Chemistry and Physics of
Chemistry and Physics of Lipids 80 (1996) 117 132
Messenger functions of phosphatidic acid Denis English ~'b,*, Yi Cui ~'b, Rafat A. Siddiqu? b ~'Bone Marrow Transplantation Laboratory, Methodist Hospital of huliana, 1701 N. Senate. Rm. 1417 MPC. hulianapol~', IN 46202, USA bSchool ~(' Allied Health Sciences, Indiana University School 01' Medichw, Indianapolis. IN. ~"SA
Abstract Under physiological conditions, phosphatidic acid (PA) is an anionic phospholipid with moderate biological reactivity. Some of its biological effects can be attributed to lyso-PA and diacylglycerol generated by the action of cellular hydrolases. However, it is clear that the parent compound exhibits biological activities of its own. Early studies implicated PA in the transport of Ca + ~ across plasma membranes as well as in the mobilization of intracellular stored calcium. Both responses may be induced as a consequence of other cellular processes activated by PA, as opposed to being directly mediated by the lipid. PA may be involved in the activation of certain functions confined to specialized groupings of cells, such as the neutrophil superoxide-generating enzyme or actin polymerization. Recent studies implicate PA as an activator of intracellular protein kinases, and a PA-dependent superfamily of kinases involved in cellular signalling has been hypothesized. Deployed on the outer surface of the plasma membrane, PA potentially provides a method of communication between cells in direct contact. This review will explore the known functions of PA as an intracellular mediator and extracellular messenger of biological activities and address ways in which these functions are potentially regulated by cellular enzymes which hydrolyse the phospholipid.
Keywords: Phosphatidic acid; Second messengers: Cellular signalling: Phospholipase D; Phosphatidic acid phosphohydrolase
1. Introduction In resting cells, phosphatidic acid (PA) consti-
Ahbreviations: IP 3, inositol trisphosphate; PA, phosphatidic acid: Lyso PA, lysophosphatidic acid; PI, phosphatidylinositol: PIP> phosphatidylinositol bisphosphate; PC, phosphatidylcholine. * Corresponding author, Bone Marrow Transplantation Laboratory, Methodist Hospital of Indiana, 1701 N. Senate, Rm. 1417 MPC, Indianapolis, IN 46202, USA. Tel.: 317-9292663, Fax: 317-929-2021
tutes a m i n o r portion o f the total phospholipid pool. Its lifetime there is transient, owing to its involvement as an intermediate in lipid synthesis and its availability as a substrate for potent cellular phosphatases which rapidly generate diglycerides at the expense o f the parent c o m p o u n d (see Brindley, this volume). U p o n stimulation o f cells with certain metabolic agonists, m e m b r a n e PA levels rapidly increase, a result that led to early speculation that the lipid stimulated key processes involved in cellular signalling. In 1953, H o k i n and Hokin observed that certain h o r m o n e s markedly
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D. English et al. Chemisto' and Physics o[' Lipids 80 (1996) I 17 132
potentiated the incorporation of 32-p-inorganic phosphate into cellular phosphatidylinositol (PI) and PA . Michell implicated this phenomenon, termed the phospholipid effect, as the basis for increased transmembrane conveyance of Ca + + in response to neurotransmitters and hormones . Indeed, cellular stimuli that induce such effects on phospholipids seemed more likely than others to also increase intracellular free Ca + + . Putney and coworkers demonstrated the apparent ability of PA to mediate the transport of radioactive calcium from the aqueous to the organic phase of the suspending media, a property shared by the ionophore A23187 . The authors concluded that PA formed in cells as a consequence of induction of the phospholipid effect may mediate the movement of cellular calcium that results from ligation of surface receptors.
2. Calcium influx Although the direct ionophoretic properties of PA probably resulted from contaminant (see below), the phospholipid may indeed play an important, yet indirect, role in both the transport and mobilization of cellular calcium, as these responses may result from cellular processes induced by the phospholipid. Exogenous PA has been demonstrated to induce a calcium influx in a number of biological tissues, including isolated platelets [4,5], parotid gland , liver cells [7,8], nerve terminals , and epithelium . In studies with neural tissue, Harris et al.  observed that PA was unique among membrane phospholipids in stimulating both Ca + + influx and neurotransmitter release and thus may be a natural mediator of depolarization. In renal brush border membranes, PA was found to be more potent than either PI or phosphatidylcholine (PC) in stimulating calcium influx . The results of these early studies were consistent with the view that PA stimulated cellular processes that led to the transmembrane conveyance of extracellular calcium.
