Abstract
The lipid mediator platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) has been shown to elicit several important biochemical signaling responses in mammalian cells, including polyphosphoinositide hydrolysis, arachidonic acid release/eicosanoid production, and protein tyrosine phosphorylation. In the present study, the roles of Ca2+ and protein kinase C (PKC), two signaling components of the phospholipase C pathway, in AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation, were investigated in cultured rat Kupffer cells. AGEPC at nanomolar concentrations induced an increase in intracellular calcium concentration ([Ca2+]i), stimulated membrane PKC activity, and resulted in protein tyrosine phosphorylation. The maximal increase in [Ca2+]i and membrane PKC activity in response to AGEPC were observed within 30-50 s, whereas the AGEPC-induced protein tyrosine phosphorylation reached maximal levels within 2-5 min. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) but not 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of calcium release from intracellular compartments, nearly abolished the AGEPC-induced increase in [Ca2+]i suggesting involvement of extracellular calcium influx in this event. Both EGTA and TMB-8 abolished or inhibited AGEPC-stimulated protein tyrosine phosphorylation and eicosanoid formation, respectively. The calcium ionophore A23187 alone stimulated eicosanoid production and protein tyrosine phosphorylation with an identical pattern to that of AGEPC. Phorbol myristate acetate (PMA), an activator of PKC, which did not affect [Ca2+]i, mimicked the actions of AGEPC, stimulating eicosanoid production and promoting tyrosine phosphorylation of a set of proteins similar to those phosphorylated following AGEPC stimulation. AGEPC-enhanced tyrosine phosphorylation of some of the protein substrates and eicosanoid production were inhibited in cells "down-regulated" for PKC. Furthermore, both PMA- and AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation were attenuated or abolished by at least one of the PKC inhibitors, staurosporine, and calphostin C. Taken together, these results are consistent with the conclusions that: (a) AGEPC stimulates the phospholipase-mediated arachidonic acid release/eicosanoid synthesis cascade and protein tyrosine phosphorylation through extracellular Ca(2+)-dependent and PKC-dependent and -independent mechanism(s) and (b) the Ca(2+)-PKC interaction determines the efficacy of the AGEPC-stimulated cellular events.
Highlights
The lipid mediator platelet-activating factor (1-0- cascade and protein tyrosine phosphorylation through alkyl-2-acetyl-sn-glycero-3-phosphocholineA,GEPC) extracellular Ca2+-dependent and protein kinase C (PKC)-dependent and has been shown to elicit several importantbiochemical -independent mechanism(s) and ( b ) the Ca2+-PKCinsignaling responses in mammalian cells, including po- teraction determines theefficacy of the AGEPC-stimlyphosphoinositidehydrolysis,arachidonicacid re- ulated cellular events
Trhoeles of Ca2+and protein kinase C (PKC), two signalingcomponents of the phospholipase C pathway, in AGEPC-stimulated Platelet-activating factor is a phosphoglyceridewhich poscpesi(n[ueihcCcllolotraserse.2ydaai+AlinnsanIioeGttinrii)aEdo,scptnPier,mCpollrtwuuoaelltdieaanruntreecatdtnyiioomrnomnvesemoisnaltbaenirrgdpapancrhtooeeoctdnaesPcplicneKithniynuoCrtmrrocyaasultcilainttotciueinvoorsiernntadyc.itne,TKndahtnuurepdcaphetfmiodrfoaeesannr--xi-ctsmsheenoisl-slssgsetlps(yosoacefteeverniaoRttrs-ileei3fpbt-syiip.do1holmo-fo4sgbe,piidhcoifoalaoolctrghoeoirrcflefwaivelnacifeestu(swAnfoscvG)uti.iaEnosTdPpnheCsteoc)iin’bcfi(hedc5ei-1fm7f-)e0ri.cre-eAaacnlleGktpsytEttirolsPu-rs2scCu-teauwescrlhieeacitnicyotdhlsfmal increase in [Ca2’Ii and membrane PKC activity in usually are located on the surface of cells andare widely response to AGEPC were observed within 30-50 s, distributed in various tissues [8].Signal transduction between wheretahse
The results demonstrate and stimulation of PKC [48,49]. iIst of interest to determine that AGEPC stimulatesprotein tyrosine phosphorylationand whether calcium ionophore A23187-stimulated eicosanoid for- cyclooxygenase-derived eicosanoid production through extramation and protein tyrosine phosphorylation in Kupffecrells cellular Ca2+-and PKC-dependent and -independent pathwere secondary to PKCactivation
Summary
Effect of AGEPC on Intracellular Levels of Calcium in Cul- mediated protein tyrosine phosphorylation and eicosanoid tured Kupffer Cells-Intracellular Ca2+concentration, [Ca'+], production in Kupffer cells, the effect of PKC inhibitors on was quantitated by measuring the fluorescence intensity of the AGEPC-elicited responses was examined. The PKC inhibitor staurosporine at 1.0 p~ inhibited almost completely AGEPC-induced protein tyrosine phosphorylation. It was found that PMA caused more than a 10-foldincrease in the production of PGEz andTXB, (Table I), and the stimulatory effect of PMA was inhibited by calphostin C in a concentration-dependent fashion with an IC60of approximately 0.5 p~ (Fig. 6). Similar tothe AGEPC-stimulated protein tyrosine phosphorylation, the PMA-promoted tyrosine phosphorylation was inhibited almost completely by staurosporine and was only slightly inhibited by calphostin C (Fig. 7B).
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