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Phosphatidic acid (PA) is produced rapidly in agonist-stimulated cells, but the physiological function of this PA is unknown. We have examined the effects of PA on distinct isoforms of protein kinase C (PKC) using a new cell-free assay system. Addition of PA to cytosol from COS cells overexpressing PKC-alpha, -epsilon or -zeta differentially-activated all three isotypes, as shown by PKC autophosphorylation, and prominent phosphorylation of multiple endogenous substrates. In the absence of Ca2+, the diacylglycerol-insensitive zeta-isotype of PKC was most strongly activated by both PA and bisPA, a newly identified product of activated phospholipase D, with each lipid inducing its own profile of protein phosphorylation. BisPA was also a strong activator of PKC-epsilon, but a weak activator of PKC-alpha. Ca2+, at > or = 0.1 microM, inhibited PA and bisPA activation of PKC-zeta, but did not affect PKC-epsilon activation. In contrast, PKC-alpha was strongly activated by PA only in the presence of Ca2+. BisPA-induced phosphorylations mediated by PKC-zeta could be mimicked in part by other acidic phospholipids and unsaturated fatty acids. PA activation of PKC-zeta was unique in that PA not only stimulated PKC-zeta-mediated phosphorylation of distinctive substrates, but also caused an upward shift in electrophoretic mobility of PKC-zeta, which was not observed with other acidic lipids or with PKC-alpha or -epsilon. We have presented evidence that this mobility shift is not caused by PKC-zeta autophosphorylation, but it coincides with physical binding of PA to PKC-zeta. These results suggest that in cells stimulated under conditions where intracellular Ca2+ is at (or has returned to) basal level, PA may be a physiological activator of PKC-zeta.

The mechanism by which cAMP inhibits growth factor-induced DNA synthesis in fibroblasts is not understood. Here we show that in Rat-1 fibroblasts, cAMP-raising agents inhibit p21(ras)-mediated mitogen-activated protein (MAP) kinase activation induced by either epidermal growth factor or lysophosphatidic acid. Under the same conditions, however, epidermal growth factor- or lysophosphatidic acid-induced protein tyrosine phosphorylation, Ca2+ mobilization, and activation of Na+/H+ exchange are not attenuated. In ras-transformed Rat-1 cells, 8-bromo-cAMP rapidly deactivates constitutively active MAP kinase without reducing p21(ras)·GTP levels; long term 8-bromo-cAMP treatment of these cells leads to growth arrest and reversion of the transformed phenotype. These results show that elevation of intracellular cAMP levels abrogates the p21(ras) MAP kinase pathway at a step downstream of p21(ras) activation. This finding provides a molecular basis for the growth-inhibitory action of cAMP in normal and transformed fibroblasts.

Lysophosphatidic acid (LPA), a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6), showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1) being 10-fold more potent than acyl-LPA(18:1). The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA) but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2), LPA(5) and LPA(6) receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5) receptor (GPR92), which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5) as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.

Introduction L’autotaxine est une lysophospholipase D secretee responsable de la synthese du mediateur lipidique : l’acide lysophosphatidique (LPA). L’autotaxine est secretee par le tissu adipeux et son expression est augmentee chez les individus obeses/insulino-resistants. L’objectif de la presente etude etait d’etudier le role de l’autotaxine adipocytaire dans l’expansion du tissu adipeux lors d’une obesite nutritionnelle, et sa contribution aux taux plasmatiques de LPA. Materiels et methodes Nous avons etabli une lignee transgenique murine (FATX-KO) dont le gene de l’autotaxine a ete delete des exons codant le site catalytique specifiquement dans le tissu adipeux par une approche Cre-lox. Les souris FATX-KO et controles de la meme portee ont ete soumises a un regime standard ou a un regime gras (45 % de lipides) durant 13 semaines et analyses. Resultats L’expression de l’autotaxine est fortement diminuee (jusqu’a 90 %) dans le tissu adipeux blanc et le tissu adipeux brun des souris FATX-KO, mais demeure inchangee dans les autres organes exprimant l’autotaxine. Les taux plasmatiques de LPA sont reduits de 38 %. Sous regime gras, l’augmentation de la masse grasse est plus importante chez les souris FATX-KO que chez les souris controles alors que la prise alimentaire reste inchangee. Ceci est associe a une augmentation de l’expression de PPARγ2 et des genes cibles de PPARγ (aP2, adiponectine, leptine, glut-1) dans le tissu adipeux sous-cutane, ainsi qu’a une augmentation de la tolerance au glucose. Conclusion Ces resultats montrent que l’autotaxine adipocytaire regule negativement le developpement du tissu adipeux en reponse a un regime gras, et qu’elle contribue aux taux plasmatiques de LPA.

