@Pergamon

P in Neurobiology, V o l5 0p p5 8t 5 91 9C o p y r i g h tG 1 9 9 6E l s e v i e rS c i e n cL t dA lr i g hr e s e

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PILS0301-0082(96)00047-O

P L A T E L E T - A C T I V A T I N GF A C T O RI T HC N

KARYN M. MACLENNAN*, PAUL F. SMITH* andCYNTHIA L. DARLINGTONt

* DP hS M S U O M SD N Z a ~ P a t N R C

U O D N Z

( J 1

A b s t r a c t - P l a t e l e t - a c t i v a t i n gf a c t o r(PAF)is a phospholipidsynthesizedin a varietyof cellsthroughoutt h eb o d y .P l a t e l e t - a c t i v a t i n gf a c t o rh a sb e e ni d e n t i f i e di nt h eC N Sa n dh a sa n u m b eo d i v e rp h y s i o l o g i c a la n dp a t h o l o g i c a lf u n c t i o n s .I th a sb e e ns h o w nt ob ea m o d u l a t o ro fm a nC Np r o c e s sr a n g i n gf r o ml o n g - t e r mp o t e n t i a t i o n( L T P )t on e u r o n a ld i f f e r e n t i a t i o n .E x c e s s i v el e v e lo P Aa p p et op l a ya ni m p o r t a n tr o l ei nn e u r o n a lc e l li n j u r y ,s u c ha st h a tr e s u l t i n gf r o mi s c h a e m i a ,i n f l a m m a t ih u m a ni m m u n o d e f i c i e n c ys y n d r o m e( H I V )a n dm e n i n g i t i s .T h eb e n e f i c i a le f f e c t so P Ar e c e pa n t a g o n i s t sa r em a n ya n dg i v er i s et op o s s i b l et h e r a p e u t i cs t r a t e g i e sf o rn e u r o t r a m u a .C o p y r i g h0 1 9E l s e v i e rS c i e n c eL t d .

CONTENTS1 .S y n t h e s i so fP A F2 .S t r u c t u r a ld i v e r s i t yo fP A F3 .P r e s e n c eo fP A Fi nt h eC N S4 .P A Fr e c e p t o r s5 .P A Fr e c e p t o rs i g n a lt r a n s d u c t i o n6 .C N Se f f e c t so fP A F

6 . 1 .C a l c i u m6 . 2 .I m m e d i a t ee a r l yg e n e s6 . 3 .L o n g - t e r mp o t e n t i a t i o n6 C e r e b r o v a s c u l a re f f e c t s6 . 5 .N e u r o p e p t i d e s6 . 6 .N e u r o n a lm o d u l a t i o n

7 .P A Fa n dn e u r o n a lc e l li n j u r y7 . 1 .I s c h a e m i a7 . 2 .I n f l a m m a t i o n7 . 3 .H u m a ni m m u n o d e f i c i e n c yv i r u s( H I V )7 . 4 .M e n i n g i t i s

8 .P A Fa n t a g o n i s t s8 . 1 .N a t u r a l l yo c c u r r i n gP A Fa n t a g o n i s t s8 . 2 .S y n t h e t i cd e r i v a t i v e sf r o mn a t u r a lp r o d u c t s8 . 3 .S t r u c t u r a la n a l o g u e so fP A F8 . 4 .S y n t h e t i cc o m p o u n d s

9 .C o n c l u s i o n sA c k n o w l e d g e m e n t sR e f e r e n c e s

ABBREVIATIONSA C T Ha d r e n o c o r t i c o t r o p h i ch o r m o n eI E GA P R L

i m m e d i a t ee a r l yg e n ea n t i - h y p e r t e n s i v ep o l a rr e n a ll i p i dL P Sl i p o p o l y s a c c h a r i d e

C B Fc e r e b r a lb l o o df l o wL T Pl o n g - t e r mp o t e n t i a t i o nC N Sc e n t r a ln e r v o u ss y s t e mN On i t r i co x i d eD A Gd i a c y l g l y c e r o l P A FF F A

p l a t e l e t - a c t i v a t i n gf a c t of r e ep o l y u n s a t u r a t e df a t t ya c i dP K Cp r o t e i nk i n a s eC

H I Vh u m a ni m m u n o d e f i c i e n c yv i r u sT N F - ut u m o u rn e c r o s i sf a c t o r - a

5555555555555555555555555

Benvenisteet a (1972)isolated a lipid factor released called this solublesubstanceplatelet-activatingfactorfrom immunoglobulinE-stimulated basophils which (PAF). Muirhead (1980) identified a similar lipidcaused potent aggregation of rabbit platelets. They factor in the kidney that lowered blood pressure,

which they named anti-hypertensivepolar renal lipid*Authorfor correspondence.Tel: ( 6 4 )3 - 4 7 9 - 7 2 5 3 ;F a x :(APRL). The two lipid factors were pursued

( 6 4 )3 - 4 7 9 - 9 1 4 0 ;e - m a i l :p a u l . s m i t h @ s t o n e b o w .o t a g o . a c . n z .s e p a r a t e l yu n t i l1979,when it was determined that

5 8 5

5 8 6K .M .M a c l e n n a ne ta

separately until 1979,when it was determined thatPAF and APRL had an identical chemical structure,1 - alkyl -2- acetyl-m-glycero-3- phosphocholine(Snyder, 1995).When syntheticpreparations of PAFbecame available, research increased and it wasdiscoveredthat PAF is produced in a variety of cellsthroughout the body; PAF can act at picomolarconcentrations and is now believed to have amultitude of pathologicaland physiologicalfunctions(for reviews,see Braquet a 1987;Venable a1993).Therefore,whilethe name PAF has remained,it has become rather misleadingand inappropriate.

1. SYNTHESIS OF PAF

Platelet-activating factor can be synthesized viatwo pathways (see Fig. 1), the n pathway andthe remodeling pathway, both of which have beenreviewedextensively(seeSnyder, 1994,1995).Then pathway begins with l-alkyl-2-lyso-sn-glycero-3-phosphatewhich, through a seriesof reactions, canbe converted to PAF or complex membrane etherlipids. The conversionto PAF takes three enzymaticsteps. Firstlyj it is acetylated by an acetyltransferaseto form alkylacetylglycerol-phosphate. Followingdephosphorylationby a phosphohydrolase,choline-Pis added by the dithiothreitol (DTT)-insensitivecholinephosphotransferase to form PAF (Snyder,1994).

