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Introduction Membrane Phospholipid Hydrolysis Following Neurotrauma PUFAs and PAF as Second Messengers or Mediators of Cerebral Ischemia and Seizure PAF and Neurotransmission PAF and Immediate-Early Genomic Responses Immediate-Early Genomic Responses to Neurotrauma Role of PAF, PUFAs, and IEGs in Long-Term Potentiation Summary References
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Membranes serve as physical delimiting boundaries of cells, provide microenvironments for correct protein function, and provide phospholipid substrates for multiple signal transduction pathways. Components of signal transduction include the release of diacyl-glycerols and subsequent activation of C-type protein kinases, the hydrolysis of inositol phosphates from phosphatidyl precursors and the intracellular receptormediated Ca 2+ ionization by inositol phosphates, and the oxidative metabolism of free arachidonic and other polyunsaturated fatty acids. Neural cell membrane lipids, by virtue of their intrinsic role as sites of second messenger synthesis, are susceptible to extensive hydrolysis during the unregulated neural stimulations that follow neurotrauma. These resulting physical and chemical changes in cell membranes contribute to neural damage and, ultimately, to loss of brain function. Under physiological conditions, lipid second messengers derived from membranes serve as intermediaries between the extracellular environment *Author to whom all correspondence and reprint requests should be addressed. and the cytoplasmic and nuclear metabolic machinery. Transduction of neurotransmission to the nucleus to initiate synthesis of new proteins is an important mechanism in neural processes, including maintenance of neural integrity, memory formation and synaptic plasticity, and repair following trauma. An integral component of this transduction is the rapid stimulation of immediate-early genes following neural stimulation. The proteins encoded by these inducible genes regulate expression of the cellular proteins that effect long-term neural adaptations. The functioning of lipid messengers in transduction of surface-to-nucleus phenomena in neural cells make them important potential targets of unregulated neurotransmission following trauma and cognitive impairment, as well as integral components during learning and memory formation. This paper summarizes current information regarding the role of membrane-derived lipids, in particular platelet-activating factor, in the regulation of gene expression induced through neurotransmission during physiological and pathophysiological processes.
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Neural stimulation involves the degradation of membrane phospholipids by phospholipases through diverse and selective receptor-mediated events. Many products of these enzymes are intra-and extracellular mediators of the initiating receptor stimulation. Under pathological conditions (e.g., ischemia, convulsions, and other trauma), an overstimulation of receptors leads to the synthesis and accumulation of such biologically active lipid mediators in brain. A further consequence of pathological phospholipid hydrolysis is disruption of intrinsic membrane homeostasis and symmetry, which may leave cells susceptible to toxic alterations in membrane-associated biochemical events and may lead to the release of toxic levels of phospholipid rnetabolites.
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Certain phospholipids of synaptic membranes are rapidly deacylated by phospholipase A 2 during convulsions, leading to the accumulation of free arachidonic and docosahexaenoic acids (Bazan, 1970;Bazan et al., 1986;Bazan and Birkle, 1987), the most prevalent polyunsaturated fatty acids (PUFAs) in neural tissue. Ischemia and other forms of brain injury, such as cryogenic brain edema, also lead to the accumulation of free PUFAs (Bazan, 1970;Aveldano and Bazan, 1975a,b;Cenedella et al., 1975;Bosisio et al., 1976;Yoshida et al., 1980;Seisj6 et al., 1982;Lazarewicz et al., 1983;Ikeda et al., 1986;Politi et al., 1985). Moreover, the ischemia-induced accumulation of free fatty acids preferentially takes place in grey matter as compared with white matter (Bazan, 1971). In retina, similar responses occur during anoxia (Aveldano and Bazan, 1974;Giusto and Bazan, 1983) or depolarization (Birkle and Bazan, 1984). An enhanced production of lipoxygenase products takes place under these conditions in the retina as well as in brain synaptosomes. Analysis of subcellular fractions from the brains of rats undergoing bicuculline-induced status epilepticus shows that the synaptosome fraction, and not endoplasrnic reticulum fractions, is the prevalent site of phospholipase A 2 activation and accumulation of lipoxygenase reaction products (Birkle and Bazan, 1987).