Lysophosphatidic acid (LPA) is a water-soluble phospholipid with hormone-like and growth-factor-like activities. LPA activates a putative G-protein-coupled receptor in responsive cells, but the natural source of exogenous LPA is unknown. Here we show that LPA is present in mammalian serum in an active form (bound to albumin) at concentrations of 1-5 microM, but is not detectable in platelet-poor plasma, suggesting that LPA is produced during blood clotting. We find that thrombin activation of platelets prelabelled with [32P]Pi results in the rapid release of newly formed [32P]LPA into the extracellular environment. We conclude that LPA is a novel platelet-derived lipid mediator that may play a role in inflammatory and proliferative responses to injury.

The naturally occurring phospholipid lysophosphatidic acid (LPA) can induce a number of physiological responses in vertebrate cells, including platelet aggregation, smooth muscle contraction, and fibroblast proliferation. LPA is thought to activate a specific G-protein-coupled receptor, thereby triggering classic second messenger pathways such as stimulation of phospholipase C and inhibition of adenylate cyclase. Here we report that 1-oleoyl-LPA, at submicromolar concentrations, evokes a chemotactic response in amoebae of the cellular slime mold Dictyostelium discoideum. LPA-induced chemotaxis is specific in that other lysophospholipids, phosphatidic acid, and monoacylglycerol have no effect. We show that the response to LPA is not secondary to the accumulation of extracellular cAMP, a well-established chemoattractant for nutrient-starved D. discoideum. Compared with cAMP-induced chemotaxis, LPA-induced chemotaxis has a somewhat lower efficiency and is not accompanied by the characteristic cellular elongation and orientation along the gradient. These results indicate that LPA has a previously unsuspected role as a chemoattractant for D. discoideum and imply that its biological function as a "first messenger" is not restricted to vertebrate cells.

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.

Phospholipids are currently attracting much interest in studies on cellular signalling and growth control. It is now well established that the breakdown products of several plasma membrane phospholipids act as signal molecules, i.e. as intracellular second messengers or as agonists that modulate cell function. Well known examples of phospholipid-derived signaling molecules include diacylglycerol, inositol trisphosphate and prostaglandins, which are all rapidly generated upon receptor stimulation. The simplest natural phospholipid, lysophosphatidic acid (LPA or monoacylglycerol-3-phosphate), is a particularly intriguing case in that it not only serves a critical precursor role in de novo lipid biosynthesis but also shows striking hormone- and growth factor-like activities when added exogenously to appropriate target cells, as if LPA binds to and thereby activates its own G-protein-coupled receptor(s). Although LPA’s lipid precursor role has been known for many decades, the possibility that this simple phospholipid may have an additional role as a signaling molecule is being appreciated only since a few years. In this chapter, we briefly summarize our recent findings on the various actions of LPA, with particular emphasis on the signal transduction pathways triggered by LPA and the identification of a candidate cell surface receptor.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with hormone- and growth factor-like activities. Exogenous LPA stimulates GTP-dependent phosphoinositide hydrolysis and inhibits adenylate cyclase in its target cells, but the site of action of LPA is unknown. We now report the identification by photoaffinity labeling of a putative LPA membrane receptor in various LPA-responsive cell types. A 32P-labeled LPA analogue containing a photoreactive fatty acid, [32P]diazirine-LPA, labels a membrane protein of apparent molecular mass of 38-40 kDa in various cell types, including neuronal cells, brain homogenates, carcinoma cells, leukemic cells and normal fibroblasts. Labeling of the 38-40 kDa protein is competitively inhibited by unlabeled 1-oleoyl-LPA (IC50 approximately 10 nM), but not by other phospholipids. Specific labeling is not detected in rat liver membranes or in human neutrophils, which are physiologically unresponsive to LPA. Suramin, an inhibitor of both early and late events in the action of LPA, completely inhibits the binding of photoreactive LPA. We suggest that the 38-40 kDa protein represents a specific LPA cell surface receptor mediating at least part of the multiple cellular responses to LPA.

Lysophosphatidic acid (3-sn-lysophosphatidic acid; LPA) can activate cells similar to hormones and growth factors. We have considered the question whether metabolic conversion of LPA taken up by the cell could be of any importance in this activation. Addition of [14C-glycerol]LPA to quiescent Rat-1 fibroblasts resulted in rapid formation of [14C]monoacylglycerol (MG), closely followed by accumulation of [14C]triacylglycerol. Only very little [14C]diacylglycerol and [14C]phosphatidic acid was formed (approx. 100-fold less than MG). MG, when added exogenously to cells, lacks detectable biological activity. The results suggest that LPA itself, rather than one of its metabolites is the biologically active principle.

Publisher Summary This chapter discusses the multiple biological effects of phosphatidic acid (PA) and lysophosphatidic acid (LPA), with particular emphasis on their growth factor-like activities. LPA and PA are the key intermediates in the early steps of phospholipid biosynthesis. They can exert their own biological effects. In particular, exogenous PA and LPA stimulate DNA synthesis and cell division in fibroblasts and epithelial cells, with LPA being a more potent growth stimulant than the corresponding PA species. The effect of LPA and PA on cell proliferation appears to be highly specific. All common lipids, other than PA and LPA, are incapable of stimulating DNA synthesis in quiescent fibroblasts. The relative potencies of various LPA and PA analogs on fibroblast proliferation have recently been determined. As LPA is a potent activator of certain G-protein-coupled effector systems, it may serve as a convenient probe to identify and dissect various biological responses in different cell systems. LPA is used to monitor cellular effects and activate second-messenger pathways in various cell types.