Alternatively, the synthesis of PAF via theremodeling pathway requires the conversion ofl-alkyl-2-acyl-sn-glycero-3-phosphocholineinto lyso-PAF, the immediate precursor of PAF. For manyyears, it was believed that phospholipase Az wasdirectly responsiblefor the production of lyso-PAF,however, such activity has never been provenexperimentally.Recent resultshave demonstrated theinvolvementof a coenzymeA-independenttransacy-lase, in the production of lyso-PAF (Uemura a1991).Phospholipid A, generates the release of thefree polyunsaturated fatty acid, arachidonic acid,from plasmalogensor glycerophospholipidsand thes acyl group from l-alkyl-2-acyl-sn-glycerol-3-phosphocholine (the membrane-bound precursor ofPAF), is then transferred to form lyso-PAF.The finalstep is catalysed by a specific acetyl-coenzymeA:lyso-PAFacetyltransferase,and involvesthe additionof an acetate to the lyso-PAF to form PAF. Thedegradation of PAF requires its deacetylation byPAF acetylhydrolase to form lyso-PAF. This lipidlacks biological activity and is either modified intoother lipidproducts within the cellor secretedoutsidethe cell (Snyder, 1994).

The properties of enzymes involved in these twopathways differ from each other notably. Perhaps acritical difference,however, is that the remodelingpathway is onlyinitiated whenthe cellsare stimulatedby a particular agonist such as calcium or thrombin(Imaizumi a 1995). Enzymes in the npathway are not activated by such inflammatorystimuli. Snyder has proposed, therefore, that then pathway is involved in the production of PAFto sustain physiological levels and the remodelingpathway may be responsiblefor the abnormal levels

of PAF occurring in pathological conditions (Yueand Feuerstein, 1994).

PAF is thought to be synthesized within cellularmembranes, therefore, the movement of PAF frommembrane to membrane as it travels to its intra- orextracellular destination is of interest. Membranetransfer of PAF has been shown to occur via acytosolicprotein-catalysedprocessand rapid transfercan occur between phospholipid vesicles or or-ganelles.The mechanismsof cell surface release andsecretionof PAF are not well understood at present,although a protein named PAF-releasing factor hasbeen characterized recently (Snyder, 1995).

2. STRUCTURAL DIVERSITY OF PAF

It has nowbeenestablishedthat PAF is not a singleentity but is made up of a family of s acetylated,choline-containingphosphoglyceridesthat are struc-turally related but differmarkedlyin biologicalactionand potency (Pinckard a 1994).PAF has beenseparated into three subclasses, alkyl-, alkenyl- andacyl-PAF, on the basis of biologically significantdifferences in the chemical linkages of the fattyacid/alcohol residuesin the sn-1 position of the PAFglycerolbackbone. Amongthese subclasses,there arefurther molecular species of PAF that bear sn-1chains of varying length and degree of saturation(Pinckard et a 1994).

A considerable amount of PAF research to datehas concentrated primarily on two molecular speciesof PAF (16:0- and 18:0-alkyl-PAF),including thedevelopment of PAF receptor antagonists. Atpresent, the biological activities and importance ofother members of the PAF family remain to bediscovered.The molecular composition of PAF can

I REMDDELLINGPA’PHWAY I

IRIC+PIKJIP=Az-rdeneoffreeptyuwur.idhllyadds

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1-tiyl-2-lymn-@y_%p~h&oti(lysOPAP)

IA & i i t i m o f m i a t e tia.tyl.m.4:1ywPAFacetyleamferaw

l-alkyI-2-acetyl-sn-glycerc-3-phaephocholine

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1-dkyl-2-lysemgly-2-phosphate(Anintermdiateinthesynawisofethdinkdrmlbme phqholipids)

DENOVOPATHWAY

F i g .1 .T h eP A Fb i o s y n t h e s i sv it hr e m o d e lan p a t h w a y s

P A F i nt h eC N S 5

vary depending on cell or tissue type and the natureof the activating stimulus. There are also differencesin the composition of PAF produced in the same celltype from different species. This should be animportant considerationwhenapplyingfindingsfromanimal models of diseaseto humans (Pinckard et a1994).

3. PRESENCE OF PAF IN THE CNS

It has been demonstrated that the rat brainpossessesall the enzymesrequiredfor the synthesisofPAF both by the n and the remodelingpathways (Goracci and Francescangeli, 1991). En-zymes involved in PAF generation have also beenfound in rabbit cerebral cortex (Baker and Chang,1993)and human brain astrocytes(Stephensonet a1994).

In a quantitative study of PAF in the rat brain,Tokumura et a (1992)discoveredan age-dependentdecrease in PAF levels beginning at 8 weeks.Tiberghien a (1991)used a radioreceptor assay toquantify levels of PAF and lyso-PAF in differentareas of rat brain. While PAF levels were low,lyso-PAF levelswere very high in the hippocampuswith lower levels in the cerebellumand cortex.

PAF production has been demonstrated incultured rat cerebella granule cells (Yue et a1990),and human foetal neurons and glial cells inculture (Sogos et a 1990). PAF synthesis inneurons and glial cells is increased followingacetylcholinestimulation and loweredby atropine, amuscarinic cholinergic receptor antagonist. Kumaret a (1988)have also evaluated synthesisof PAF inrat brain and found that, whilePAF basal levelswerelow, levels could be increased by injection ofchemoconvulsant drugs and electroconvulsion.Thepresence of PAF in the CNS, therefore, has beenclearly demonstrated, however, it is more prevalentin its inactive form.