2.1. Relationship of PA generation, Ca + + mobilization and junctional activation The potential relationship between PA generation, calcium mobilization and functional activation in stimulated cells was apparent in some of the earliest studies on the subject [6,11]. In these studies, PA synthesis in stimulated cells was found to hold characteristics compatible with its hypothesized role as a primary mediator of membrane calcium gating and did not occur as consequence of receptor-driven calcium mobilization. Mechanisms involved in membrane calcium gating are still unsettled, subject to lively speculation and likely to be quite complex (for example, see ). Early theories that endogenous PA acts as an ionophore to mediate calcium influx into stimulated cells were laid to rest when Holmes and Yoss  demonstrated that PA-mediated calcium transport across liposomes was due to the presence of contaminating oxidized fatty acids; freshly prepared PA preparations were inactive in this respect, However, a number of studies subsequently assessed calcium uptake by cells after treatment designed to increase endogenous levels of PA. Incubation of brush border membranes with Mg-ATP, for example, increased endogenous levels of PA and the rate of calcium uptake by these membranes in parallel . Neutrophilic leukocytes were found to generate PA prior to releasing inflammatory mediators after stimulation . In an investigation of the relationship of phosphatidylinositol bisphosphate (PIP2) hydrolysis to calcium influx, neutrophils were treated with a stimulus that did not cause release of intracellular stored calcium but was known to evoke influx of the cation from extracellular stores . PA generation occurred in two distinct phases after neutrophil stimulation. The initial phase of PA generation, which correlated with the kinetics of calcium influx, was not inhibited by chelation of extracellular calcium, and was thus apparently not a result of calcium-dependent activation of cellular phospholipases. Subsequent studies indicated that this initial calcium-independent phase of PA generation resulted from activation of phospholipase D . Later PA generation was, however, dependent on the presence of extra-
D. English et al. / Chemistry and Physics ~[ Lipids 80 (1996) 117 132
cellular calcium and may have resulted from activation of calcium-dependent cellular responses. Neutrophil functional activation was similarly dependent on the presence of extracellular calcium under the conditions used. Thus, in neutrophilic leukocytes, phospholipase D mediated generation of membrane-associated PA apparently promotes calcium transport across the plasma membrane resulting in potentiation of membrane phospholipid changes and eventual functional activation. Kiesel and Catt  postulated a similar relationship of functional activation to PA generation in pituitary cells stimulated to release luteinizing hormone by gonadotropin-releasing hormone. In these cells, the incorporation of 3~p into the total phospholipid pool was significantly potentiated by gonadotropin-releasing hormone, with specific increases in labeling noted for both PI and PA. Stimulation of luteinizing hormone release by exogenous PA was highly dependent on extracellular calcium, increasing by over one order of magnitude as the calcium concentration in the media was increased from zero to 0.5 raM. The authors concluded that endogenous PA, formed in response to receptor activation, may participate in the stimulation of hormone-induced responses in endocrine cells. Ohata and colleagues studied the mechanisms of carbachol-induced contraction in Guinea-pig taenia coli [19,20]. Exogenous PA induced contraction as it promoted uptake of calcium into the tissue t¥om the extracellular media; depletion of extracellular calcium inhibited these responses. Carbachol increased, in a concentration dependent manner, the mass of PA within the tissue, apparently as a result of immediate activation of both phospholipase C and diglyceride kinase. The resultant increase in PA, the authors suggested, contributed to the sustained phase of agonist-induced contraction by promoting calcium influx from extracellular sources. 2.2. Mobilization (~[ intracellular stored calcium The reports cited above appeared coincident with the emergence of results that clearly implicated other important functions for the phospholipid. The first of these to be recognized was the