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During reperfusion after ischemia, the renewed availability of oxygen in the presence of abnormally large PUFA pools leads to enzyme-mediated oxygenation of arachidonic acid and other PUFAs and to peroxidation of membrane lipids (Braughier and Hall, 1992). In the pathogenesis of brain damage, degradation of certain excitable membrane phospholipids, effects concomitant with accumulation of free PUFAs or their metabolites, may contribute to functional impairment (Siesj6 et al., 1982;Bazan, 1976;Bazan and Rodriguez de Turco, 1980). These early responses of the nervous system may result from overstimulation of receptors and increased intracellular Ca 2 § an activator of cellular phospholipases (Bazan and Birkle, 1987;Bazan, 1976;Bazan and Rakowski, 1970). The membrane phospholipid components that are primarily degraded by phospholipases are thought to be ethanolamine plasmalogen (Edgar et al., 1982), inositol lipids (Reddy and Bazan, 1987), and phosphatidylcholine (Marion and Wolfe, 1978). The ischemic involvement of inositol lipids is further documented, since these lipids decrease in ischemia and convulsions concomitant with the accumulation of stearoylarachidonoyl-sn-glycerol, the predominant diacylglycerol byproduct of inositol lipid hydrolysis in brain (Bazan, 1970;Aveldano and Bazan, 1975a;Aveldano de Caldironi and Bazan, 1979;Ikeda et al., 1986).
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The ether-linked molecular species of phosphatidylcholine, 1-O-alkyl-2-acyl-sn-glycero-3phosphocholine (alkyl-acyl-GPC) that is a precursor of platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) may also function as a source of free PUFAs that accumulate in the nervous system under ischemic conditions and during epilepsy. The synthesis of PAF is triggered in several tissues in response to injury, inflammation, or during immune reactions through different biosynthetic routes (reviewed in Snyder, 1990; Fig. 1): 1. A remodeling pathway, initiated by phospholipase A 2 activation that releases the PAF precursor lyso-PAF from membrane alkyl-acyl-GPC, or 2. A de novo pathway involving choline transfer to 1-alkyl-2-acetyl-sn-glycerols. It has been suggested that brain cells synthesize PAF through the latter de novo pathway, because acetyltransferase activity has not been detected at appreciable levels in brain tissue (Francescangeli and Goracci, 1989). However, brain does contain a Ca2 § lysophospholipase D (Kawasaki and Snyder, 1987;Lee et al., 1988) that will generate de novo pathway substrate 1-O-alkyl-2-1yso-sn-glycerol from lyso-PAF released by the action of phospholipase A 2 on membrane phospholipid. Lysophospholipase D may represent a "neural shunt" of PAF synthesis between remodeling and de novo pathways.
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During pathological conditions such as seizure and ischemia, Ca2 § phospholipase A 2 activation (Bazan, 1970;Bazan and Rodriguez de Turco, 1980) is likely a major source of PAF liberated from cell membrane precursors (Bazan et al., 1991). In addition, the observations that Ca 2+ inactivates all enzymes of de novo pathway of PAF synthesis (Lee et al., 1986;Woodard et al., 1987;Lee et al., 1988) suggest that phospholipid remodeling initiated through Ca2+-activated phospholipase A 2 is the source of PAF synthesis during ischemic and epileptic Ca 2+ fluxes.
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Since the remodeling route involves an initiating phospholipase A 2 activation, it has been suggested that PAF synthesis may be accompanied by the generation of free PUFAs following ischemia or convulsions (Panetta et al., 1987;Birkle et al., 1988). Furthermore, the alkyl-acyl-GPC, on which phospholipase A 2 acts to release the PAF precursor lyso-PAF, is known in several cells and tissues to be enriched in arachidonoyl groups at the sn-2 position (Chilton et al., 1984;Ramesha and Pickett, 1986;Suga et al., 1990). Recently, synthesis of PAF through phospholipase A 2 digestion of ethanolamine plasmalogen has been demonstrated (Nieto et al., 1991). A CoA-independent transacylase functions to transfer arachidonic acid from alkyl-acyl-GPC to the lyso-plasmalogen product of the initiating phospholipase A 2 activity, generating the PAF precursor, lyso-PAF. Since ethanolamine plasmalogen is among the earliest phospholipids hydrolyzed by phospholipase A 2 activity following neurotrauma (Edgar et al., 1982), discovery of this route of PAF synthesis further suggests involvement of phospholipid remodeling as the major route of PAF synthesis following neurotrauma.