4. PAF RECEPTORS

Although PAF is a phospholipid, surprisingly itexerts its effectsvia specificreceptors. PAF receptorshave been identifiedin a number of cellsand tissues,however, CNS PAF receptors were not discovereduntil the late 1980s(see Chao and Olson, 1993,for areview).Junier a (1988)discoveredthe presenceof two populations of PAF binding sites in rat brainhypothalamic membranes. The populations weredistinguished by high and low affinities. Hosford

a (1990)investigatedthe presenceof PAF bindingsites using gerbil brain membrane homogenates.Upon examining discrete brain areas, they foundmaximal specific binding in the midbrain andhippocampus,with lessbindingin the olfactory bulb,frontal cortex and cerebellum. Three distinct PAFbinding sites are present in rat cerebral cortex, twohigh affinity intracellular sites on microsomalmembranes and one low affinitybinding site locatedin synaptic plasma membranes (Marcheselli et a1990). High and low affinity PAF binding popu-

lations also have been reported in neuroblas-toma x glioma hybrid NG 108-15cells (Chau et a1992).Activation of the high affinityPAF receptorsin these cells led to polyphosphoinositide turnover,whereas low affinity receptor activation resulted incalcium influx from extracellular stores.

Following much difficulty in determining thestructure of the PAF receptor, it was first cloned byHonda a (1991) using guinea pig lung. Thereceptor has also been cloned in human tissue (Yeet a 1991)and is predicted to be a member of the‘serpentine’ receptor family, due to the proteinspanning the membrane seven times, as has beendemonstrated for G-protein-linked receptors(Shukla, 1992).

The cloning of the PAF receptor provided a newtool for investigatingits existencein the CNS. PAFreceptor mRNA have beendetected in the hypothala-mus, medulla-pens, olfactory bulb, hippocampus,cerebralcortex, spinal cord, thalamus and cerebellumin rat brain. Expressionof the PAF receptor is seenin both neuronal and glial cells and on hippocampalneurons some PAF receptors co-localize withIV-methyl-D-aspartate(NMDA) receptors(Bito a1992).Specificbinding sites for PAF have sincebeenlocalized on differentiated neuroblastoma NIE-115cells (Lalouette a 1995)and there is evidencefora functional PAF receptor in microglial cells (Righi

a 1995).

5. PAF RECEPTOR SIGNAL TRANSDUCTION

Following receptor activation, PAF stimulatespolyphosphoinositide (PPI) turnover via phospho-lipase C, phospholipaseA, and phospholipaseD. Asa result, inositol trisphosphate (IP~)is generated andservesas a second messengermobilizingintracellularcalcium. A concomitant increase in diacylglycerolsoccurs, leading to activation of protein kinase C(PKC). Phosphoinositide breakdown also activatestyrosine kinase. These processeshave been shown tobe receptor-dependent,as they are inhibited by PAFreceptor antagonists (Shimizu a 1992;seeFig. 2).

Asidefrom the structure of the PAF receptor beingsimilar to that of G-protein-coupledreceptors, manystudies have indicated that G-proteins play a role inPAF receptor signal transduction. PAF stimulatesGTPase activity (the hydrolysis of the activeguanosine 5’-triphosphate (GTP) to the inactiveguanosine5’-diphosphate(GDP)), GTP causesa shiftin PAF binding (Shukla, 1992)and an inactive GTPanalogue injected into oocytes has been shown toinhibit over Too/o of PAF-elicited chloride currents(Shimizu a 1992). While it has been demon-strated that the PAF receptor is coupled to effectersystems through G-proteins, the identities of theG-proteins involvedhave not been characterized andmay differ from cell to cell (Izumi a 1995).

6. CNS EFFECTS OF PAF

6.1. Calcium

Subsequentto findingthat PAF acts on the clonedneuronal cell linesNG108-15and PC12,raising levels

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1 SIGNALLINGPATISWAYS

“ m ” g p h ” ’ p = c‘w’b”v” - — [C.z+]i— PKC

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MODULATSONOF

‘[Ca2+]i.IEGtmmnptiori.LTP.CerSmdblcalflowandmetaholim.NeUmpepdda.Neumnaldiffemmiatim.Neumuaumdtlerrdeaw. C y t o k i n e s

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m o d u l a t i o no fv a r i o u sC N Sp r o c e s s e s .

of intracellular calcium and increasing dopaminerelease,Kornecki and Ehrlich (1988)investigatedtheeffect of PAF on dissociated cells obtained fromembryonic mouse CNS. In neostriatal and corticalcells loaded with aequorin, PAF induced a dose-de-pendent increasein calciummobilizationat micromo-lar concentrations. PAF also increased calcium inadult rat brain synaptosomes at concentrations aslow as 9.9 nanomolar (Kornecki and Ehrlich, 1991).

PAF-induced calcium increase also has beendemonstrated in neurohybridNCB-20cellspreloadedwith the calciumindicator Fura-2 (Yue et a 1991a).PAF applicationproducesa fast, transient increaseincalcium, followed by a decline to basal level. Thisconcentration-dependenteffectwas inhibitedby PAFreceptor antagonists. The calcium channel blockers,nifedipineand diltiazem,did not affectPAF-inducedcalcium elevation, however, the increase was attenu-ated by the intracellular calcium blocker, 8-(N,N-di-ethylamine)octy13,4,5-trimethoxytenzoatehydrochloride (TMB-8). Following depletion ofextracellular calcium, the PAF-generated increase incalciumwas largelyreduced. It seems,therefore, thatPAF increases calcium primarily via receptor-oper-ated calcium channels and to a lesserextent throughrelease from intracellular stores. These results alsohave been demonstrated in neurohybrid NG108-15cells (Yue a 1991b) and bovine cerebralmicrovascular cells (Lin and Rui, 1994).

6.2. ImmediateEarly Genes

Immediate early genes (IEGs) undergo fast andtransient activation via numerous extracellularsignals.The IEGs studied in the CNS include cc and k (alsoknown as zz~/268),whichgiverise to proteins that regulate the transcription ofother genes responsible for long-term changes incellular function (Doucet and Bazan, 1992).Squinto

a (1989)discovereda PAF-induced activation ofc and c in human SH-SY5Y neuroblastomacells. PAF (1 KM) elicited rapid and transienttranscription of these IEGs, with c-~osmRNA levelsincreasingsevenfoldwithin 15min and c mRNAelevating eight-fold in 60 min. PAF-induced IEGexpressionis inhibitedby pre-treatment with the PAFantagonist, ginkgolide B. PAF can activate genetranscription through an AP-1 transcriptional el-ement. Followingdeletion of the c promoter, forPAF to activate c expression, generation ofadenosine 3’,5’-cyclicmonophosphate (cAMP) and/or increasedintracellularcalciumis required (Squintoet a 1989).