ability of PA to promote the release of stored intracellular calcium. In fact, prior to studies of the potential role of PA in mediating calcium influx, Gerrard et al. demonstrated that the phospholipid elicited release of calcium stored on the inner surface of platelet membranes . The authors suggested that the phospholipid may have an important role intracellularly in promoting calcium movements. Moolenaar and associates  studied the relation of calcium mobilization to the growth factorlike activities of PA, including stimulation of DNA synthesis and the expression of certain proto-oncogenes. While PA preparations were shown to elicit a short-lived rise in intracellular free calcium, this transient resulted not from influx of the cation but from its release from intracellular stores. Unlike an ionophore, the authors concluded, PA exerted its effect by stimulating another cellular response, namely hydrolysis of phosphoinositides, with consequent formation of calcium mobilizing second messengers. Subsequent studies by these investigators identiffed contaminating lyso-PA as the stimulus of tile cellular responses observed [23,24]. Lyso-PA is now known to evoke a wide range of cellular responses, apparently by ligating a G-proteinlinked receptor . However, the original study by Moolenaar and associates set the stage for numerous subsequent investigations which implicated phosphatidic acid in stimulation of phosphoinositide metabolism and consequent release of intracellular stored calcium in several systems. Dunlop and Larkins demonstrated that PA directly effected PIP 2 hydrolysis in isolated islet cell membranes . In intact cells, this effect was associated with a transient rise m intracellular fi'ee calcium. McGhee and Shoback  demonstrated that exogenous PA promoted the release of intracellular stored calcium as it stimulated tile formation of inositol trisphosphate ill bovine parathyroid cells. Kawase and Suzuki observed that exogenous PA stimulated internal calcium release and IP3 generation in cultured osteoblasts [28,29]. These responses, which apparently resuited from PA-dependent activation of phospholipase C, were linked to proliferation of these cells induced by PA in vitro. Kurz el al. postulated a
D. English et al. / Chemistry and Physics of Lipids 80 (1996) 117-132
similar reaction sequence in cardiac myocytes exposed in vitro to PA . In this system, as in the others cited above, phospholipase C activation and consequent mobilization of stored calcium by exogenous PA was mediated by generation of IP3. Contaminating lyso-PA was not responsible for the results observed. PA may activate pathways that result in the mobilization of intracellular stored calcium independent of IP3 generation. Breittmayer and colleagues have reported that the Jurkat line of cultured T cells responds to PA with a burst of calcium mobilization that is not inhibited when extracellular cation is chelated [31,32]. This phosphatidate-sensitive calcium pool was depleted when the cells were first exposed to antibodies to CD3, the T cell receptor molecular complex. Moreover, CD3-stimulated cells also generated phosphatidic acid at kinetics which were consistent with a role for the phospholipid in stimulated calcium release. Neither the PA-induced calcium release nor calcium release engendered by cellular stimulation could be ascribed to the presence or production of lysoPA. In this respect, it is of interest to note that Jurkat cells, like neutrophils, fail to respond to lyso PA with mobilization of intracellular stored Ca + + . These cells are thereby useful to define PA-specific responses. Moreover, under conditions that resulted in intracellular calcium mobilization, PA failed to increase the generation of IP3 in Jurkat cells. Breittmayer et al. concluded that, in the system used, PA activates cellular pathways that result in calcium release in the absence of IP3 generation [31,32]. In summary, PA has been implicated in mediating both the influx of calcium and release of intracellular stored calcium in stimulated cells. While the ionophoretic properties of the phospholipid may not be physiologically relevant, generation of endogenous PA parallels calcium influx in several systems. Thus, PA may exert effects that result in calcium influx independent of its function Cellular responses to lyso-PA are beyond the scope of this review; the interested reader is referred to the excellent review by Jalink et al.  for an in depth discussion of this interesting topic.
as an ionophore. By stimulating cellular metabolism, PA elicits the release of intracellular stored calcium. While activation of phospholipase C has been implicated in this response, other pathways may be involved as well.