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Alkyl-acyl-GPC and ethanolamine plasmalogen may provide substantial sources of both PUFAs and PAF, following neural trauma. Production of PUFAs and PAF may have potent The remodeling pathway is initiated by the activity of phospholipase A 2 on alkyl-acyl-glycerophosphocholine (alkyl-acyl-GPC), a species of membrane phospholipid that is enriched in arachidonoyl chains at the sn2 position. The products of the phospholipase A 2 reaction are free arachidonic acid, the precursor of bioactive eicosanoids, and the PAl: precursor lyso-PAF. The subsequent activity of a specific acetyltransferase generates PAF (alkyl-acetyl-GPC). The de novo pathway of PAF synthesis involves the transfer of phosphocholine from CDP-choline to alkyl-acetyl-glycerol. PAl: interaction with specific membrane binding sites results in the activation of phospholipases and other second messenger systems. PAF can be inactivated by the action of an acetylhydrolase followed by reacylation. The de novo route is believed to provide endogenous PAF required for physiological functions. Cellular stimulation during pathophysiological conditions is believed to generate PAF through the remodeling pathway. biological consequences. PUFAs are substrates for oxygenating enzymes, which produce the most biologically potent mediators of cellular swelling and edema, the eicosanoids. Levels of free PUFAs and eicosanoids increase following seizure, ischemia reperfusion, and trauma (reviewed in Bazan and Birkle, 1987). One of the potential effects of PUFAs released by the activity of phospholipase A 2 is the uncoupling of oxidative phosphorylation (Hillered and Chan, 1990). Suppression of mito-chondrial ATP production is associated with formation of free radicals, which damage membranes through the formation and propagation of membrane-disruptive lipid peroxides (Braughler et al., 1985;Braughler and Hall, 1992).
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Although the metabolic fates of arachidonic acid and other PUFAs released during ischemia and convulsions are not completely known, some effects associated with PUFA release may be attributed directly to arachidonic acid, as opposed
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to its bioactive oxygenated metabolites. Arachidonic acid is released from hippocampal slices through stimulation by NMDA and glutamate (Pellerin and Wolfe, 1991). Arachidonic acid potentiates channel current through NMDA receptors in rat cerebellar granule ceils (Miller et al., 1992), and inhibits glutamate uptake in glial cells (Barbour et al., 1989). These effects are mediated by arachidonic acid itself and apparently not its lipoxygenase or cyclooxygenase metabolites. It is conceivable that arachidonic acid exerts these effects either through direct binding to glutamate receptors or through physical alteration of membrane fluidity surrounding these proteins. Thus, release of free arachidonic acid affects excitatory neurotransmission by enhancing glutamate activity at the synapse and may contribute to neuronal death during ischemia through this mechanism.
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Massive free fatty acid release following ischemia-evoked phospholipase A 2 may affect other receptor systems. Phospholipase A 2 treatment of adult rat synaptic membranes increases the affinity of AMPA binding to neural membranes (Massicotte and Baudry, 1990;Baudry et al., 1991). Increased membrane polyunsaturation in retinoblastoma cells through enrichment of the culture medium with arachidonic or docosahexaenoic acids increases high affinity uptake mechanisms for choline and taurine (Hyman and Spector, 1982;Yorek et al., 1984). This is clear evidence that PUFAs released from phospholipid sequestration in cell membranes will affect ligand binding processes in cell membranes. Docosahexaenoic acid is the other major fatty acid released following seizure and ischemia (Bazan, 1970;Bazan et al., 1986). Although the function of this PUFA is largely unknown, its release from phospholipids has been demonstrated to inhibit PAF-induced Ca2+-influx (Weber et al., 1991). This inhibition of PAF activity is not observed with other PUFAs and may occur through a lipoxygenase metabolite of docosahexaenoic acid. Lipoxygenase metabolites of docosahexaenoic acid have previously been described in retina (Bazan et al., 1984).
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PAF has been implicated as a modulator of neural activity, particularly during the initiation of various forms of neural trauma. PAF precursors are abundant in neural tissue (Blank et al., 1981). Convulsions, induced either electrically or through the administration of picrotoxin or bicuculline, are accompanied by PAF synthesis inbrain (Kumar et al., 1988). PAF directly impairs brain metabolism (Kochanek et al., 1988), and PAF antagonists reduce cellular damage and improve metabolism in brain following ischemia (Panetta et al., 1987;Spinnewyn et al., 1987;Oberpichler et al., 1990;Lindsberg et al., 1990;Gilb6e et al., 1991). PAF apparently does not traverse the blood-brain barrier (Kumar et al., 1988 ), and, hence, PAF accumulation in brain, following neural trauma, must be derived from neural metabolism.