Background Lysophosphatidic acid (LPA) is a lipid mediator that acts through specific G protein-coupled receptors to stimulate the proliferation, migration and survival of many cell types. LPA signaling has been implicated in development, wound healing and cancer. While LPA signaling pathways have been studied extensively, it remains unknown how LPA affects global gene expression in its target cells. Results We have examined the temporal program of global gene expression in quiescent mouse embryonic fibroblasts stimulated with LPA using 32 k oligonucleotide microarrays. In addition to genes involved in growth stimulation and cytoskeletal reorganization, LPA induced many genes that encode secreted factors, including chemokines, growth factors, cytokines, pro-angiogenic and pro-fibrotic factors, components of the plasminogen activator system and metalloproteases. Strikingly, epidermal growth factor induced a broadly overlapping expression pattern, but some 7% of the genes (105 out of 1508 transcripts) showed differential regulation by LPA. The subset of LPA-specific genes was enriched for those associated with cytoskeletal remodeling, in keeping with LPA's ability to regulate cell shape and motility. Conclusion This study highlights the importance of LPA in programming fibroblasts not only to proliferate and migrate but also to produce many paracrine mediators of tissue remodeling, angiogenesis, inflammation and tumor progression. Furthermore, our results show that G protein-coupled receptors and receptor tyrosine kinases can signal independently to regulate broadly overlapping sets of genes in the same cell type.

Epidermal growth factor (EGF)-induced receptor dimerization may provide a mechanism for activation of the receptor protein tyrosine kinase and for initiation of post-receptor signalling pathways. We have examined whether second messengers and agents that modulate EGF receptor function act at the level of receptor dimerization. Both the Ca2+ ionophore ionomycin and the tumour promotor tetradecanoylphorbol acetate (TPA), added shortly before EGF, inhibit EGF receptor protein tyrosine kinase activity in intact cells. In permeabilized cells, elevation of Ca2+ similarly inhibits EGF receptor function. The inhibitory effect of Ca2+, unlike that of TPA, appears not to be dependent on protein kinase C activity. Neither ionomycin nor phorbol ester affects EGF-induced receptor dimerization, as shown by cross-linking and immunoblotting techniques, although the phosphotyrosine content of both monomeric and dimeric receptors is strongly decreased. Furthermore, we show that EGF receptor dimerization is not affected by increases in cyclic AMP or intracellular pH, nor by changes in transmembrane potential, medium osmolarity or the glycosylation state of the receptor. These result suggest that modulation of EGF receptor function occurs at a step other than receptor dimerization.

The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP beta S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTP gamma S, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTP gamma S. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.

The regulation of phospholipase C has been investigated in both intact and streptolysin-O permeabilized human HeLa carcinoma cells. Stimulation of phospholipase C by histamine and guanosine-5'-O-thiotriphosphate (GTP[S]) requires the presence of at least 10 nM free Ca2+, but is not significantly further increased by raising [Ca2+]i to greater than 10(-6) M. The pH optimum of the inositol phosphate response is at pH 6.8, while small changes in intracellular pH, as occur during hormonal stimulation (0.2-0.4 unit) attenuate the histamine/GTP[S]-induced stimulation of phospholipase C. Increasing cellular cAMP levels, either through addition of cell permeable cAMP analogues to intact cells or by stimulation with isoproterenol, does not affect histamine responsiveness, arguing against cross-talk between both signalling pathways. In contrast, we found that the response to histamine and/or GTP[S] is largely inhibited after brief pretreatment of the cells with phorbol esters or synthetic diacylglycerol prior to permeabilization, suggesting that protein kinase C exerts feedback inhibition at the level of, or downstream from, the putative GTP-binding protein.

In human HeLa carcinoma cells, histamine causes a dose-dependent formation of inositol phosphates, production of diacylglycerol and a transient rise in intracellular [Ca2+]. These responses are completely blocked by the H1-receptor antagonist pyrilamine. In streptolysin-O-permeabilized cells, formation of inositol phosphates by histamine is strongly potentiated by guanosine 5′-[gamma-thio]triphosphate and inhibited by guanosine 5′-[beta-thio]diphosphate, suggesting the involvement of a GTP-binding protein. Histamine stimulates the rapid but transient formation of Ins(1,4,5)P3, Ins(1,3,4)P3 and InsP4. InsP accumulates in a much more persistent manner, lasting for at least 30 min. Studies with streptolysin-O-permeabilized cells indicate that InsP accumulation results from dephosphorylation of Ins(1,4,5)P3, rather than direct hydrolysis of PtdIns. The rise in intracellular [Ca2+] is biphasic, with a very fast release of Ca2+ from intracellular stores, that parallels the Ins(1,4,5)P3 time course, followed by a more prolonged phase of Ca2+ influx. In individual cells, histamine causes a rapid initial hyperpolarization of the plasma membrane, which can be mimicked by microinjected Ins(1,4,5)P3. Histamine-induced hyperpolarization is followed by long-lasting oscillations in membrane potential, apparently owing to periodic activation of Ca2+-dependent K+ channels. These membrane-potential oscillations can be mimicked by microinjection of guanosine 5′-[gamma-thio]triphosphate, but are not observed after microinjection of Ins(1,4,5)P3. We conclude that H1-receptors in HeLa cells activate a PtdInsP2-specific phospholipase C through participation of a specific G-protein, resulting in long-lasting oscillations of cytoplasmic free Ca2+.