Albani a (1993)investigatedc and kexpression in rat astroglial cell cultures followingtreatment with PAF and a PAF analogue, l-O-oc-tadecyl-2-O-methoxy-glycero-3-phosphocholine(ET-18-0CH3). Both compounds (2 pM) produced arapid and transient elevation of c and kmRNA, an effect almost completely inhibited bypre-treatment with a PAF receptor antagonist.Pre-administrationof the PAF antagonist, BN-50730,has been shownto inhibit electroconvulsiveshock-in-duced expression of the IEGs, c and k(Marcheselli and Bazan, 1994).The results of thesestudies suggest that PAF is involved in short- andlong-termresponsesof cellsto stimulation or trauma.

6.3. Long-TermPotentiation

Long-term potentiation (LTP) is a long-lastingenhancement of the postsynaptic response of aneuron as a result of increased synaptic input and isbelieved to be a cellular mechanism for memoryformation (Bliss and Collingridge, 1993). Theinduction of LTP occurs postsynaptically, therefore,a retrograde messengeris required to travel from thepostsynaptic cell to the presynaptic cell in order tosignal the increase in transmitter release which is thebasis of LTP maintenance. The latest candidate forthis messengeris PAF (Goda, 1994;Kato a 1994;Medina and Izquierdo, 1995).

The first indication that PAF was involvedin LTPoccurredwhen Del Cerro a (1990)found that thePAF antagonist, 2,5-bis(3,4,5-trimethoxyphenyl)-l,3-dioxolane(BTD), inhibited the developmentof stablepotentiation. It has been found since that PAF caninduce LTP in hippocampal slices, an effectblockedby pre-administration of the high affinity PAFreceptor antagonists, BN-52021 and WEB-2086(apafant). PAF induction of LTP prevents sub-sequent high frequency stimulation-induced LTP,and vice versa, suggestingthe two have a commonpathway ~ieraszko a 1993). Recently, Kato

a (1994)have shownthat the involvementof PAFin LTP is specificto receptors localized to synaptic

P A Fi nt h eC N S 5

regions. When applied to hippocampal slices, thesynaptosomal PAF antagonist, BN-52021, blocksLTP, whereas application of the microsomal PAFantagonist, BN-50730,has no effecton postsynapticenhancement. Upon postsynaptic application ofMC-PAF, a non-hydrolysable PAF analogue, anincreasein transmitter releaseoccurs,and is inhibitedcompletely by extracellular administration of BN-52021.From these results, PAF appears to have allthe requirements of a retrograde messenger.

Consistent with this is the finding that the PAFanalogue, carbamyl-PAF (c-PAF), enhances excit-atory postsynaptic currents (epscs)in rat hippocam-pal neurons (Clark e~a 1992;Bazan et a 1993).c-PAF has no effect on inhibitory postsynapticcurrents (ipscs) and enhanced synaptic transmissioncan be blocked by BN-52021.PAF has also beenshown to cause depolarization of some myentericneurons in culture (Willard, 1992), although. ac-PAF-induced, dose-dependent inhibition of epscshas been reported in a patch-clamp investigation ofthe neostriatum (Jiang et a 1993).Further supportfor PAF involvementin LTP is the reported memoryenhancement in behavioral tasks followinginfusionof MC-PAF into the hippocampus, amygdala andentorhinal cortex (Izquierdo et a 1995).

6.4. CerebrovaacularEffects

Following 15min of intracarotid infusion of PAF,cerebral blood flow (CBF) decreases and the globalcerebral metabolic rate for oxygen (CMR02) iselevated (Kochanek a 1988). Similarly, PAFadministration can cause a dose-dependentdecreasei ns p i n a lc o r db l o o df l o w ,a ne f f e c tb l o c k e db yt h ePAF receptor antagonist WEB-2170 (Faden andHalt, 1992).To overcome difficultiesin quantifyingPAF concentrations, Kochanek a (1990) em-ployed the PAF receptor antagonists, BN-52021andWEB-2086, to investigate the role of endogenousPAF on CBF and CMROZ.Neither drug had anyeffect on these variables, indicating that, undernormal circumstances, levels of PAF are belowthreshold and do not modulate CBF or metabolism.

PAF’s influence on CBF is unlikely to occur viadirect action on brain microvasculature,sincethere isno apparent effect on lumen diameter of ratintracerebral arterioles v (Edwards a 1991).In the isolated, perfused rat brain, synthetic PAFperfusion causes no change in cerebral metabolism,however, a decreased perfusion pressure and in-creased lactate output has been observed.WhilePAFdoes not cross the blood–brain barrier (BBB)itself,these changes are possibly due to a PAF-inducedchange in BBB permeability (Kumar a 1988).Administration of PAF to cerebral microvesselsresults in an elevation of protein kinase C (PKC) inmembrane fractions, and a decreasein cytosolicPKClevels.The ability of PAF to induce translocation ofPKC may be involved in PAF-induced changes inBBB permeability (Catalan a 1993).

6.5. Neuropeptides

PAF has been shownto elevateplasma adrenocor-ticotrophin levels following intra-third ventricular

(Hashimoto a 1993), intra-lateral ventricular(Rougeot et a 1990)and intravenous (Bernardino

a 1989) administration in rat. PAF adminis-tration can also increaselevelsof plasma ~-endorphinand corticosterone (Rougeot a 1990),although,long-term treatment with PAF via a cannulaconnected to the jugular vein results in decreasedlevelsof plasma and adrenal corticosterone(Blasquez

a 1990).Central administration of the neuropep-tide, a-melanocyte-stimulating hormone (a-MSH),prevents the PAF-induced inflammation that occursfollowingintradermal PAF injection (Ceriani a1994).PAF has been shown to inhibit the release ofluteinizinghormone-releasinghormone (LHRH) andsomatostatin from rat median eminence, but notanterior pituitary (Junier a 1988).

