Platelet-activating factor stimulates phosphoinositide turnover in neurohybrid NCB-20 cells: involvement of pertussis toxin-sensitive guanine nucleotide-binding proteins and inhibition by protein kinase C.
Platelet-activating factor (PAF) is an unusually potent phospholipid known to be produced by neuronal cells and to modulate cerebral blood flow and metabolism. In previous studies with NCB-20 cells, we reported that PAF induced a significant mobilization of intracellular free Ca2+ ([Ca2+]i), which was inhibited by PAF antagonists. The increase was the result of release from intracellular stores and influx from extracellular sources. The present study was designed to characterize further PAF receptor-mediated cellular signal-transduction mechanisms in myo-[3H]inositol-labeled cells. PAF induced a concentration-dependent increase in phosphatidylinositol (Pl) metabolism, with EC50 values of 1.96 +/- 0.62 nM and 1.12 +/- 0.50 nM for inositol trisphosphate (IP3) and inositol monophosphate (IP1) formation, respectively (four experiments). The maximal production of IP3 and IP1 induced by 50 nM PAF was 254 +/- 34% and 178 +/- 25% over the basal, respectively (four experiments). PAF-induced Pl metabolism was concentration-dependently inhibited by the PAF antagonist BN50739, with an IC50 value of 6.48 +/- 0.52 nM (four experiments). The protein kinase C (PKC) activator phorbol 12,13-dibutyrate concentration-dependently inhibited PAF-induced Pl metabolism and [Ca2+]i mobilization in NCB-20 cells, of NCB-20 cells with pertussis toxin (PTX) resulted in a concentration-dependent inhibition of PAF-induced IP3 production and intracellular Ca2+ release, with a maximal reduction of 66.9 +/- 3.5% and 63 +/- 6.1%, respectively, at 300 ng/ml PTX. PTX in the presence of [32P]NAD specifically [32P]ADP-ribosylated a 38-kDa protein in membranes prepared from NCB-20 cells. Pretreatment of the cells with PTX resulted in a concentration-dependent inhibition of subsequent 32P-labeling of the toxin substrate in the membranes and correlated with the uncoupling of PAF-induced IP3 formation. PAF (0.01-10 nM) elicited a concentration-related stimulation in guanosine 5'-O-(3-[35S]) triphosphate ([35S]GTP gamma S) binding to G alpha i(1,2) proteins, which was inhibited by the PAF antagonist BN50739. PAF at 10 nM also increased [35S]GTP gamma S binding to G alpha s and G alpha o. PAF-evoked activation of G alpha i(1,2) and G alpha o was reduced by preincubation with PTX. Our results reveal that neuronal cells possess PAF receptors linked through guanine nucleotide-binding proteins to phospholipase C and receptor-operated Ca2+ channels that are regulated by PKC. Both PTX-sensitive and -insensitive guanine nucleotide-binding proteins appear to couple the PAF receptor to activation of phospholipase C and the increase in [Ca2+]i. These results contribute to the further understanding of the mechanisms behind PAF actions on neuronal cells.
Yue
T L
TL
Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Stadel
J M
JM
Sarau
H M
HM
Friedman
E
E
Gu
J L
JL
Powers
D A
DA
Gleason
M M
MM
Feuerstein
G
G
Wang
H Y
HY
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000187:drug effects* / Q000502:physiology; D006822:Hybrid Cells / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D037342:Pertussis Toxin; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011493:Protein Kinase C / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013268:Stimulation, Chemical; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D008565:Membrane Proteins; D010716:Phosphatidylinositols; D010972:Platelet Activating Factor; D010566:Virulence Factors, Bordetella; D015240:Phorbol 12,13-Dibutyrate; D000244:Adenosine Diphosphate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D002118:Calcium