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PAF, the cognate phospholipase A 2 product of PUFA liberation, may exert a unique spectrum of detrimental effects during neural trauma. Choline lysophospholipids, including PAF, induce effiux of Ca 2+ from mitochondria, and this may represent a mechanism by which phospholipase A2, through lysophospholipid generation, and impaired mitochondrial function, may mediate ischemic tissue damage (Rustenbeck et al., 1991). Several PAF antagonists improve neural metabolism following ischemia (Panetta et al., 1987;Spinnewyn et al., 1987;Oberpichler et al., 1990;Lindsberg et al., 1990;Gilb6e et al., 1991), suggesting an activity of PAF in tissue damage through disruption of metabolism.
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PAF antagonists suppress the rapid accumulation of brain free fatty acids following ischemia and electrically or pharmacologically-induced seizure (Panetta et al., 1987;Birkle et al., 1988). The suppression of free fatty acid release by PAF antagonists suggests inhibition of a phospholipase A 2 activity, as evidenced by the fact that arachidonic and docosahexaenoic acids are the prevalent PUFAs liberated under these conditions (Bazan, 1970).
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In addition, the ischemia-reperfusion-associated generation of free radicals appears to be sensitive to PAF antagonism (Braquet et al., 1990;Gilb6e et al., 1991). It is likely that neural free radicals generated during ischemia are metabolites of PUFAs, the phospholipase A2-mediated release of which is sensitive to PAF antagonists.
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From these biochemical events resulting from cerebral injury, the sensitivity to PAF antagonists implicate PAF as a mediator in component cellular processes. PAF, in addition, has been identified as a potent chemotactic agent for leukocytes, which are recruited to sites of injury and produce rapid and profound effects on the vascular system (Braquet et al., 1987), including alterations of blood-brain barrier permeability (Kumar et al., 1988). The protective effects of PAF antagonists on neural metabolism following trauma may reflect participation of endothelial and leukocytic cells.
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At high concentrations, PAF is a membrane perturbant in vitro (Sawyer and Anderson, 1989) and is toxic to at least some neural cell types in vitro (Kornecki and Ehrlich, 1988;Lustig et al., 1992). Although it is not known if PAF could accumulate to such high perturbant or neurotoxic concentrations in vivo, even during ischemia and convulsions, it is easy to envision local membrane changes accompanying the synthesis of PAF. Both PAF and its remodeling pathway precursor, lyso-PAF, localize to the plasma membrane following their respective syntheses (Record et al., 1989;Vallari et al., 1990). Accumulation of PAF and lyso-PAF, owing to relatively small molecular densities at sn2, likely alter membrane homeostasis among the 2-acyl-sn-glycero-3-phospholipids (Harris et al., 1985). In this respect, transacylation of phospholipids and reacylationinactivation of PAF function to preserve membrane integrity following stimulus-evoked hydrolysis of 2-acyl PAF precursors.
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PAF is a potent activator and recruiter of leukocytes during the inflammatory response (reviewed in Braquet et al., 1987). Such actions imply that PAF derived from intracellular metabolism is made available externally. But the observation that leukocytes and endothelial cells retain a substantial mass of PAF following cell stimulation (reviewed in Henson, 1987) has led to speculation that PAF functions as an intracellular messenger. In these cell types, PAF antagonists will block the activities of endotoxin, chemotactic peptide (fMLP), and bradykinin, indicating that PAF mediates the intracellular activities of these cell stimulators (Stewart et al., 1989;Stewart and Phillips, 1989;Stewart et al., 1990). In neutrophils, exogenous PAF itself induces synthesis of intracellular PAF through the remodeling pathway (Tessner et al., 1989;Doebber and Wu, 1987).