It has been reported that long-term PAFadministration can cause a reduction in adrenocorti-cotrophic hormone (ACTH) concentration in theanterior pituitary v This effectwas reversedbyconcomitant treatment with BN-52021 and asignificant ACTH elevation was seen in the distallobe. PAF had no effect, however, on anteriorpituitary fragments v when applied atmicromolar concentrations (Blasquez a 1990).Bernardinoet a (1989)found an increase in ACTHsecretion from cultured pituicytes which could beinhibited by the PAF antagonists, alprazolam andBN-52021.The differencein these findings may bedue to the PAF concentrations used, since increasedACTH levelswere observed only with a nanomolarconcentration.

It has been demonstrated that, followinginjury tothe meninges, mRNA levels for the neuropeptidecholecystokinin are enhanced in the underlyingcortex. Gotz a (1993)have shown that injectionof the PAF antagonists, WEB-2086or brotizolam,30 min before injury in rats, results in a significantdecrease in cholecystokinin mRNA. These resultsindicate that PAF is involved in the change inneuropeptide gene expressionfollowingmeningocor-tical damage.

6.6. N M

P may act as a neuromodulator in thecholinergic system, since its application inhibits thepotassium-evokedrelease of acetylcholine (ACh) inrat brain slices of cortex and hippocampus. Thiseffect is blocked by various PAF antagonists,pertussis toxin and anti-Gai,l, antiserum, indicatingthat ACh release is inhibited via a Gai,,j protein-me-diated action (Wang a 1994).

PAF has been shown to regulate neuronaldifferentiation in enriched cultures derived from ratcerebra (Veal a 1991).Following application of4 pM PAF to culture medium, neuronal growth andan increase in dendritic and axon-like developmentwas observed. A dose-dependent increase in theactivity of the neuron-associatedenzyme acetylchol-inesterase, and increased total protein content, wasalso noted. Stimulation was inhibited by the PAFantagonist, triazolam, and there was no apparentplasma membrane damage.

PAF-induced neuronal differentiation has alsobeen found in cultured cells of the cholinergicclone,

5 K .M .M a c l e n n a na

NG108-15.Application of PAF for 24 days resultedin arrested growth, morphologicaldifferentiationandneurite extension. These processes were concen-tration- and time-dependent, with the maximallyeffectivedose being 2.5 ~M (Kornecki and Ehrlich,1988).Higher doses (3.5–10pM) were found to beneurotoxic; however,Lustig et a (1992)found that10VM PAF applied to cortical cultures resulted inonly a gradual, sustainedrise in intracellular calcium.

Inclusionof PAF in the mediumcontainingmurineneuroblastoma NM-2a cells results in an earlyinduction of neurite outgrowth, although, within2,hrthe number of cellswith neurites is similarto controls(Herrick-Davis a 1991). PAF also has beenreported to stimulate nerve growth factor (NGF)secretion in enriched astrocyte cultures. Stimulationof this neurotropic factor was blocked by the PAFantagonist, WEB-2086.Concomitant administrationof PAF and tumour necrosisfactor-a (TNF-a) led toa synergistic elevation of NGF, suggesting aninteraction betweenPAF and TNF-a (Brodie, 1995).

7. PAF AND NEURONAL CELL INJURY

7.1. Ischaemia

It has been proposed that oxygen free radicalattack on phospholipids containing a polyunsatu-rated fatty acyl residuein the WI-2position, results infragmentation of the residue, leading to theunregulated production of mediators that have theapproximate structure and mimic the biologicalaction of PAF (Zimmerman et a 1995). Theseproducts, which are lesspotent than PAF, effectivelystimulate the PAF receptor and their effects areinhibited by PAF receptor antagonists and PAFacetylhydrolase.Suchphospholipidshave beenfoundin injured brain extracts (Tanaka a 1993),therefore it is probable that, in conditions such asischaemia,both PAF and PAF-likephospholipidsareof importance (Imaizumi a 1995).

There is a considerable amount of researchindicatingPAF involvementin ischaemicinjury,withmuch indirect evidence coming from the beneficialeffectsof PAF receptor antagonists (for reviews,seeLindsberg a 1991;Yue and Feuerstein, 1994).Following bilateral carotid artery occlusion in thegerbil, the PAF antagonists, BN-52021,BN-52020,BN-52022 and BN-52024, improved stroke indexscores (determined by symptoms ranging frompiloerection to seizures) and mitochondrial respir-ation when administered prior to, or at the time of,the occlusion. Administration of BN-52021or twounrelated PAF antagonists, kadsurenone and broti-zolam, 1 hr post-occlusion,also resultedin reversalofcerebral impairment (Spinnewyn a 1987).

During ischaemia,the activation of phospholipasesA, and C resulted in free polyunsaturated fatty acid(FFA) and diacylglycerol (DAG) accumulation,respectively.Panetta a (1987)have demonstratedthat BN-52021, administered at the onset ofreperfusion, leads to an improved cerebral bloodflow, a reduction in FFA accumulation andconsistently lower (although not significantlylowerstatistically), levels of DAG. Attenuation of FFA

accumulation has also been reported in rat followingtreatment with BN-52021(Birkle a 1988) andBN-50739(Sun and Gilboe, 1994).

The PAF receptor antagonists BN-50726 andBN-50739, promote metabolic recovery duringpost-ischaemic reperfusion in dogs (Gilboe a1991).A similarbeneficialeffecthas been observedinrats pre-treated with the ginkgolide-containingGinkgob extract, EGb-761, following hypoxia(Karcher et a 1984)and unilateral embolization(LePoncin Lafitte et a 1980). (Na+ ,K+) ATPaseactivity is believed to be reduced during ischaemiaand it is possible that this effectis mediated by PAF.PAF inhibits (Na+ ,K+) ATPase activity in thecerebral cortex both dose- and time-dependently.This inhibition is blocked by the PAF antagonist,PCA-4248(Catalan a 1994).