3. Potential involvement of PA in cellular signalling
3.1. Activation of neutrophilic leukocTtes A variety of methods have been used to investigate specific roles of PA in cell signalling. These involve, for the most part, assessing the effects of the phospholipid on isolated enzymes or other components of signalling pathways in cell free systems. These studies, discussed below, have been prompted by studies with intact cells which implicate PA in the signalling process. Many of these early studies were carried out with neutrophilic leukocytes, which respond quickly to metabolic stimulation by a variety of diverse mediators with a wide array of physiologically relevant responses, ranging from superoxide release to hyperadherence and degranulation. After initial reports of the presence of phospholipase D in mammalian cells appeared in 1987 [33-36], many of the initial studies implicating a role for this enzyme in signal transduction were carried out with neutrophils [37-48]. The potential involvement of PA in functional activation in these cells was immediately apparent. These studies also led to an immediate recognition of the possible role of PA phosphohydrolase, an enzyme that converts the phospholipid to diacylglycerol, in the regulation of functional activation . Various PA phosphohydrolase inhibitors were subsequently used in attempts to differentiate the relative roles of the substrate and product of the enzyme in responses of isolated cells to metabolic stimuli. Propranolol was found to be relatively useful for this purpose, since it is water soluble and relatively nontoxic. Thus, propranolol decreases generation of diacylglycerol while preserving elevated levels of PA in stimulated cells [17,41,49,50]. Propranolol-mediated inhibition of receptor-dependent phosphoinositide hydrolysis
D. English et al. ~ ChemL~'try and Physics ol Lipids 80 (1996) I 17 132
in mast cells, differentiation in pre-ovulatory granulosa cells and secretion in hormone-responsive cells have each been traced to inhibition of PA phosphohydrolase [51-53]. However, the drug is known to inhibit the activity of other enzymes as well  and caution is needed in assessing the basis of inhibitory effects observed in the presence of this agent. Used at relatively high levels (150-170 tiM), propranolol markedly enhanced functional activation of neutrophils stimulated with certain receptor-dependent stimuli, including the chemotactic peptide FMLP and the lipid mediator, leukotriene B4 [49,55,56]. Responses to other stimuli were inhibited [49,56-58]. Functional potentiation induced by pretreatment of cells with propranolol was attributed to enhanced levels of PA, resulting from inhibition of PA phosphohydrolase. On the other hand, both Perry et al. and Suchard et al. correlated propranolol-mediated inhibition of neutrophil activation with diminished generation of diglycerides [57,58]. Under other conditions of stimulation, Kanaho et al.  and Zohu et al.  found little influence of propranolol on functional activation. Both groups interpreted this result to indicate that PA produced on activation of phospholipase D is more important than its respective diglyceride in mediating neutrophil function. To further confuse the issue, Meshulam et al. recently observed potentiation and inhibition by propranolol of neutrophil functional responses, depending, again, on the manner of stimulation . However, propranolol-treated neutrophils did not mount a normal respiratory burst under conditions in which accumulation of neither PA (mediated by phospholipase D) nor diacylglycerol (mediated by phospholipase C) were altered. Thus, other factors are required in the triggering of neutrophil functional responses, and the availability of these factors is diminished in the presence of propranolol as well. 3.2. Correlation oJ second messenger availability and cellular ./unction A number of other approaches have been employed which implicate PA in the activation of specitic functions of neutrophilic leukocytes. For
example, Korchak et al.  demonstrated that while a number of neutrophil stimuli that increased diglyceride levels also promoted granule enzyme release, only a few induced release of superoxide anion, an event that results from activation of the cells' membrane-bound NADPH oxidase (more on this enzyme, below). Unlike other stimuli, those agents which did effectively induce superoxide release also induced generation of PA. Using kinetic considerations and pharmacological agents to probe the response, Gelas et al. concluded that phospholipase D activation in FMLP-treated neutrophils is a two step process with functional activation linked to the late phase of PA generation . Kessels and colleagues related neutrophil respiratory burst and phospholipase D activation on the basis of the similarities of both responses to the availability of Ca ~ + . Mori et al. concluded that potentiation of respiratory burst activation by the protein kinase C inhibitor staurosporine was a consequence of phospholipase D activation . Meshulam et al. noted that PA increased prior to superoxide generation in yeast-stimulated neutrophils, consistent with a role of the lipid in functional activation . Primary alcohols have been used to investigate the rote of PA in neutrophil activation. These agents reduce phospholipase D-dependent PA generation by providing substrates for the unique transphosphatidylation activity that the enzyme possesses (see Cockroft, this volume). The resulting phosphatidyl-alcohols are metabolically inert. Rossi et al. observed that butanol inhibited the formation of PA as it inhibited induction of the respiratory burst in FMLP-treated neutrophils . Bonser and associates demonstrated that ethanol and butanol blocked PA and superoxide generation in parallel in stimulated cells [65,66]. Kanaho et al. demonstrated an inhibitory effect of ethanol on neutrophil degranulation and likewise traced the inhibition to attenuation of PA generation . In addition to direct metabolic stimulation, PA has been linked to the priming of neutrophil functions by a variety of agonists and growth factors . Bourgoin et al. provided evidence that the mechanism of GM-CSF induced priming
