Multiple polyphosphoinositide pathways regulate apoptotic signalling in a dorsal root ganglion derived cell line

The polyphosphoinositides play important roles in transmembrane signalling but are also involved in anchoring cell surface proteins, organellar transp ort, cytoskeleton organization, and cell survival. The polyphosphoinositide s synthesized by phosphatidylinositol-3 kinase (PI-3K), (Ptd(3,4)InsP(2), a nd PtdIns(3,4,5)P-3), appear to play a critical role in cell survival by me mbrane recruitment and activation of Akt kinase. Inhibitors of PI3K, wortma nnin, 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 en hanced both caspase-3 activation and cell death induced by PI3K inhibitors, whereas a nonhydrolyzable cAMP analog (Bt(2)cAMP), lowered CPP32 and was p rotective We stably transfected the F-11 cells with the constitutively acti ve p110 catalytic subunit of PI-3 kinase and observed resistance to both ca spase-3 (CPP32) activation and subsequent apoptosis induced by either wortm annin or LY294002. Treatment of F-11 cells with bradykinin (BK) stimulated the hydrolysis of a different polyphosphoinositide, PtdIns(4,5)P-2, and enh anced both wortmannin-induced caspase-3 (CPP32) activation and subsequent a poptosis. PtdIns(4,5)P-2 is also a precursor of the anti-apoptotic PtdIns(3 ,4,5)P-3 and towering cAMP levels with opioid agonists for 30 min enhanced both the hydrolysis of PtdIns(4,5)P-2 and cellular apoptosis. The enhanceme nt was opioid dose-dependent and opioid antagonist (naloxone)- reversible a nd was also seen following 24-hr exposure to opioids such as U69,593 and Da la(2), Dleu(5) enkephalin (DADLE). However, unlike the bradykinin stimulati on of PtdIns(4,5)P-2 hydrolysis following activation of phospholipase C, th e opioid-enhanced hydrolysis was independent of external Ca2+ and was block ed by pertussis toxin, suggesting a different mechanism involving G(l), G(o ), or beta gamma-subunits. In summary, both the receptor-mediated lowering of cAMP levels and the hydrolysis of 4,5-polyphosphoinositides have no dire ct effect on caspase-3 activity or apoptosis but do exacerbate the activati on of caspase-3-like activity and subsequent cell death by apoptosis induce d by inhibitors of 3-polyphosphoinositide synthesis. We suggest that multip le polyphosphoinositide pathways are involved in the regulation of apoptosi s. (C) 2000 Wiley-Liss, Inc.