Ischaemia-induced neuronal damage is reducedfollowing treatment with the PAF antagonist,TCV-309,prior to cold brain injuryin rats (Tokutomi

a 1994). In rats with middle cerebral arteryocclusion, infarct volume reduction was observedfollowingpre- or post-occlusionadministration of thetriazolobenzodiazepine, apafant. This protection islikelyto occur via direct action on tissue as there wasno effect on local cerebral blood flow (Bielenberg

a 1992).Combined pre- and post-treatment withginkgolidesA and B in rats with transient forebrainischaemia, resulted in reduced neuronal damage inthe hippocampus and neocortex, These drugs alsoreducedexcitotoxicdamage in cultured chickembryotelencephalic neurons overexposed to glutamate(Prehn and Krieglstein, 1993).

Cerebral ischaemia results in increased levels ofPKC and an overexpressionof ornithine decarboxy-lase (ODC). Zablocka a (1995)demonstrated thatpre-administration of the PAF receptor antagonist,BN-52021,attenuates the post-ischaemicinduction ofPKC and ODC. Theseproducts werealso blocked byadministration of the NMDA receptor/channelantagonist, ([ + ]-5-methyl-10,1l-dihydro-5H-diben-zo[a,d] cyclohepten-5,10-iminemaleate) (MK-801)and the nitric oxide (NO) synthase inhibitor,N-nitro-L-arginine methyl ester (L-NAME).Zablocka a (1995)suggest that NMDA receptoractivation may stimulate the releaseof NO and PAF.In turn, PAF and NO could mediate the ischaemia-inducedincreasein PKC and ODC by amplifyingthecalcium signal generated by NMDA receptoractivation.

In a clinical study, Satoh a (1992)found thatblood PAF and PAF-like lipid levelsappeared higherin patients who had undergone ischaemic stroke,when compared to age-matched, healthy controls.Recently, a study quantifying levels of PAF andlyso-PAF in the ischaemicbrain was carried out byNishida and Markey (1996). Following bilateralcarotid artery occlusion in gerbils, PAF levelsincreased in the hippocampus, cortex and thalamusafter 1 hr, but not 6-96 hr, of reperfusion.

Pettigrew a (1995)employed v microdialy-sis to examinelevelsof thromboxane and PAF in thehippocampal extracellular space of ischaemic rats.Thromboxane levels maximally increased at 20 minpost-ischaemia; this was followed by delayed actionof PAF at 140min post-ischaemia. These authors

P A Fi nt h eC N S 5

concluded that PAF is releasedinto the extracellularspace from neurons with dysfunctional plasmamembranes and thromboxane synthesis may beenhanced by intracellular PAF. Recently, it has beendemonstrated that antagonists of PAF (Y-24180)andthromboxane (S-1452)attenuate the increased pro-duction of oxygen-free radicals during ischaemiareperfusion (Matsuo a 1996).

A reduction in neurological deterioration due totraumatic brain injury or subarachnoid hemorrhagehas also been reported following administration ofthe PAF antagonists, BN-52021 or WEB-2170(Faden and Tzendzalian, 1992) and CV-6209(Hirashima a 1993),respectively.Past research,therefore, indicates an important role for PAF in thepathophysiology of ischaemia and other braintraumas. The ability of PAF to modulate apoptosisin a human T cell line (Azzouzi a 1993)raises thepossibility of a PAF role in programmed cell deathas well as necrosis.

7.2. Inflammation

There is substantial evidencesupportingthe role ofPAF as a regulator of cytokines in inflammatoryresponses (see Bonavida and Mencia-Huertaj 1994for a review).TNF-a is a cytokinecapable of causingnecrosis and regression of tumours. Hunyadi a(1993) examined the effect of the PAF receptorantagonists, BN-52021 and BN-50730, on TNF-ctcytotoxicityin murine L929tumour cells.As proteaseinhibitors block the synthesisand releaseof PAF, theproteinase inhibitor, aprotinin, was also tested. Allthree drugs dose-dependently inhibited TNF-ct-in-duced cytotoxicityin this model, indicating that thisprocessis dependenton the production and releaseofPAF.

In order to determine whether stroke risk factorsare associated with an increase in release ofpro-inflammatory mediators, Siren a (measured CNS levels of TNF-ct and PAF in youngand old rats. Following intracerebroventricular(i.c.v.) administration of lipopolysaccharide (LPS),an activator of cytokine- and PAF-producing cells,both TNF-Ix and PAF levels increased moresignificantly in rats with the stroke risk factor ofadvanced age. The authors propose that a possiblereason for the increased levels in older rats is toprepare the endothelium for thrombosis or hemorr-hage.

NO is generated by the enzyme NO synthase(NOS), which is induced by various cytokines andLPS. Excessproduction of NO has been associatedwith hypotension and endotoxic shock (Dominiczakand Bohr, 1995). The PAF receptor antagonists,BN-50739and E6123, have been shown to reduceLPS-induced NOS activity in cultured mousemacrophages.While PAF alone had no effecton theactivity of NOS, a combination of LPS and PAFresulted in enhanced LPS-induced NOS activity(Arthur a 1995).Inhibition of LPS-inducedNOSalso has been demonstrated v using the PAFreceptor antagonist, WEB-2086(Szabo a 1993).

PAF stimulates leukotriene synthesis, an actionassociated with the pathogenesis of inflammatoryprocesses.Hyneset a (1991)investigatedwhether or

not this pathogenic process also occurs in the catCNS. Firstly, it was determined that pepti-doleukotrienes, primarily LTC,, are found incerebrospinal fluid (CSF). It was then determinedthat these levels could be increased by i.c.v.administration of PAF, an effectreversedby the PAFantagonist, BN-52021.