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.

The sudden addition of Na+ to mouse neuroblastoma cells suspended in Na+-free medium causes a rapid but transient increase in the rate of H+ release from the cells. Li+ can substitute for Na+, but addition of choline, K+, or Ca2+ has no effect. This process has the following properties: it is distinct from metabolic acid production, it does not require ATP, and it saturates at about 40 mM external Na+; it is independent of membrane potential and can be mimicked by addition of the Na+/H+ ionophore monensin to cells in Na+-containing media. In contrast, a net uptake of protons is observed when Na+-loaded cells are suddenly exposed to Na+-free medium. Na+-induced H+ extrusion is accompanied by a rise in intracellular pH, as inferred from an enhanced net uptake of weak acids and from direct pH measurements on lysed cells. Conversely, Na+ uptake by the cells is stimulated upon lowering the intracellular pH with externally applied acetate. Na+-dependent proton transport, intracellular alkalinization, and acetate-stimulated Na+ uptake are completely inhibited by the diuretic amiloride (0.2 mM) and do not occur in digitonin-permeabilized cells. It is concluded that the plasma membrane of neuroblastoma cells contains an electroneutral Na+/H+ exchange system which is involved in the regulation of intracellular pH.

One decade ago it was reported that within the first minutes after fertilization, sea urchin eggs show a striking increase in intracellular pH (pHi)' apparently due to an amiloride-sensitive exchange of extracellular Na + for intracellular H+ across the plasma membrane (26). There is now good evidence that this rise in pHi is a necessary signal for the initiation of growth and development of the egg (10, 74). The studies of the pHi response to fertilization in sea urchin eggs were the first to suggest that a rise in pHj, mediated by N a + IH + exchange, might be a common event in the metabolic activation of quiescent animal cells. In recent years, a convincing role for Na+ /H+ exchange and changes in pHi in the action of extracellular growth stimuli has been documented with a variety of cell types in culture. The purpose of this review is to describe the effects of growth factors on Na + /H+ exchange and pHj, with emphasis on those studies that have examined the mechanisms by which the Na + /H+ exchanger is activated. Because of space limitations, only the most pertinent results can be discussed. A review of the role of pHi in the regulation of cell metabolism and growth can be found elsewhere (to; W. B. Busa, this volume) and is beyond the scope of this chapter.

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells1,2. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3–6), due to activation of a Na+/H+ exchanger in the plasma membrane4, and this alkalinization is necessary for the initiation of DNA synthesis7. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger8–10. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli11,12. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12–15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.

The regulation of cytoplasmic pH (pHi) has been examined in normal human foreskin fibroblasts (HF cells) using a fluorometric technique for continuously monitoring rapid pHi transients. We previously reported that pHi in HF cells is rapidly raised by growth factors due to activation of a Na+/H+ exchange mechanism in the plasma membrane ( Moolenaar , W. H., Tsien , R. Y., van der Saag , P. T., and de Laat , S. W. (1983) Nature (Lond.) 304, 645-648). Here we characterize the ionic basis of pHi homeostasis in quiescent HF cells. When HF cells are acid-loaded by externally applied weak acids or by pretreatment with NH4+, pHi immediately recovers toward its resting value (approximately 7.05). pHi recovery follows an exponential time course and is accompanied by enhanced Na+ influx and net H+ extrusion. Recovery of pHi and concomitant Na+/H+ fluxes are reversibly inhibited by amiloride (half-maximal effect at approximately 0.1 mM). The rate of pHi recovery from an acid load depends on external Na+ (half-maximal rate at approximately 35 mM), but is independent of external anions (HCO3-, Cl-) and is not affected by membrane depolarization. Li+ can substitute for Na+ in pHi recovery. In Na+-free media, pHi spontaneously falls to a new resting value, from which it rapidly recovers after readdition of Na+. A stepwise increase in external pH (pHo) accelerates pHi recovery from an acid load and raises the resting pHi by approximately 50% of the pHo shift. The response of pHi to alkaline pHo shifts is abolished by amiloride and by Na+ removal. It is concluded that pHi in HF cells is closely regulated by an amiloride-sensitive, reversible Na+/H+ exchanger, which is driven by the transmembrane concentration gradients for Na+ and H+. Under normal conditions, the exchanger appears to be relatively inactive, while its rate is increasingly stimulated by lowering pHi or by raising pHo.

