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

TL Yue, JM Stadel, HM Sarau, E Friedman, JL Gu, DA Powers, MM Gleason, G Feuerstein and HY Wang

Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.

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.

Volume 41, Issue 2, pp. 281-289, 02/01/1992
Copyright © 1992 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				S. L. Brown, V. R. Jala, S. K. Raghuwanshi, M. W. Nasser, B. Haribabu, and R. M. Richardson Activation and regulation of platelet-activating factor receptor: role of gi and gq in receptor-mediated chemotactic, cytotoxic, and cross-regulatory signals. J. Immunol., September 1, 2006; 177(5): 3242 - 3249. [Abstract] [Full Text] [PDF]

				Q. Zhuang, Y. Bastien, and B. D. Mazer Activation Via Multiple Signaling Pathways Induces Down-Regulation of Platelet-Activating Factor Receptors on Human B Lymphocytes J. Immunol., September 1, 2000; 165(5): 2423 - 2431. [Abstract] [Full Text] [PDF]

				J. Chen and S. N. Giri J. Pharmacol. Exp. Ther., June 1, 1997; 281(3): 1047 - 1058. [Abstract] [Full Text]

				M. Mori, M. Aihara, K. Kume, M. Hamanoue, S. Kohsaka, and T. Shimizu Predominant Expression of Platelet-Activating Factor Receptor in the Rat Brain Microglia J. Neurosci., June 1, 1996; 16(11): 3590 - 3600. [Abstract] [Full Text] [PDF]

				T. van Biesen, B. E. Hawes, J. R. Raymond, L. M. Luttrell, W. J. Koch, and R. J. Lefkowitz G(o)-protein alpha-Subunits Activate Mitogen-activated Protein Kinase via a Novel Protein Kinase C-dependent Mechanism J. Biol. Chem., January 19, 1996; 271(3): 1266 - 1269. [Abstract] [Full Text] [PDF]