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Phosphatidic acid (PA), an intriguing phospholipid that is rapidly produced during receptor-stimulated breakdown of phosphoinositides, has often been proposed to function as a Ca2+ ionophore in activated cells. The PA-ionophore hypothesis is supported by the fact that exogenously applied PA stimulates Ca2+ uptake in various cells and can evoke Ca2+-mediated physiological responses, but it is not known whether PA accumulation affects cytoplasmic free Ca2+ concentration ([Ca2+]i). Here we report that PA elicits a transient rise in [Ca2+]i in cultured cells, not by stimulating Ca2+ influx, but, surprisingly, by releasing Ca2+ from intracellular stores. We further show that PA evokes growth factor-like effects in that it raises cytoplasmic pH, induces expression of the c-fos and c-myc proto-oncogenes and stimulates DNA synthesis. Our results indicate that, unlike an ionophore, PA acts by triggering the hydrolysis of phosphoinositides, with consequent formation of second messengers such as inositol trisphosphate signalling Cai2+ release. Furthermore, our data strengthen the notion that any Ca2+-mobilizing stimulus acting through phospholipase C may ultimately function as a growth factor.

The proliferation of cells in vivo and in culture is regulated by polypeptide growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Growth factors initiate their action by binding to specific cell surface receptors. Receptor occupancy triggers a cascade of physiological changes in the target cell which ultimately lead to DNA synthesis and cell division. Immediate consequences of receptor activation include tyrosine-specific protein phosphorylations, a sustained increase in cytoplasmic pH (pH,) and a transient rise in free Ca2+. The rise in pH, has a permissive effect on DNA synthesis and is mediated by an otherwise quiescent Na+/H+ exchange mechanism in the plasma membrane, which is turned on by protein kinase C, the cellular receptor for phorbol esters. The rapid Ca2+ signal is due to either release from internal stores (PDGF) or net entry via a voltage-independent channel in the plasma membrane (EGF). Phorbol esters, acting via kinase C, inhibit the growth factor-induced Ca2+ signals without affecting resting Ca2+ levels. Monoclonal antibodies against the human EGF receptor can act as partial agonists in that they activate the tyrosine-specific protein kinase without inducing any of the ionic signals. These antibodies fail to induce DNA synthesis when added to quiescent fibroblasts, indicating that the Ca2+ and pH, signals can be dissociated from tyrosine kinase activity and suggesting that these signals are indispensable for the stimulation of cell proliferation.

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.

The cell surface plays a crucial role in the regulation of cell metabolism in general and in the regulation of cell proliferation in particular. Receptor proteins in the plasma membrane receive and transduce growth-stimulatory signals that trigger a cascade of biochemical and physiological changes in the cell. Ultimately, this chain of events culminates in replicative DNA synthesis and cell division. Among the most extensively studied mitogens are the polypeptide hormones epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Although much has been learned about the interaction of EGF and PDGF with their specific receptors on the cell surface, relatively little is known about the ensuing intracellular signals transmitting the mitogenic response from the plasma membrane to the cell nucleus. In the search for putative mitogenic signals, much attention has been focused on the earliest detectable cellular reactions following the addition of growth factors to appropriate target cells. Immediate consequences of growth factor-receptor interaction include tyrosine-specific protein phosphorylations (Ushiro and Cohen, 1980; Ek et al., 1982; Cooper et al., 1982), breakdown of inositol phospholipids (Sawyer and Cohen, 1981; Habenicht et al., 1981), and increased transport of Na+, K+, and Ca2+ across the plasma membrane (Rozengurt and Heppel, 1975; Moolenaar et al., 1981a, 1982a,b; Sawyer and Cohen, 1981).

Among the immediate consequences of growth factor-receptor interaction are the activation of various ion transport systems in the plasma membrane and changes in intracellular ionic composition. Mitogen-induced alterations in the levels of cytoplasmic free Ca2+ ([Ca2+]i) and H+ are of special interest, because these ions are thought to serve as second messengers that trigger and regulate cell proliferation. During the past few years our understanding of the ionic changes in growth factor-stimulated cells has increased dramatically. In particular, the molecular mechanisms responsible for a transient rise in [Ca2+]i and for a sustained increase in cytoplasmic pH (pHi) have been largely unraveled, and much effort is now being devoted to determining how these ionic signals might contribute to a proliferative response. This review describes the Ca2+ and pH signals generated by growth factor receptors, with emphasis on the activation of Na+ /H+ exchange and the resultant rise in pHi, which has a permissive effect on the initiation of DNA synthesis in mitogenstimulated cells.