D. English et al. / Chemistry and Physic,~" o/ Lipids 80 (1996) 117 132
of neutrophils involved the activation of phospholilSase D and consequent generation of PA . Subsequent studies from this group indicated that priming was associated with PA-dependent increase in mobilization of intracellular free calcium . Bauldry et al. correlated PA production with priming of superoxide generation in cells pretreated with tumor necrosis factor ~ . Using a system that allowed differentiation of effects induced by PA from those associated with diglycerides, a strong correlation was observed between PA generation and priming of FMLP-induced superoxide release. The authors thereby suggested a link between PA generation and NADPH oxidase activation in human neutrophils. Garland hypothesized that priming results when a cytoplasmic protein tyrosine kinase increases the efficiency of coupling between membrane receptors and phospholipase D . PA generated as a result of this enzyme's activity potentially acts as a second messenger by direct activation of the cells' NADPH oxidase. Interconversion of PA and diacylglycerol catalyzed by PA phosphohydrolase and diacylglycerol kinase regulates the individual roles of these lipids in the priming response. Several subsequent studies have documented the involvement of tyrosine phosphorylation in neutrophil activation and priming pathways [71-75]. However, despite considerable speculation (for example, see [76,77]), the mechanism by which these kinases exert their effects remains unknown. 3.3. Permeabilized cells
Obvious limitations to the interpretations of the above results illustrate the problems inherent in studies designed to define specific roles of putative mediators in signalling networks in intact cells. Apart from kinetic analyses and the use of propranolol and primary alcohols, few alternative approaches have been available to explore the role of PA in cellular activation, since molecular techniques applicable for this purpose have not become available. One approach that has generated some informative data is the use of permeabilized cells. From studies with electropermeabilized neu-
trophils, Kessels concluded that phospholipase D activation was important, but not necessary, for respiratory burst activation; Ca ÷ ÷ chelation strongly inhibited activation of phospholipase D but a respiratory burst could still be elicited . Studies with bacterial toxin permeabilized cells led Bauldry et al. to conclude that diglyceride is not a second messenger in the signal transduction pathway leading to activation of the neutrophil NADPH oxidase . However, under a variety of conditions, there was a close correlation between neutrophil activation and generation of PA with generation of the phospholipid preceeding oxidase activation in permeabilized cells stimulated with the G-protein activating agent, GTP-7S. The study by Bauldry et al. indicates that PA is generated in response to some cellular process activated by a guanine nucleotide regulatory protein, such as the G proteinqinked to the activation of PLD in stimulated neutrophils [17,7982]. In another study with permeabilized cells, Mitsuayama et al. provided evidence that PA stimulates a neutrophil G-protein that leads to superoxide generation . Thus, PA directly stimulated superoxide generation in permeabilized cells and this effect was inhibited by GDP-fl-S, which specifically blocks G protein activation. The inability of protein kinase C inhibitors to block the effect of PA in permeabilized cells led the authors to conclude that the site of action of the lipid lay somewhere between activation of the kinase and the oxidase. Results of a subsequent study supported the notion that PA is a direct activator of the neutrophil NADPH oxidase , an event that is described in more detail below. 3.4. N A D P H oxidase activation
While the studies discussed above do not provide a unified model that defines the role of PA in neutrophil activation, they do indicate that the lipid is involved somewhere in the activation scheme. The pattern that emerges is certainly confusing, but these results provide a compelling rationale for studies designed to identify functions of the phospholipid in various cell-free systems designed to mimic events that occur when intact
D. English et al./ Chemistry and Physics e~l' Lipi~t*' 80 (1996) 117 132