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Direct measurements of PAF synthesized in retina (Bussolino et al., 1986(Bussolino et al., ,1988)), cerebellar granule cells (Yue et al., 1990), and fetal brain cells (Sogos et al., 1990), have provided indications that an intracellular mediator role for PAF also exists in neural cells. Stimulation of retina with acetylcholine and dopamine, but not glycine, GABA, or glutamate, induces de novo synthesis of PAF (Bussolino et al., 1986), an effect mediated through muscarinic cholinergic and D2 dopamine receptors, respectively (Bussolino et al., 1989). The acetylcholine and dopamine effects are more profound following synapse maturation, indicating a physiological association of neurotransmitter-evoked PAF synthesis with the synapse (Bussolino et al., 1988). Acetylcholine is also an agonist of intracellular PAF synthesis in fetal brain cells (Sogos et al., 1990). Bradykinininduced PAF synthesis in leukocytes is not without ramifications as a potential mechanism in neural tissue as well, since this mediator of inflammation is a potent stimulator of phospholipid hydrolysis in neural tissue, and may, in fact, participate in the progression of neurotrauma (Francel, 1992).
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In addition to being a product of cholinergic and dopaminergic transmission, PAF is implicated in the functioning of other neurotransmitters. Exogenous PAF affects secretion of neurohormones from the hypothalamus (Junier et al., 1988;Rougeot et al., 1990;Blasquez et al., 1990), and excitatory neurotransmitters from presynaptic hippocampal neurons (Clark et al., 1992). In addition, PAF induces secretion of PC 12 cell adenosine triphosphate, which is stored and released with catecholamines during stimulation of PC12 cells, through a Ca2*-dependent mechanism (Kornecki and Ehrlich, 1988). The nonbenzodiazepine PAF antagonist BN52021 (a ginkgolide terpene) enhances GABA-dependent chloride uptake in cortical synaptoneurosomes through a noncompetitive mechanism at the GABA A benzodiazepine site (Miller et al., 1991). Although activity of PAF at GABA receptors has not been demonstrated, triazole-substituted benzodiazepines are both potentiators of GABAergic neurotransmission and potent PAF antagonists (Kornecki et al., 1984).
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In rat brain, two kinetically distinct high-affinity intracellular PAF binding sites are present on microsomal membranes, in addition to a lower affinity synaptic binding site (Marcheselli et al., 1990). A synaptic PAF receptor may be linked to presynaptic enhancement of excitatory neurotransmission (Clark et al., 1992). The piperidinothieno diazepine BN50730 (Braquet et al., 1990) exhibits high affinity and selectivity for the intracellular sites (Marcheselli and Bazan, 1991). Localization of high-affinity PAF binding sites to intracellular membranes in brain suggests that cellular retention of PAF is functionally linked to metabolic events subsequent to its stimulusevoked synthesis.
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The structure of PAF itself suggests its candidacy as an intracellular messenger, particularly during the phospholipase activations and membrane degradation that accompany neurotrauma. The acetyl group at sn2 does not make it a preferred substrate of phospholipase A2, if it can be a substrate for that enzyme at all. Indeed, an acetylhydrolase highly specific for phospholipids with short chains at sn2 utilizes PAF as a substrate during PAF degradation (Blank et al., 1981;Wardlow et al., 1986). An ether linkage at snl imparts resistance to esterases such as phospholipase A 1. Although the significance of phospholipase A 1 activation in neural tissue is not fully understood despite its broad intracellular distribution (Bazan, 1971), the major fatty acids liberated from neural membranes following ischemia and seizure, in addition to arachidonic acid, are saturated fatty acids (Bazan, 1970), typically esterified at snl in membrane phospholipids. In addition, there is evidence that the phospholipase C metabolite of PAL 1-alkyl-2acetyl-sn-glycerol, retains PAF activity (Stoll et al., 1989). Thus, particular structural features of PAF, unique among cellular phospholipids, impart intrinsic resistance to phospholipasemediated hydrolysis following cell stimulation and suggest a role as an intracellular mediator of signal transduction.
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Although the identity and location of the initiating phospholipase A 2 in PAF synthesis is not clear, the other enzymes of PAF synthesis localize to intracellular membranes (Record et al., 1989;Vallari et al., 1990). Following synthesis, PAF relocalizes from the endoplasmic reticulum to the plasma membrane (Vallari et al., 1990). Although factors determining either intracellular retention of PAF or its liberation as an agonist are unknown, studies in erythrocyte membranes suggest that both membrane phospholipid asymmetry as well as presence of acceptor molecules inside and outside the cell determine the fate of intracellular PAF (Bratton et al., 1991).
