmembrane signalling but are also involved in anchoring cell surface proteins, organellar transport, cytoskeleton organization, and cell survival. The polyphosphoinositides synthesized by phosphatidylinositol-3 kinase (PI-3K), (Ptd(3,4)InsP2, and PtdIns(3,4,5)P3), appear to play a critical role in cell survival by membrane recruitment and activation of Akt kinase. Inhibitors of PI3K, wortmannin, and LY294002, induced a time-dependent activation of caspase-3 (CPP32), with a peak at 6 hr, leading to subsequent cell death by apoptosis in a dorsal root ganglion cell line (F-11). Lowering cyclic AMP (cAMP) levels enhanced both caspase-3 activation and cell death induced by PI3K inhibitors, whereas a nonhydrolyzable cAMP analog (Bt2cAMP), lowered CPP32 and was protective. We stably transfected the F-11 cells with the constitutively active p110 catalytic subunit of PI-3 kinase and observed resistance to both caspase-3 (CPP32) activation and subsequent apoptosis induced by either wortmannin or LY294002. Treatment of F-11 cells with bradykinin (BK) stimulated the hydrolysis of a different polyphosphoinositide, PtdIns(4,5)P2, and enhanced both wortmannin-induced caspase-3 (CPP32) activation and subsequent apoptosis. PtdIns(4,5)P2 is also a precursor of the anti-apoptotic PtdIns(3,4,5) P3 and lowering cAMP levels with opioid agonists for 30 min enhanced both the hydrolysis of PtdIns(4,5) P2 and cellular apoptosis. The enhancement was opioid dose-dependent and opioid antagonist (naloxone)-reversible and was also seen following 24-hr exposure to opioids such as U69,593 and Dala2, Dleu5 enkephalin (DADLE). However, unlike the bradykinin stimulation of PtdIns(4,5)P2 hydrolysis following activation of phospholipase C, the opioid-enhanced hydrolysis was independent of external Ca2+ and was blocked by pertussis toxin, suggesting a different mechanism involving GI, GO, or betagamma-subunits. In summary, both the receptor-mediated lowering of cAMP levels and the hydrolysis of 4,5-polyphosphoinositides have no direct effect on caspase-3 activity or apoptosis but do exacerbate the activation of caspase-3-like activity and subsequent cell death by apoptosis induced by inhibitors of 3-polyphosphoinositide synthesis. We suggest that multiple polyphosphoinositide pathways are involved in the regulation of apoptosis.
authors.affiliation: Department of Pediatrics, University of Chicago, Illinois 60637, USA.
authors.forename: R
authors.initials: R
authors.lastname: Goswami
authors.forename: S A
authors.initials: SA
authors.lastname: Dawson
authors.forename: G
authors.initials: G
authors.lastname: Dawson
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D001920:Bradykinin / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000378:metabolism*; D009294:Narcotics / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011957:Receptors, Opioid / Q000187:drug effects; D015398:Signal Transduction / Q000502:physiology*; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D001920:Bradykinin; D053148:Caspase 3; D020169:Caspases; D000242:Cyclic AMP; D004791:Enzyme Inhibitors; D009294:Narcotics; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011957:Receptors, Opioid; D000077191:Wortmannin