cells are activated. Development of a cell-free system for activation of the neutrophil superoxide-generating enzyme was immensely important in this respect. Dormant in resting cells, this membrane-bound enzyme provides substrates for several potent bactericidal and cytocidal systems when the cells are activated. It is an enzyme essential for adequate host defense; its absence or defective activation in chronic granulomatous disease predisposes patients to severe, life-threatening bacterial infections. Its activation mechanism has been extensively studied in a cell-free system, and the emerging data demonstrate that activation is a dynamic process that results from the interaction of several cytosolic components with the dormant membrane-associated enzyme. This interaction may be promoted by PA. Early studies of the neutrophil superoxide generating system demonstrated that plasma membranes derived from activated cells effectively mediated the generation of the superoxide free radical in the presence of oxygen and NADPH, an effect ascribed to the presence of an active N A D P H oxidase (for review see ). Little or no activity was present in membranes of resting neutrophils, or in membranes of activated cells from patients with chronic granulomatous disease, indicating that stable activation of the enzyme resulted from cellular stimulation. For many years, attempts at activating the enzyme in resting membranes met with limited success, until Heyneman and Vercauteren demonstrated its activation in membranes of resting horse neutrophils upon exposure to cytosol in the presence of certain fatty acids . Similar activation was quickly documented in Guinea pig  and human neutrophils [88,89] in similar systems. Bellavite et al. demonstrated that PA could replace both fatty acid and cytosol in the activation of this enzyme in plasma membranes of pig neutrophils . Subsequently, Agwu et al. demonstrated that PA was an effective, albeit weak, activator of the human neutrophil enzyme when used with cytosol . Subsequent studies by McPhail and colleagues demonstrated that this property was markedly enhanced in the presence of low concentrations of diglycerides . Thus, PA generated by the action of phospholipase D during neutrophil stimu-
lation, in the presence of diacylglycerol generated by PA phosphohydrolase-mediated dephosphorylation of its substrate, may play an important role in the activation of the cells' superoxide-generating enzyme. How the lipid mediates this effect is now the subject of intense study, The nature of the cytosolic involvement in NADPH oxidase activation has proven to be quite complex. At least three components are involved, each of which impinge upon the inactive enzyme during cellular stimulation . One of these components is the small molecular weight G-protein, Rac2 . Rac2 is active in its GTPbound form. Post-translational isoprenylation facilitates its interaction with regulatory proteins that stimulate the exchange of GTP for GDP . In resting neutrophils, Rac2 is exclusively confined to the cytosol as a complex with GDP dissociation inhibitor (GDI) . When cells are activated, this complex is disrupted, allowing Rac to translocate to the plasma membrane and participate in NADPH oxidase activation. Like arachidonic acid, PA effectively disrupts the RacGDI complex, which may explain the role of the phospholipid in neutrophil activation. However, further investigation will be necessary to substantiate this hypothesis, since the physiologic relevance of NADPH oxidase activation by PA in comparison to other potential activators is not clear. In addition, it is not known if the ability of the phospholipid or other agents to activate the oxidase in the presence of unfractionated cytosol derives exclusively from disruption of Rac-GDI or if other factors are additionally involved, However, if PA- promoted Rac-GD! complex disassociation is involved in neutrophil oxidase activation in intact cells, similar processes may govern second messenger functions of PA in other systems as well.
4. Effects of PA in other cell flee systems 4.1. Activation ~['phospholipase C
The effects of PA have been studied in other cell free systems, although not as extensively as NADPH oxidase activation. Early studies docu-
D. English et al. / Chemistry and Physics o/'Lipids 80 (1996) 117 132
mented the activation of phosphoinositide-specific phospholipase C by PA . In the system used, diacylglycerol was without effect. In addition to direct stimulation, PA potentiated receptor agonist and guanine nucleotide-dependent stimulation of phospholipase C. Jones and Carpenter documented the activation of native and tyrosine phosphorylated phospholipase C-7 by PA and concluded that the lipid was an allosteric modifier of the enzyme . Jacob et al. investigated activation of Xenopus laevis oocyte plasma membrane phospholipase C by PA . Activity of this phosphoinositide-specific phospholipase C was stimulated about two-fold by PA.
4.2. Protein kinase activation by PA PA has effects on other enzymes that may participate in signal transduction. Bocckino et al. documented PA-dependent protein phosphorylation catalyzed by soluble extracts from a number of tissues, including liver, brain, lung and testis . PA-dependent phosphorytation required Ca + + and differed from that induced by the protein kinase C activators, phosphatidylserine and 1,2-diolein. Ca + +-independent phosphorylation of a 30 kDa protein in soluble extracts of heart tissue was also observed upon addition of PA. The second messenger activities of PA in cells may, in part, thus be mediated by activation of this protein kinase. Several investigators have studied the influence of PA on protein kinase C. Epland and colleagues reported that PA can replace phosphatidylserine in the activation of this kinase [100,101]. Stasek et al. studied the activation of endothelial cell protein kinase C by the phospholipid . Both dioleoyl and l-stearoyl, 2-arachidonyl-PA activated the purified enzyme in a concentration dependent manner. Activation was not a result of contaminating diglycerides or diglycerides generated from PA by the action of PA phosphohydrolase. The results indicated that PA and diglycerides activate protein kinase C by different mechanisms which may be differentially involved in mediating responses of cells to metabolic stimuli.