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Extracellular PAF acts as an agonist coupled to multiple phospholipases, including types A2, C, and D (reviewed in Shukla, 1992). Intracellular PAF synthesized following cell stimulation may also be coupled to phospholipase A 2 activation. PAF antagonists block free radical and eicosanoid generation in leukocytes and endothelial cells following stimulation, suggesting its involvement as a second messenger (Stewart et al., 1989(Stewart et al., ,1990;;Stewart and Phillips, 1989). Generation of PAF through the phospholipase A 2 remodeling of neural membranes, in addition to activation of phospholipase A 2 by intracellular PAF, suggests an amplification circuit that may account for the protective effects of PAF antagonists against trauma-evoked free fatty acid release and cellular damage (Birkle et al., 1988;Spinnewyn et al., 1987).
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Cell stimulation is transduced to the nucleus though mechanisms involving the generation of second messengers and stimulation of target protein kinases. The immediate-early genomic response is the earliest transcriptional activity following cell stimulati0n (reviewed in Herschman, 1989;and Morgan and Curran, 1991). The stimulus-evoked transcription is both transient and independent of de novo protein synthesis. Nuclear machinery is poised for a burst of new gene expression following cell stimulation. The components of the immediate-early genomic response include members of thefos, jun, fra, myc, and TIS immediate-early gene (lEG) families (reviewed in Herschman, 1991). The structures of these lEG products suggest proteins of various inferred functions, but the majority of those discovered and by far the most well-studied are nuclear-localized, DNA-binding transcription factors. The transduct'ion of extraceUular stimuli to the generation of transcription factors suggests a long-term mechanism that mediates cellular fate, in the event of stimulus, through synthesis of new proteins.
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The best-studied IEGs in neural tissue include fos, jun, and zif/268.fos and jun encode DNA-binding transcription factors with leucine-zipper structures that allow formation of DNA-binding dimers (reviewed in Ransone and Verma, 1990). Fos-Jun and Jun-Jun dimers constitute sequencespecific AP-1 transcription regulatory activity. Zif/268 is also a transcription factor, but it binds DNA as a monomer through zinc-finger structures at sequences distinct from those bound by AP-1 proteins (Christy et al., 1988;Christy and Nathans, 1989). An immediate-early genomic response to PAF has been reported in leukocytes (Ho et al., 1987;Squinto et al., 1989;Mazer et al., 1991;Schulam et al., 1991), epidermal carcinoma cells (Tripathi et al., 1991), and neural cells (Squinto et al., 1989;Doucet and BaTan, in preparation). Further evidence of an immediate-early genomic response to PAF is provided by the cycloheximide-sensi-tive stimulation of prostaglandin E 2 in macrophage-like cells (Glaser et al., 1990). It is interesting to note that cyclooxygenase (Maier et al., 1990), and possibly prostaglandin endoperoxide synthase (Herschman et al., 1991), are encoded by immediate-early genes. In primed leukocytes, stimulated synthesis of PAF itself, likely through a phospholipase A2-mediated pathway, is also dependent on new protein synthesis (Wirthmueller et al., 1990). Since exogenous PAF is an activator of phospholipase A 2 and intracellular lyso-PAF is a product of the enzyme, it is likely that de novo proteins are regulators of phospholipase A 2 stimulation.
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In NG108-15 neurohybrid cells, picomolar PAF elicits expression of the zif/268 (Egr-1, TIS 8, NGFI-A) immediate-early gene (Doucet and Bazan, in preparation). This potency of PAF suggests that, it is an important component of the immediateearly genomic response in neural cells. The effectiveness of low concentrations of PAF in eliciting an immediate-early genomic response may reflect the activity of low concentrations of PAF in priming cells for enhanced responses to subsequent stimuli, an effect described in leukocytes and epithelial cells (reviewed in Braquet et al., 1989) that may be dependent on de novo synthesis of proteins (Wirthmueller et al., 1990). The immediate-early genomic response to PAF in NG108-15 cells is suppressed by the potent intracellular PAF antagonist BN50730, suggesting that the immediate-early transcription is transduced through an intracellular PAF receptor. In addition, the immediate-early genomic response to acetylcholine in these cells is suppressed by the intracellular PAF antagonist. In neuroblastoma cells (Squinto et al., 1989;Squinto et al., 1990), PAF elicits both (1) transcription of both los and jun immediate-early genes and (2) stimulation of transcription from promoters containing multiple AP-1 binding sequences. This indicates that the immediate-early transcriptional stimulation by PAF is physiologically coupled to corresponding protein activity. In addition, deletion mutagenesis of the los promoter has revealed the requirement of a calcium-response
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Volume 6, 1992 sequence (CARE) for PAF-stimulatedfos expression (Squinto et al., 1989). Thefos CARE, coincident with the cyclic AMP-response sequence, locates upstream of thefos transcription initiation site and functions as a binding site for a transcription factor during cyclic AMP-and Ca2+-stimulatedfos expression (Sheng et al., 1988;Sheng et al., 1990). The finding that PAF utilizes the los CaRE for transcriptional regulation is supported by studies demonstrating cytoplasmic influx of Ca 2+ stimulated by PAF from both intracellular and extracellular stores in neural cells (Yue et al., 1991a,b). The presence of the CaRE in transcriptional promoter regions of other immediate-early genes, in particular zif/268 (Christy et al., 1988), which is co-regulated withfos under many types of cell stimulations including neural cell depolarization (Sukhatme et al., 1988;Bartel et al., 1989), suggests that PAF and other stimulators of Ca 2+ mobilization may regulate multiple components of the immediate-early genomic response through a common mechanism.