7.3. Human ImmunodeficiencyVirus (HIV)

Direct HIV-1 infection of the brain results inneuropathogenesis known as HIV-l-associated de-mentia complex. This complex, associated withHIV-1 infected cells, astrocyte proliferation andneuronal loss occurring in the absence of viralinfection of neurons, results in cognitive and motordysfunction. It is possible that HIV-l-infectedmacrophages secrete products which are responsiblefor this neurotoxicity (for a review, see Dewhurst

a 1996).To investigate whether PAF is a neurotoxin

involved in HIV-1 infection, Gelbard a (1994)measured PAF levels in co-cultures of HIV-infectedmonocytesand astroglia. At varying times followingco-culture, PAF was quantified using radioimmuno-assay and elevatedPAF levelswere observed from 3to 20 min. Following this finding, Gelbard a(1994)compared PAF levels in the CSF of HIV-1infected patients with those of controls. PAF wasfound to be increasedin HIV-l-infected patients withimmunosuppressionand signsof CNS dysfunction.Itis of interest that PAF levels were also high inpatients with other medical ailments such as multiplesclerosis,leukaemia and disseminated cancer, whereCNS symptoms were also occurring.

In order to determinewhether elevatedPAF levelsplay a role in CNS dysfunction,Gelbard a (1994)applied PAF to neuronal foetal cultures, atconcentrations greater than or equal to thoseobserved in the co-cultures. These concentrations(5&6000pg/ml) were toxic in a dose-dependentmanner, an effect partially blocked by the NMDAantagonist, MK-801.

From this study it appears that PAF is anHIV-l-induced neurotoxin, a notion further sup-ported by Nottet a (1995),who demonstrated anoverexpressionof PAF by LPS-stimulated,HIV-l-in-fectedmonocytes.In this study, it was also found thatmonocyte secretionis down-regulatedby addition ofprimary human astrocytes, an action attributed toscavengingability rather than secretionof regulatorymolecules.

As elevated PAF levels are found in the CSF ofHIV-l-infected patients and it is likely that PAFplays a role in HIV-associated dementia complex,Nishida a (1996)investigatedCNS PAF levelsinmice infected with the LP-BM5 murine leukaemiavirus. These mice develop murine AIDS andencephalopathycharacterizedby spatial learning andmemory impairments.

PAF levelswere elevated in the frontal cortex andhippocampus, at both 6 and 12weeks post-inocu-lation, and in the striatum and cerebellum at12weeks post-inoculation. It was proposed that, asconcentrations of the NMDA receptor agonist,quinolinic acid, are increased in the brain during

5 K .M .M a c l e n n a ne [a l .

murine AIDS, an increase in intracellular calcium,resulting from NMDA receptor activation, couldlead to the synthesisof PAF. To investigatethis, ratsreceived the NMDA receptor/channel antagonist,MK-801,via a minipump for 5 days. Levelsof PAFwere significantlyreduced and it was concluded thatNMDA receptor activation may be required forincreased PAF levelsin LP-BM5 murine leukaemia-infected mice.

PAF increasesin the brain have also beenobservedin guinea pigs followinginfection with the Pichindevirus, a virus that may lead to fever, electrolyteimbalance, body wasting, cardiopulmonary dysfunc-tion and death (Guo a 1993).On post-infectionday 12, PAF levels had increased by 81, 110 and147~0 in the cerebrum,cerebellumand diencephalon-brainstem, respectively,indicating that PAF may, atleast in part, be responsible for the Pichinde virussyndrome

7.4. Meningitis

During bacterial meningitis, the pathophysiologi-cal changes that occur lead to cerebral dysfunction.It is likely that CNS damage is induced by thebacterial products and the inflammatoryresponsesbythe host. As the effects of LPS, contained in thebacterial cellwall, are mediated by TNF-cYand PAF,concentrations of these factors were measured in theCSF of children with bacterial meningitis (Arditi

a 1990). On average, PAF levels weresignificantlyelevated in children with meningitis ascompared with age-matchedcontrols. Levelsof PAFwere stronglycorrelated with concentrations of LPS,TNF-a and bacterial counts. These results indicatethat PAF may play a role in the inflammatory

responseand resultingCNS damage during bacterialmeningitis.

The bacterium S p can causesepsis,pneumoniaand meningitis.Pneumococcuscanadhere to endothelial cells, but is only observedintracellularly following inflammatory activation ofthese cells. Cundell a (1995) investigated howinflammatory activation brings about this transloca-tion. It was found that activation led to expressionofPAF and PAF receptors and internalization ofpneumococciwas inhibited by the PAF antagonist,WEB-2086.Theseauthors believethat this is the firstinstance of a bacterium entering a eukaryotic cellaided by a G-protein-coupledreceptor, and proposethat the PAF receptor is critical for the progressionof pneumococcal infection.

8. PAF ANTAGONISTS

Consideringthe various pathophysiologicaleffectsof PAF, the possible clinical applications of PAFantagonists are many. PAF receptor antagonists canbe separated firstly into natural and syntheticcompounds,and then further on the basisof chemicalstructure (see Table 1). This reviewwill not attemptto describe all of the currently known PAF receptorantagonists; there are a large number of PAFantagonists and they have been reviewedextensivelyby others (Koltai a 1994;Hosford and Braquet,1990).

8 N O P A

Ginkgolides are terpenoids found in the leaves ofG b an ancient tree which, for centuries,the Chinesehave held sacredfor its health-promoting

T a b l e1 .P A FR e c e p t o rA n t a g o n i s t sa n dT h e i rO r i g i n s

A n t a g o n i s tt y p eO r i g i nP h a r m a c e u t i c a lc o m p a

1 .N a t u r a l l yo c c u r r i n gP A Fa n t a g o n i s t sG i n k g o l i d eA ( B N - 5 2 0 2 0 )G bG i n k g o l i d eB ( B N - 5 2 0 2 1 )G bG i n k g o l i d eC ( B N - 5 2 0 2 2 )G bG i n k g o l i d eJ ( B N - 5 2 0 2 4 )G bG i n k g o l i d eM ( B N - 5 2 0 2 3 )G bK a d s u r e n o n eP fB u r s e r a nB mY a n g a m b i nO d

S y n t h e t i cd e r i v a t i v e so fn a t u r a lc o m p o u n d sF R - 9 0 0 4 5 2S pT H C - 7 - o i ca c i dC a n n a b i sm e t a b o l i z e