Addition of epidermal growth factor (EGF) to quiescent confluent cultures of human foreskin fibroblasts causes a rapid, nearly 2-fold stimulation of unidirectional Na+ influx and a doubling of the rate of the Na+,K+ pump, whereas K+ efflux remains unaltered. The diuretic amiloride, an inhibitor of Na+/H+ exchange, completely blocks EGF-induced Na+ influx, Na+,K+-pump activity, and DNA synthesis without affecting the cellular binding, visible clustering, and internalization of 125I-labeled and fluorescent EGF. In the absence of EGF, the induction of amiloride-sensitive Na+ influx and Na+,K+-pump activity can be mimicked by exposing the cells to weak acids. Neither the rapid stimulation of Na+ influx by EGF nor its inhibition by amiloride is accompanied by a detectable change in membrane potential (mean value of -66 mV), as evidenced by direct intracellular recording. In contrast, a rapid but transient membrane depolarization of about 50 mV, due to an unselective permeability increase, is observed in response to serum-growth factors. These results (i) indicate that EGF rapidly activates an electroneutral, previously inactive Na+ transport system in the plasma membrane of quiescent fibroblasts, and (ii) suggest that EGF-induced Na+ influx occurs in exchange for intracellular protons. The data further imply that early changes in membrane potential are not necessary for the initiation of a mitogenic response.

Many cell types display two classes of epidermal growth factor receptor (EGFR) as judged from EGF binding studies; i.e., a major class of low affinity EGFR and a minor class of high affinity EGFR. We have studied their respective contribution to the cascade of events elicited by EGF in human A431 carcinoma cells, using anti-EGFR mAb 2E9. This antibody specifically blocks EGF binding to low affinity EGFR, without activating receptors in intact cells, and thus enables us to study the effects of exclusive EGF binding to high affinity EGFR. We show that blocking of low affinity EGFR by mAb 2E9 has almost no effect on the activation of the receptor protein-tyrosine kinase by EGF, suggesting that EGFR kinase activation occurs exclusively through the subclass of high affinity EGFR (5-10%). In addition, we provide evidence that high affinity EGFR exists both in monomeric and dimeric forms, and that cross-phosphorylation of low affinity EGFR by high affinity EGFR may take place in dimers of both receptor types. We demonstrate that the following early cellular response to EGF are also unimpaired in the presence of mAb 2E9: (a) inositol phosphate production, (b) release of Ca2+ from intracellular stores, (c) rise in intracellular pH, (d) phosphorylation of EGF on threonine residue 654, (e) induction of c-fos gene expression, and (f) alteration in cell morphology. As possible nonspecific side effects, we observed that the EGF induced Ca2+ influx and fluid-phase pinocytosis were inhibited in A431 cells in the presence of mAb 2E9. We conclude, therefore, that the activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR.

The mechanisms by which growth factors stimulate metabolism and cell proliferation are largely unknown. Recent evidence suggests that mitogens rapidly activate a Na+/H+ exchange mechanism in the plasma membrane of their target cells, implicating cytoplasmic pH (pH1) as a potential 'messenger'. Indeed, growth stimulation of quiescent fibroblasts leads to intracellular alkalinization at approximately 1 h after mitogen addition, as measured by weak-acid distribution methods. We have used an internalized fluorescent pH1 indicator to examine the pH1-regulating mechanisms in diploid human fibroblasts and to obtain the first continuous pH1 recordings of the response to growth factors. We report here that (1) pH1 in human fibroblasts is controlled by a membrane-bound Na+/H+ exchanger, which rapidly restores pH1 after an acute cytoplasmic acidification, and (2) epidermal growth factor (EGF) and serum factors induce a rapid and persistent elevation of pH1 by modifying the pH1 sensitivity of the Na+H+ exchanger. We conclude that in addition to having a basic role in pH1 regulation, Na+/H+ exchange may function as a transmembrane signal transducer in the action of growth factors.

Addition of platelet-derived growth factor, epidermal growth factor, or serum to quiescent human fibroblasts, loaded with the fluorescent Ca2+ indicator quin-2, causes an immediate, up to 3-fold, rise in cytoplasmic free Ca2+ concentration [( Ca2+]i). In contrast, insulin and tumor-promoting phorbol ester have no effect on [Ca2+]i. The mitogen-induced [Ca2+]i response is initiated within a few s, reaches a maximum by 20-40 s, and then slowly declines to a new steady level. The [Ca2+]i response is not prevented by removal of external Ca2+ and is independent of the transmembrane Na+ gradient and membrane potential. It is concluded that platelet-derived growth factor, epidermal growth factor, and serum rapidly mobilize Ca2+ from intracellular stores, presumably due to the prior breakdown of inositol phospholipids, and that the resulting rise in [Ca2+]i may function as an initial signal in growth factor action.