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Although the mechanism through which PAF elicits genomic effects is not completely understood, certainly a role for Ca 2+ influx stimulated by exogenous PAF is implicated in the stimulation of los. The finding that PAF stimulates histone H1 kinase (p34r activity in nonproliferating cells (Samiei et al., 1991) suggests a broader mechanism of transcriptional control for PAF. Histone H1 regulates genomic transcription by affecting chromatin compactation and maintaining a transcriptionally repressed state (reviewed in Zlatanov, 1990). In addition, histone H1 kinase phosphorylates the product of thefos immediate-early gene (Fos) on residues important for negative regulation of transcription by the protein (Abate et al., 1991).
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In the nervous system, the immediate-early genomic response is elidted by physiological and pathological conditions (reviewed in Doucet et al., 1990;and Morgan and Curran, 1991). These eliciting events include ischemia, seizure, injury, and establishment of long-term potentiation. The existence of stimulus-evoked expression of transcription factors in neural tissue suggests that long-term changes in cellular activity and function, including reparative changes following neurotrauma and changes during memory formation, derive from primary, short-term genomic responses.
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PAF is implicated in the regulation of the immediate-early genomic response to seizure. Expression of los and several other immediate-early genes is stimulated by pharmacologically-or electrically-induced seizure (Morgan et al., 1987;Saffen et al., 1988;Cole et al., 1990;reviewed in Doucet et al., 1990;and Morgan and Curran, 1991). The potent intracellular PAF antagonist BN50730 (Marcheselli and Bazan, 1991) suppressesfos and zif/268 expression induced by electroconvulsive shock (Marcheselli et al., 1991;Marcheselli and Bazan, 1992). This suggests that (1) the endogenous PAF generated through seizure is functionally coupled to the stimulation of a immediate-early genomic response and that (2) the coupling of PAF to IEG expression may be an intracellular event in neural tissue.
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Both the activation of phospholipase A 2 following ischemic onset (Bazan, 1970;Bazan and Rodriguez de Turco, 1980) and the protective effects of PAF antagonists against cellular damage in brain (Panetta et al., 1987;Spinnewyn et al., 1987;Birkle et al., 1988;Oberpichler et al., 1990;Frerichs et al., 1990;Gilb6e et al., 1991) implicate PAF as a mediator of ischemic damage. Immediate-early genornic responses to i~hemia have been described (Onodera et al., 1989;Jorgenson et al., 1989;Kindy et al., 1991). In both experimentally evoked seizures and ischemia, increased immediate-early gene activity in dentate gyrus and hippocampal neurons is particularly pronounced, and specific PAF binding sites are concentrated in these brain structures (Domingo et al., 1988).
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In vitro PAF exerts intrinsic cytotoxicity in certain neural cells (Kornecki and Ehrlich, 1988;Lustig et al., 1992). As a neural Ca 2 § agonist (Yue et al., 1991a,b), PAF may contribute to neural damage by altering Ca 2+ homeostasis. Hippocampal neurons are sensitive to Ca 2+ fluxes during brain injury (Dienel, 1984;Siesj6 and Bengtsson, 1989;Siesj6, 1990;Young, 1992), and the accumulation of PAF during pathological conditions may affect extracellular and cytoplasmic Ca 2 § mobilizations through mechanisms mediated by possibly both specific PAF receptors and by membrane perturbations (Sawyer and Anderson, 1989;Young, 1992).