Kahn et al. described a novel, calcium-independent protein kinase that was activated by PA in triton extracts of human platelets . This enzyme was concluded to be distinct from any of the known isotypes of protein kinase C based on several criteria, including differential phospholipid activation profiles, substrate range and immunologic reactivity. The authors suggested that the enzyme is a member of a superfamily of phospholipid-dependent protein kinases that potentially play important roles in mediating cellular effects of PA, including mitogenesis. Limatola et al. recently assessed the effects of PA on distinct isoforms of protein kinase C in a novel cell-free assay system . PA activated three isoforms of protein kinase C when added to the cytosol of cells overexpressing each of the enzymes. In the absence of Ca + +, the diacylglycerol-insensitive zeta isotype of protein kinase C was most potently activated by the lipid; activation of other isotypes was Ca + +-dependent. Thus, PA may be a physiologically important activator of protein kinase C-zeta in cells where Ca + + is at basal levels. Finally, Bell and Ghosh have recently reported that PA binds Raf-1 kinase , a serine/ threonine kinase that is activated in many cells upon growth factor stimulation. Binding appeared to occur within the catalytic domain of raf-1. Binding of PA to raf-I may thus be a signal-regulated event evoked by activation of phospholipase D. Such binding may induce conformational changes in raf-l, ultimately leading to its activation at the plasma membrane. Activated in this manner, raf-I may initiate cell proliferation, possibly by mediating activation of MEK, the enzyme that catalyzes the phosphorylation of MAP kinase (see  for a review of this topic). Siddiqui and Yang recently demonstrated the activation of MAP kinase in cells treated with PA . Earlier studies demonstrated that PA effectively increased the activity of the ras GTPase-inhibiting protein and inhibited the activity of the ras GTPase-activating protein [108,109]. Thus, PA may regulate upstream effects that lead to Raf and MEK activation as well.
D. English et al. .' Chemistry and Pit v.sics 01 Lipi~£ 80 (I 996) / 17 1t2 4.3, Lipid kinases
PA has been reported to activate PI-4-phosphate-5-kinase, an enzyme that provides substrates for PlP2-specific phospholipase C . This effect was observed with both exogenous and endogenously generated PA. Jenkins et al. recently studied the influence of PA on specific isoforms of phosphatidylinositol phosphate kinase . PA stimulated the activity of the type I kinase purified from erythrocyte membranes as well as two immunoreactive isoforms of type I kinase in rat brain. Under similar conditions, the phospholipid had little effect on type II kinase. 4.4. Inr, oh,ement q f PA in growth-fiwtor induced cell prol(feration
When applied to cells exogenously, PA exerts potent mitogenic effects [112-117]. As discussed above, some of the observed mitogenic effects may be attributed to contamination with lyso-PA. However, endogenous PA has been implicated in proliferative responses to certain receptor-dependent agonists, including epidermal growth factor , platelet derived growth factor [119,120], interleukin 2 [121,122], interleukin 1 [123,124] and interleukin 11 . In addition, PA apparently mediates the mitogenic effects of sphingosine and sphingosine l-phosphate [125,126]. Marcoz et al. found that the mitogenic lectin concanavalin A induced a 4- 5-fold increase in thymocyte PA that preceded the proliferative response . The phospholipid potently stimulated two forms of a cyclic-AMP specific phosphodiesterase in cell-free extracts of thymocyte cytosol, a property that was not shared by diacylglycerol. The authors concluded that PA formed during the initial phase of mitogenic stimulation may thereby induce cyclic AMP degradation. Consequent lowering of intracellular cyclic AMP levels may set the stage for initiation of the first steps of cellular proliferation. Siddiqui and Yang associated growth factor-induced cellular proliferation with PA dependent actiwttion of MAP kinase, an event resulting from upstream induction of the tyrosine kinase, MAPkinase or MEK . In this system, lyso-PA was without effect. Thus, phospholipid-dependent ty-
rosine kinases may mediate some of the second messenger effects of PA in stimulated cells. However, to date, a PA-dependent protein tyrosine kinase has not been identified in a cell-fi'ee system, although the search for such a kinase continues. PA may alternatively have exerted its influence at some earlier point in the signal transduction sequence leading to proliferation. For example, as discussed above, MAP kinase activation may result from activation of Raf-I kinase by the phospholipid. 4.5. Other extracellular qff~,cts o f phosphatic acid