Three biologically active monoclonal antibodies against the human epidermal growth factor (EGF) receptor (2E9, 2D11 and 2G5) have been used to analyse the interrelationship between various cellular responses to EGF. Antibody 2E9 (IgG1) is directed against the protein core of the receptor, close to or at the EGF binding site, while 2D11 (IgG3) and 2G5 (IgG2a) recognize blood-group A-related carbohydrate determinants of the receptor. These antibodies have EGF-like effects in that they can activate the receptor tyrosine kinase both in vitro and in vivo. Cross-linking of the receptor-bound antibodies by a second antibody mimics EGF in inducing a rapid aggregation of receptors on the cell surface. However, all three antibodies fail to mimic EGF in raising cytoplasmic pH and free Ca2+ and do not stimulate DNA synthesis in quiescent fibroblasts, even after external cross-linking of the occupied receptors. It is concluded that EGF-R tyrosine kinase activity as well as substrate specificity can be modulated by ligands other than EGF, even if they bind to sites distinct from the EGF binding domain; activation of the receptor tyrosine kinase, receptor clustering and induction of the ionic signals are causally unrelated events; and tyrosine kinase activation and receptor cross-linking are not sufficient for stimulation of DNA synthesis.

Addition of growth factors to responsive cells in HCO3- -free media results in a rapid rise in cytoplasmic pH (pHi) caused by activation of Na+/H+ exchange. In this paper, we have examined how pHi regulation and growth factor responsiveness are affected by HCO3(-)using quiescent mouse MES-1 fibroblastic cells as a model. When cells are exposed to 25 mM HCO3-, 5% CO2, steady-state pHi reaches a new more alkaline level (by 0.25 unit) within 10 min. This rise in pHi is both Na+- and HCO3- -dependent, does not occur in Cl(-)-depleted cells, and is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by 5-(n,n-dimethyl)-amiloride, indicating the involvement of Na+-dependent HCO3-/Cl- exchange. Furthermore, the recovery of pHi from acute acid loads is accelerated by HCO3- in a Na+-dependent and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive manner and is blocked in Cl(-) -depleted cells. Similar results were obtained for mouse 3T3 cells and human fibroblasts. In the presence of HCO3-/CO2 (pH 7.35), mitogens and phorbol esters fail to induce a detectable rise in pHi. However, when steady-state pHi is artificially lowered by approximately 0.4 unit, growth factors evoke significant increases in pHi due to activation of Na+/H+ exchange. In the absence of HCO3-, mitogen-induced alkalinizations are readily detectable but not when pHi is artificially elevated to the value normally observed in HCO3- media. From these results we conclude that: 1) Na+-dependent HCO3-/Cl- exchange determines steady-state pHi and acts in parallel with Na+/H+ exchange to stimulate pHi recovery from acid loading; 2) Na+-dependent HCO3-/Cl- exchange raises steady-state pHi to a level beyond the operating range of the Na+/H+ exchanger and thereby prevents growth factors from alkalinizing the cytoplasm any further. The results also imply that, unlike Na+/H+ exchange, Na+-dependent HCO3-/Cl- exchange is not activated by mitogens.

Lactate production measurements during the cell cycle of synchronized populations of Dictyostelium discoideum cells reveal cyclic variations in glycolysis which correspond with pH i oscillations which were discovered by us previously [(1985) Cell, in press]. Aerobic lactate production varies about 6-fold during the cell cycle and the lactate maxima correlate with (~ 0.25 pH unit) cyclic increases in pH. However, artificially altering pH i using weak acids or bases does not influence the rate of lactate production in asynchronous cell populations. This result suggests that the cyclic variations in pH i and those in glycolytic rate are not causally related events.

We have examined the functional properties and growth factor responsiveness of the plasma membrane Na+/H+ exchanger in pluripotent P19 embryonal carcinoma (EC) cells and in a differentiated mesodermal derivative (MES-1) by analyzing the recovery of cytoplasmic pH (pHi) from an acute acid load under bicarbonate-free conditions. In the absence of exogenous growth factors, the mean steady-state pHi of undifferentiated P19 cells (7.49 +/- 0.03) is 0.55 unit higher than the value of differentiated MES-1 cells (6.94 +/- 0.01). In both cell types, recovery of pHi from an NH+4-induced acid load follows an exponential time course and is entirely mediated by the amiloride-sensitive Na+/H+ exchanger in the plasma membrane. Kinetic analysis indicates that the higher steady-state pHi in P19 EC cells is due to an alkaline shift in the pHi sensitivity of the Na+/H+ exchange rate, as compared to that in MES-1 cells. The Na+/H+ exchanger of MES-1 cells is responsive to epidermal growth factor, platelet-derived growth factor, serum, phorbol esters, and diacylglycerol, as shown by a rapid amiloride-sensitive rise in pHi of 0.15-0.35 unit. This mitogen-induced alkalinization is attributable to an alteration in the pHi sensitivity of the exchanger. In contrast, the Na+/H+ exchanger of P19 EC cells fails to respond to any of these stimuli. Similarly, hypertonic medium rapidly activates the Na+/H+ exchanger in MES-1, but not in P19 EC cells. We conclude that the Na+/H+ exchanger in undifferentiated P19 EC stem cells is maintained in a fully activated state which is unaffected by extracellular stimuli, as if signal pathways normally involved in growth factor action are constitutively operative.