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The protective effects of PAF antagonists in neurotrauma suggest that PAF effects deleterious metabolic changes in traumatized neural ceils. However, as an inducer of immediate-early genomic responses, it is presumed that PAF stimulates new protein synthesis as a means of cellular repair and maintenance. The cellular logic of a mediator of damage evoking mechanisms of repair is not apparently clear. The participation of PAF in these two apparently disparate mechanisms is reflected in studies of NG108-15 cells (Kornecki and Ehrlich, 1988), where high concentrations of PAF exhibit neurotoxicity in these cells, while low concentrations stimulate neuronal maturation. Interestingly, in cerebral cortical cultures sensitive to glutamate toxicity, PAF exerted no toxic effects (Lustig et al., 1992). The magnitude of PAF synthesis and membrane hydrolysis following neurotrauma may, on a physiological scale, reflect homeostatic changes during normal cell metabolism. In this respect, it is the over-or unregulated production of PAF during neurotrauma that contributes to cellular damage. The PAF synthesis following normal neurotransmission and during the initial phases of injury are likely promoters of the new protein synthesis that direct molecular changes and cellular plasticity.
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One of the long-term events that may be established physiologically, through the activity of immediate-early transcription factors, is memory formation. Long-term potentiation (LTP) is an experimental model of memory that involves activity-dependent changes in synaptic morphology and circuitry. Long-term structural and functional modulations of synaptic connections are believed to require de novo synthesis of proteins (Davis and Squire, 1984;Montarolo et al., 1986). LTP induces immediate-early genomic responses, particularly in cells of the dentate gyms (reviewed in Abraham et al., 1992;Kaczamarek, 1992). The activity of the zif/268 transcription factor may be most specifically linked to LTP, as its expression is particularly correlative with the method of LTP induction and its duration.
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A role for PAF in the formation of LTP-mediated neuronal changes is suggested because PAF antagonist inhibition hippocampal synaptic responses during establishment of LTP (del Cerro et al., 1990;Arai and Lynch, 1992). In addition, PAF facilitates excitatory neurotransmission at a presynaptic, antagonist-sensitive site (Clark et al., 1992). Cell-generated PAF may therefore mediate plastic responses in the neuron, and this plasticity may involve the synthesis of new proteins mediated by products of a PAF-mediated immediateearly genomic response.
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Neuronal expression of zif/268 is induced by picomolar concentrations of PAF in vitro (Doucet and Bazan, in preparation), suggesting that small changes in PAF synthesis are sufficient to stimulate an immediate-early genomic response. In addition, low concentrations of PAF induce morphological differentiation of neural cells into mature neuronal (Komecki and Ehrlich, 1988), and astrocytic (Kentroti et al., 1991) phenotypes, alluding to a role for PAF during neural cell development and formation of cellular associations.
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Several lines of evidence support a role for phospholipase A 2 in the maintenance of LTP. Phospholipase A 2 inhibitors block the maintenance of LTP in certain cell populations (Linden et al., 1987;Massicotte et al., 1990;Williams and Bliss, 1989;Okada et al., 1989). Arachidonic acid directly affects the maintenance of LTP (Kato et al., 1991), possibly deriving from a post-synaptic site (Clements et al., 1991;Barbour et al., 1989).
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The interrelationship of PAF in phospholipase A 2 activity, the enzyme being both the initiator of PAF synthesis through the remodeling pathway and a target for activation by PAF, is a possible explanation of the effect of both PAF antagonists and phospholipase A 2 inhibitors in blocking LTP. In addition, Ca 2 § antagonists block LTP formation (Obenaus et al., 1989), and PAF is linked to increased intracellular cellular Ca 2+ through both a plasma membrane Ca 2+ channel and through the phospholipase C-catalyzed generation of the intracellular Ca 2+ agonist IP 3 (Yue et al., 1991a,b). Furthermore, Ca 2+ is a stimulator of phospholipase A 2 activity (Lapetina and Crouch, 1989) and may mediate the activation of phospholipase A 2 through NMDA receptors (Dumuis et al., 1988) which mediate formation of LTP (Collingridge et al., 1983). The maintenance of LTP is likely comprised of a selective, self-limiting stabilization of phospholipase A 2 metabolites in cell membranes that affects membrane structure at the synapse and physically and biochemically alters transport mechanisms and ion channels available at the plasma membranes of the synapse.