Addition of bradykinin to mouse N1E-115 neuroblastoma cells evokes a rapid but transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i rise is accompanied by a transient membrane hyperpolarization, due to a several-fold increase in K+ conductance, followed by a prolonged depolarizing phase. Pre-treatment of the cells with a Ca2+-ionophore abolishes the hormone-induced hyperpolarization but leaves the depolarizing phase intact. The transient hyperpolarization can be mimicked by iontophoretic injection of IP3(1,4,5) or Ca2+, but not by injection of IP3(1,3,4), IP4(1,3,4,5) or Mg2+ into the cells. Instead, IP3(1,3,4) evokes a small but significant membrane depolarization in about 50% of the cells tested. Microinjected IP4(1,3,4,5) has no detectable effect, nor has treatment of the cells with phorbol esters. These results suggest that, while IP3(1,4,5) triggers the release of stored Ca2+ to hyperpolarize the membrane, IP3(1,3,4) may initiate a membrane depolarization.

Aggregation of pluripotent P19 embryonal carcinoma (EC) cells in the presence of DMSO induces differentiation to various mesodermal cell types, including spontaneously contracting muscle [9]. We have established clonal cell lines from these cultures and characterized one (MES-1) in particular for its response to growth factors. In contrast to the undifferentiated stem cells, but as a number of myoblast and muscle cell lines, MES-1 cells respond to both carbachol and bradykinin by the rapid release of Ca 2+ from intracellular stores. In addition, MES-1 express receptors for and respond mitogenically to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Isolated membranes from these cells retain the capacity to bind both ligands; addition of EGF to membranes induces endogenous phosphorylation of several proteins, including the EGF receptor itself and a 38 kD protein, while addition of PDGF specifically induces phosphorylation of the PDGF receptor. By contrast, other derivatives of P19, isolated from retinoic acid (RA)-treated aggregates and resembling neuroectodermal or endodermal cell types [10] respond only to EGF; PDGF neither binds nor induces phosphorylation and a mitogenic response in these cells. During differentiation from EC cells therefore MES-1 cells developed a combination of growth factor receptor characteristics typical of somatic mesodermal cells and indicate that such receptors on EC-derived mesodermal cells are also functional.

Lysophosphatidate (LPA), the simplest natural phospholipid, is highly mitogenic for quiescent fibroblasts. LPA-induced cell proliferation is not dependent on other mitogens and is blocked by pertussis toxin. LPA initiates at least three separate signaling cascades: activation of a pertussis toxin-insensitive G protein mediating phosphoinositide hydrolysis with subsequent Ca2+ mobilization and stimulation of protein kinase C; release of arachidonic acid in a GTP-dependent manner, but independent of prior phosphoinositide hydrolysis; and activation of a pertussis toxin-sensitive Gi protein mediating inhibition of adenylate cyclase. The peptide bradykinin mimics LPA in inducing the first two responses but fails to activate Gi and to stimulate DNA synthesis. Our data suggest that the mitogenic action of LPA occurs through Gi or a related pertussis toxin substrate and that the phosphoinositide hydrolysis-protein kinase C pathway is neither required nor sufficient, by itself, for mitogenesis. The results further suggest that LPA or LPA-like phospholipids may have a novel role in G protein-mediated signal transduction.

Publisher Summary Polypeptide growth factors play a vital role in the regulation of cell proliferation and development. Several of these factors have been isolated and purified to homogeneity from various biological sources. This chapter focuses on the generation of ionic signals by growth factors, with particular emphasis on the activation of Na+–H+ exchange and the mobilization of Ca2+. Some approaches to assess the interrelationship among the various signal pathways have been described, especially the use of tumor-promoting phorbol esters and of monoclonal antibodies against the human epidermal growth factor (EGF) receptor. The ionic signals in growth factor action include (1) the activation of Na+–H+ exchange, (2) Ca2+ mobilization by growth factors, and (3) the role of inositol lipid breakdown and protein kinase C. Numerous cellular functions are regulated by changes in cytoplasmic free Ca2+ and many of them through the formation of Ca2+ calmodulin complexes. With respect to mitogen action, a rapid rise in Ca2+ seems to be a critical signal for lymphocytes to enter the mitotic cycle. Another powerful tool for the dissociation of molecular events in the mitogenic signal pathway is achieved by the availability of monoclonal antibodies against the EGF receptor.

Cytoplasmic pH (pH l ) was monitored during the cell cycle of synchronous populations of Dictyostelium discoideum by means of a pH "null point" method. There is a cycle of pH l that closely corresponds to the DNA replication cycle, with a minimum of pH 7.20 in interphase and a peak of pH 7.45 during S phase and mitosis. When DNA replication is blocked by hydroxyurea, pH l continues to oscillate with a similar period as the uninhibited division cycle. Even when protein synthesis is inhibited by cycloheximide the periodic pH l cycles persist. Artificially raising pH l in exponentially growing cells by ∼0.1 units causes a severalfold increase in the rates of protein and DNA synthesis. We conclude that an autonomous pH l oscillator exists in Dictyostelium cells, which operates independently of protein and DNA synthesis and which may have a major role in the timing and regulation of the cell cycle.