In human A431 epidermoid carcinoma cells, epidermal growth factor (EGF) rapidly stimulates the breakdown of inositol phospholipids and raises cytoplasmic free [Ca2+]. In this paper, we investigate the action of EGF on inositol phosphate metabolism, and we compare it with the previously described effects of bradykinin on the same cell system [Tilly, van Paridon, Verlaan, Wirtz, de Laat & Moolenaar (1987) Biochem. J. 244, 129-135]. In cells prelabelled with [3H]inositol, EGF slowly but persistently (for at least 30 min) stimulates the formation of [3H]inositol phosphates, whereas bradykinin causes an immediate but transient release of inositol phosphates, which lasts for only a few minutes. The EGF effect is additive to bradykinin stimulation and does not require extracellular Ca2+. In contrast, inositol phosphate formation induced by Ca2+-ionophore A23187 has an absolute requirement for external Ca2+. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate completely abolishes the response to EGF and to sub-optimal doses of bradykinin, suggesting a negative-feedback function of protein kinase C. Pretreatment of the cells with pertussis toxin has no effect on inositol phosphate formation induced by either EGF or bradykinin. Unlike bradykinin, EGF stimulates very little accumulation of inositol 1,4,5-trisphosphate, with only a small and rather variable release of Ca2+ from intracellular stores. EGF rapidly but transiently increases inositol 1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate, but the effects are much smaller than those of bradykinin. In addition, EGF increases both inositol mono- and bis-phosphate. At 10 min after EGF addition, inositol monophosphate, unlike the other inositol phosphates, is still increasing. It is concluded that the EGF-dependent pattern of stimulation is different from that observed in bradykinin-stimulated A431 cells, suggesting that there are separate mechanisms of inositol-lipid hydrolysis involved.

The transmembrane potential of Rous sarcoma virus (RSV)-infected Rat-1 cells, expressing the pp60v-src protein kinase, is markedly less negative (by approximately 30 mV) than that of their normal counterparts. By contrast, the membrane potential of Rat-1 cells infected with Kirsten sarcoma virus is virtually unaltered. The RSV-induced membrane depolarization is shown to be due to a severalfold increase in the cation permeability ratio (PNa/PK) of the plasma membrane. When cells infected with a temperature-sensitive mutant of RSV (ts LA29), encoding a src protein with heat-labile kinase activity, are shifted from the nonpermissive to the permissive temperature, a rapid and sustained membrane depolarization is observed. Conversely, thermal inactivation of the ts LA29 pp60v-src kinase activity rapidly restores the membrane potential to near normal levels. Addition of epidermal growth factor, platelet-derived growth factor, or insulin to uninfected cells fails to cause a detectable change in membrane potential. We conclude that, unlike growth factor receptor tyrosine kinases, pp60v-src can induce, either directly or indirectly, a major change in the membrane permeability to monovalent cations.

Rapid effects of serum stimulation on electrical and ionic membrane properties and their relationship to the initiation of DNA synthesis and cell division have been investigated in mouse N1E-115 neuroblastoma cells. Addition of 10% fetal calf serum to serum-deprived N1E-115 cells results in the initiation of DNA synthesis after a lag of approximately 10 hr. The earliest events following serum addition include: transient membrane potential and resistance changes, detectable within seconds and lasting 5–10 min; a persistent increase in the initial rate of 22 Na + influx, the major part of which is not of electrodiffusional origin, and which is potentiated by weak acid anions; and an external Na + -dependent increase in the rate of the Na + ,K + pump. In the absence of serum the stimulation of the Na + ,K + pump can be mimicked by increasing net Na + influx with monensin or neurotoxins. Growth-depleted serum fails to induce any of the electrical and ionic events. The diuretic amiloride (0.4 mM) inhibits serum-induced Na + influx, Na + ,K + pump stimulation and DNA synthesis, but does not affect the electrical response or the basal influx rates. The results suggest that serum growth factors act, at least in part, by stimulating an electroneutral, amiloridesensitive Na + /H + exchange mechanism. The enhanced Na + influx then results in the observed stimulation of the Na + ,K + pump, while the simultaneous efflux of protons may raise the intracellular pH.

Neuroblastoma is a pediatric embryonal malignancy characterized by impaired neuronal differentiation. A better understanding of neuroblastoma differentiation is essential for developing new therapeutic approaches. GDE2 (encoded by GDPD5) is a six-transmembrane-domain glycerophosphodiesterase that promotes embryonic neurogenesis. We find that high GDPD5 expression is strongly associated with favorable outcome in neuroblastoma. GDE2 induces differentiation of neuroblastoma cells, suppresses cell motility, and opposes RhoA-driven neurite retraction. GDE2 alters the Rac-RhoA activity balance and the expression of multiple differentiation-associated genes. Mechanistically, GDE2 acts by cleaving (in cis) and releasing glycosylphosphatidylinositol-anchored glypican-6, a putative co-receptor. A single point mutation in the ectodomain abolishes GDE2 function. Our results reveal GDE2 as a cell-autonomous inducer of neuroblastoma differentiation with prognostic significance and potential therapeutic value.