In addition to mitogenesis, exogenous PA induces a number of cellular functions, including hormone release [26,128], actin polymerization , platelet aggregation , muscle contraction [19,20] and gene transcription [131,119,121]. Analysis of the available data indicates that responses evoked by PA generally fit within one of three specific patterns: those induced by long chain molecules, those induced by diglycerides generated alter hydrolysis of short chain PA by membrane enzymes and those induced by short chain PA independent of the generation of diglycerides. In addition, as discussed above, some responses induced by PA have been attributed to contamination of commercially available preparations by lyso-PA. In most recent reports, therefore, lyso PA contamination is excluded as the cause of the response documented (for example, see [30,107,110,114,115,132,133]). Certain responses to PA are, however, known to be mediated by diglycerides generated when the stimulus is hydrolyzed by membrane phosphatases. Of these, the most thoroughly investigated is induction of superoxide generation when short chain (di-C8) PA is added to intact neutrophils. Perry et al. recently demonstrated that these somewhat soluble phospholipids are rapidly hydrolysed by a novel ecto-PA phosphohydrolase . Localized on the extracellular side of the plasma membrane, this enzyme rapidly hydrolysed short chain substrates in the absence of detergent; long chain PA was hydrolysed much less efficiently. Diacylglycerol continuously generated as a restllt of this enzyme's activity mediated
D. English et al. / Chemistry and Physics ~/' Lipids 80 (1996) 117 132
sustained activation of the neutrophil superoxide generating system, probably as a result of activation of protein kinase C. Similar activation could be achieved by addition of exogenous short chain diacylglycerol, but not by a non-hydrolyzable analogue of di-C8 PA, phosphonate 1.2 Subsequent to this report, Xie and Low demonstrated similar activity on cultured keratinocytes . In a variety of cell types, however, certain responses, such as induction of phosphoinositide metabolism [30,113,136], regulation of adenyl cyclase [136,137] and activation of phospholipase A2  seem to be induced optimally by molecules with relatively long side chains, such as dioleoyl (di-C18:l) or disteroyl (di-C20) PAs. In the absence of detergent, these substrates are very poor substrates for plasma membrane PA phosphohydrolase(s) [138,139]. Thus, these responses are probably not mediated by diglycerides generated upon hydrolysis of the inducing agent.
5. Potential PA receptor It is possible that some responses to PA result from ligation of a cellular receptor. Several groups have raised this possibility, but none have provided evidence for specific binding of PA to cellular membranes. For example, Murayama and Ui concluded that membrane receptors mediate adenylate cyclase inhibition and phospholipid hydrolysis induced by exposure of fibroblasts to PA . This conclusion was supported by the observation that pretreatment of cells with cholera or pertussis toxin inhibited the effects of PA, indicating that activation was mediated by ligation of a specific G-protein coupled receptor. Pearce et al. implicated binding of specific PA
2 This finding is not in conflict with the observation that PA activates the neutrophil N A D P H oxidase in a cell-free system. Direct activation of the oxidase may be mediated by PA, even if activation of intact cells is mediated by diacylglycerol. In fact, Perry et al. demonstrated the generation of long chain PA in neutrophils stimulated with the short chain compound , indicating the latter induced a cellular process that resulted in production of the former and, consequently, N A D P H oxidase activation.
receptors in phosphoinositide metabolism and mitogenesis induced upon exposure of cortical astrocytes to the phospholipid . Ha and Exton recently theorized that actin polymerization in fibroblasts resulted from specific binding of PA to its receptor on these cells . In addition, Ryder et al. observed that PA-stimulated phosphoinositide turnover was inhibited by pretreatment of cells with pertussis toxin, raising again the possibility that the response was initiated by ligation of a G protein coupled receptor . The responses of cells to PA observed by Ryder et al. could not be attributed to contaminating lyso-PA. In a report that may hold farreaching implications, Ferguson and Hanley demonstrated that both dioleoyl-PA and lyso-PA potently induced Ca + +-dependent electrical responses in Xenopus laevis oocytes . Similar responses could not be induced by intracellular instillation of the stimulus, indicating the involvement of a surface receptor. In other respects, responses of the oocytes to PA followed the recognized pattern of events expected upon activation of G-protein-phosphoinositidase C-coupled receptors. The authors concluded that these cells may communicate with others, such as follicular cells, through activation of PA receptors. Moreover, the physiological significance of the PA receptor system, according to the authors, may lie in transducing signals between cells in direct contact. Further studies are necessary to determine the potential of this novel agonist-receptor system in communication between adjacent cells in other systems.
Acknowledgements This work was supported by grants from the National Institutes of Health and the Phi Beta Psi Sorority awarded to Dr. English and by grants from the Indiana Affiliate of the American Heart Association awarded to Dr. Cui and Dr. Siddiqui. We also thank Drs. Luke Akard, Jan Jansen and James Thompson for their support and encouragement. The authors acknowledge Stephanie McGillem for preparing the manuscript.
D. English et al. / Chemistry and Physics q f Lipids 80 (1996) 117 132
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