3 .S t r u c t u r a la n a l o g u e so fP A FC V - 3 9 8 8A l k y l e t h e rC V - 6 2 0 9P h o s p h o l i p i dT C V - 3 0 9P y r i d i n i u mS D Z6 4 - 4 1 2A r y l - i m i d a z o i s o q u i n o l i n es a l tB N - 5 2 1 1 1D i o x o l a nR o - 7 4 7 1 9P y r i d y lc a r b o x a m i d e

4 .T r i a z a l o b e n z o d i a z e p i n e sW E B - 2 0 8 6( a p a f a n t )H e t r a z e p i n eW E B - 2 1 7 0( b e p a f a n t )H e t r a z e p i n eW E B - 2 3 4 7H e t r a z e p i n eB N - 5 0 7 2 6H e t r a z e p i n eB N - 5 0 7 2 7H e t r a z e p i n eB N - 5 0 7 3 0H e t r a z e p i n eB N - 5 0 7 3 9H e t r a z e p i n e

I P S E N - B e a u f o u rI P S E N - B e a u f o u rI P S E N - B e a u f o u rI P S E N - B e a u f o u rI P S E N - B e a u f o u rM e r c k

F u j i s a w a—

T a k e d aC h e m i c aC oT a k e d aC h e m i c aC oT a k e d aC h e m i c aC oS a n d o zI P S E N - B e a u f o u rH o f f m a n - L aR o c h

B o e h n n g e rI n g e l h e iB o e h n n g e rI n g e l h e iB o e h r i n g e rI n g e l h e iI P S E N - B e a u f o u rI P S E N - B e a u f o u rI P S E N - B e a u f o u rI P S E N - B e a u f o u r

P A Fi nt h eC N S 5

properties (Smith a 1996).BN-52020,BN-52021and BN-52022 or ginkgolides A, B and C,respectively, are all competitive antagonists at thePAF receptor, however, ginkgolide B is the mostpotent (Braquet, 1987).Ginkgolides J and M havealso been identified, with all of these compoundsdiffering in the number and position of hydroxylfunctions (Hosford and Braquet, 1990).

Kadsurenone is a benzofuranoid lignan isolatedfrom another Chinese plant, Piper fKadsurenone is a potent competitive PAF receptorantagonist, while related compounds from the sameplant have only weak activity (Handley, 1990).OtherPAF antagonist Iignans include burseran, isolatedfrom B m i(Hosford and Braquet,1990)and yangambin, a furofuran lignan from thefruit of the Brazilian plant, O d (Castro-Faria-Neto et a 1995).

8.2. Synthetic Derivativesfrom Natural Products

The PAF receptor antagonist, FR-900452,is one ofthe compounds derived from the fermentation brothof the microbe, S tp (Handley,1990). The cannabis metabolize, tetrahydrocan-nabinol-7-oicacid, has also been shownto have PAFantagonistic properties (Koltai et a 1994).

8.3. Structural Analoguesof PAF

The first synthetic PAF antagonist reported wasCV-3988 and its competitive action at the PAFreceptor has been demonstrated in many models.Followingthis came the developmentof CV-6209,aneven more potent and long-acting PAF receptorantagonist (Handley, 1990).Other compoundswith astructural resemblance to PAF include TCV-309(Terashita et a 1992),SDZ-64-412,BN-52111andRo-74719(Koltai a 1994).

8.4. Synthetic Compounds

Triazolobenzodiazepinesare well known as anxio-lytics, however, some of these, including triazolam,alprazolam and brotizolam, show PAF antagonism.A problem with using these compounds as PAFantagonists are their sedative and hypnotic effects.This led to the dissociationof their PAF antagonisticactivity and GABA~ receptor activity, allowing forthe developmentof potent PAF antagonists (CasaIs-Stenzel, 1991).

Apafant (WEB-2086) was the first compoundderived from brotizolam, and is over 50-fold morepotent than BN-52021.Newer compounds such asWEB-2170 (bepafant) and WEB-2347 have sincebeen developed, which have greater potency or alonger half-life than apafant (Ikegami a 1992;Heuer, 1991;Heuer a 1990).A further series ofcompounds, related to WEB-2086 and other tria-zolobenzodiazepines, has been developed recentlyand includes BN-50726, BN-50727,BN-50730andBN-50739(Braquet and Esanu, 1991).

9. CONCLUSIONS

PAF is a potent lipid autacoid that is synthesizedin neuronal cells throughout the CNS. Followingactivation of the CNS PAF receptors,variouseffectersystemslead to numerousCNS effects.PAF has beenshownto modulate calciumlevelsand to regulate thetranscription of IEGs. The involvement of PAF inLTP has lead to its nomination as a retrogrademessenger in this model. PAF can influenceneuropeptide levels, cerebral blood flow and metab-olism, and plays a role in neuronal differentiation.Abnormal levels of PAF, that are likely to beproduced via a remodeling pathway, are involvedinthe neuronal cell damage occurring followingischaemia and inflammation. Elevated PAF levelsinthe brain have also been reported in patients infectedwith HIV and meningitis.

It is clear that PAF is of considerable importancein CNS physiology and pathology, and thatadministration of PAF receptor antagonists can bebeneficial during neurotrauma. A more preciseunderstanding of the mechanisms underlying theseCNS PAF effects, including the seeminglycomplexcross-talk between PAF and other mediators, maylead to the clinicalusage of PAF receptor antagoniststo interrupt the PAF-induced biochemical cascadeleading to cell death.

A ct h a n kR i c h a rL a v e rf ohh e l p f u lc o m m e n t so nt h em a n u s c r i p t ,a nM u r rC r afh i sh e l pi np r e p a r i n gf i g u r e s .T h iw o rw as u p p o rbf u n d i n gf r o mt h eN e wZ e a l a n dN e u r o l o g i cF o u n d aa n dt h eU n i v e r s i t yo fO t a g oM e d i c aS c h oB e q uF ut oa u t h o r sP Sa n dC D .A u t h o rK w as u p p o rb aU n i v e r s i t yo fO t a g oP o s t g r a d u a t eS c h o l a r s h i

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