Activation of protein kinase C (PKC) in Rat-1 fibroblasts leads to rapid phosphorylation of an 80-kDa protein, a major substrate of PKC. Digiton-in-permeabilized cells perfectly supported this early response. Introduction of a PKC pseudosubstrate peptide inhibited 80 kDa phosphorylation with an IC 50 of 1 μM, while a control peptide had no effect. The results indicate that this semi-intact cell system can be used in combination with the inhibitory pseudosubstrate peptide to study the involvement of PKC in cellular processes.

Examination of ionic membrane currents in a voltage-clamped neuronal cell line derived from the mouse C1300 neuroblastoma disclosed four kinetically different components: sodium, potassium, calcium, and leakage current. The kinetics, voltage dependence, and pharmacological properties of the sodium and potassium currents qualitatively resemble those of the corresponding currents in squid giant axon and frog myelinated nerve fiber, suggesting that the molecular structures of the sodium and potassium channels in neuroblastoma are similar to those of the non-mammalian preparations.

Addition of the nonapeptide bradykinin to human A431 epidermoid carcinoma cells causes an immediate release of inositol phosphates and a rapid rise in [Ca2+]i. Half-maximal stimulation occurs at a bradykinin concentration of 4 nM. In the continuous presence of a saturating hormone concentration (1 μM) the inositol phosphate accumulation levels off within approximately 2 min; however, subsequent stimulation with histamine, another activator of phospholipase C, gives an additional increase in inositol phosphate production. Separation of the inositol phosphates by HPLC-anion exchange chromatography, reveals a rapid but transient accumulation of Ins(1,4,5)P3, followed by an increase in Ins (1,3,4,5)P4 and Ins(1,3,4)P3. Our data support a precursor/product-relationship between Ins(1,4,5)P3 and Ins(1,3,4)P3 with Ins(1,3,4,5)P4 as intermediate and argue against a messenger role of Ins(1,3,4,5)P4 and Ins(1,3,4)P3 in Ca2+ signalling.

Nerve growth factor (NGF) induces neuronal differentiation of rat pheochromocytoma cells (PC12). Here we show that NGF causes a stimulation of Na + ,K + -pump mediated K + influx, with a maximum at 30 min after addition of NGF. The stimulation of the Na + ,K + -pump is completely blocked by the Na + -flux inhibitor amiloride (0.2 mM) and can be mimicked by the Na + ionophore monensin. These results suggest that NGF causes a rapid enhancement of Na + influx leading to an activation of the Na + ,K + -pump, a mechanism similar to the action of other growth factors.

Stimulation of human A431 epidermoid carcinoma cells by bradykinin causes a very rapid release of inositol phosphates and a transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). Bradykinin-induced inositol phosphate formation is half-maximal at a concentration of 4 nM and is not affected by pertussis toxin. H.p.l.c. analysis of the various inositol phosphates shows an immediate but transient accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reaches a peak value of approx. 10 times the basal level within 15 s and slightly precedes the rise in [Ca2+]i, both parameters changing in parallel. After a lag period, bradykinin also induces a massive accumulation of Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Our data support the view that part of the newly formed Ins(1,4,5)P3 is converted into Ins(1,3,4)P3 phosphorylation/dephosphorylation with Ins(1,3,4,5)P4 as intermediate. Furthermore, A431 cells were found to contain strikingly high basal levels of two other inositol phosphates, presumably inositol pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6), representing more than 50% of the total 3H radioactivity incorporated into inositol phosphates. The presumptive InsP5 and InsP6 are only slightly affected by bradykinin. Although Ins(1,3,4)P3 and InsP4 could function as second messengers, our results suggest that, unlike Ins(1,4,5)P3, neither Ins(1,3,4)P3 nor InsP4 are involved in Ca2+ mobilization.

The EGF-receptor (EGF-R) is a transmembrane glycoprotein with intrinsic protein tyrosine kinase (TK) activity. To explore the importance of the receptor TK in the action of EGF, we have used transfected NIH-3T3 cells expressing either the normal human EGF-R or a receptor mutated at Lys721, a key residue in the presumed ATP-binding region. The wild-type receptor responds to EGF by causing inositol phosphate formation, Ca2+ influx, activation of Na+/H+ exchange and DNA synthesis. In contrast, the TK-deficient mutant receptor fails to evoke any of these responses. It is concluded that activation of the receptor TK is a crucial signal that initiates the multiple post-receptor effects of EGF leading to DNA synthesis. Furthermore, the results suggest that tyrosine phosphorylation plays a role in the activation of the phosphoinositide signalling system.