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Biomedical subjects

W H Moolenaar

Publications and source records attributed to W H Moolenaar.

At least 109 records · Page 6Linked to original sources
PubMed1986

Calcium in the action of growth factors.

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). Binding of growth factors to their specific cell-surface receptors initiates a cascade of biochemical events in the cell which ultimately leads to DNA synthesis and cell division. Immediate consequences of receptor activation include tyrosine-specific protein phosphorylations, a sustained increase in cytoplasmic pH and a transient rise in cytoplasmic free Ca2+. The PDGF-induced Ca2+ signal is due to Ca2+ release from intracellular stores, whereas EGF seems to activate a voltage-independent Ca2+ channel in the plasma membrane. Monoclonal antibodies to the EGF receptor that stimulate the tyrosine-specific protein kinase fail to raise [Ca2+]i and are not mitogenic for quiescent cells. These results suggest that activation of the EGF receptor tyrosine kinase is not sufficient for the induction of a Ca2+ signal, and that the rise in [Ca2+]i is indispensable for cell proliferation.

Animals↗
PubMed1986

Dissociation of cellular responses to epidermal growth factor using anti-receptor monoclonal antibodies.

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.

Amino Acids↗
PubMed1986

Establishment of a differentiated mesodermal line from P19 EC cells expressing functional PDGF and EGF receptors.

Aggregation of pluripotent P19 embryonal carcinoma (EC) cells in the presence of DMSO induces differentiation to various mesodermal cell types, including spontaneously contracting muscle. 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 Ca2+ 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 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.

Animals↗
PubMed1986

Ionic signalling by growth factor receptors.

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 (pHi) and a transient rise in free Ca2+. The rise in pHi 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 pHi signals can be dissociated from tyrosine kinase activity and suggesting that these signals are indispensable for the stimulation of cell proliferation.

Animals↗
PubMed1985

Cytoplasmic pH and the regulation of the Dictyostelium cell cycle.

Cytoplasmic pH (pHi) 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 pHi 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, pHi continues to oscillate with a similar period as the uninhibited division cycle. Even when protein synthesis is inhibited by cycloheximide the periodic pHi cycles persist. Artificially raising pHi in exponentially growing cells by approximately 0.1 units causes a severalfold increase in the rates of protein and DNA synthesis. We conclude that an autonomous pHi 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.

Cell Cycle↗
PubMed1985

Ionic signal transduction in growth factor action.

Growth factors are polypeptides which exert their mitogenic action through binding to specific high-affinity receptor molecules on the cell surface of target cells. This interaction leads to the rapid activation of a receptor-linked signal transduction system, involving the stimulation of an intrinsic receptor tyrosine phosphokinase activity, the breakdown of inositol lipids, and the production of ionic signals. In this contribution we have analysed the nature and origin of the ionic signals, and we have applied monoclonal antibodies against the receptor for epidermal growth factor (EGF), as well as tumour-promoting phorbol esters, to dissociate the early cellular responses to growth factors. Evidence is presented that the ionic signals are coupled to the breakdown of inositol lipids. The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) would lead to the production of 1,2-diacylglycerol (DG) and inositol triphosphate (IP3). DG production results in the stimulation of protein kinase C, which causes the activation of Na+/H+ exchange by increasing its affinity for cytoplasmic H+. Consequently, a rise in cytoplasmic pH is observed. This response can be mimicked by the tumour promoter TPA, which can replace DG in activating the protein kinase C. Independently, IP3 production leads to the rapid mobilization of Ca2+ from intracellular stores. Monoclonal antibodies against the EGF receptor differed in their ability to evoke EGF-like responses upon binding to the EGF receptor. Of the three anti-EGF receptor IgGs tested, one was directed against the EGF-binding domain (2E9), and the others (2D11 and 2G5) were directed against sugar moieties not involved in EGF binding. Receptor tyrosine kinase activity could be stimulated by 2E9 as well as 2D11, but not by 2G5. Only 2D11 induced morphological changes similar to EGF. None of the antibodies was able to trigger the production of ionic signals, which implies probably that antibody binding to the receptor, even when they bind to the EGF binding domain, is an insufficient stimulus for the breakdown of inositol lipids. Most importantly, these monoclonal antibodies were also not able to induce DNA synthesis in quiescent human fibroblasts, not even after cross-linking of the EGF receptors by a second antibody. It may thus be concluded that the stimulation of the intrinsic receptor tyrosine phosphokinase activity can be dissociated from other early responses, and that none of the identified early responses is a sufficient trigger for the mitogenic action of growth factors.

Animals↗
PubMed1984

Growth factors immediately raise cytoplasmic free Ca2+ in human fibroblasts.

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.

Aminoquinolines↗
PubMed1984

The regulation of cytoplasmic pH in human fibroblasts.

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.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1983

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells.

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.

Amiloride↗
PubMed1982

Epidermal growth factor induces electrically silent Na+ influx in human fibroblasts.

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.

Amiloride↗
PubMed1981

Sodium/proton exchange in mouse neuroblastoma cells.

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.

Amiloride↗
PubMed1981

Rapid ionic events and the initiation of growth in serum-stimulated neuroblastoma cells.

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 22Na+ 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, amiloride-sensitive 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.

Animals↗
PubMed1979

The calcium action potential and a prolonged calcium dependent after-hyperpolarization in mouse neuroblastoma cells.

1. Action potentials elicited in solutions with elevated [Ca2+] (1.8-40 mM) have been studied in differentiated cells of mouse neuroblastoma clone N1E-115 in tissue culture. 2. The action potential in high [Ca2+] solutions containing eithr Na+ or Tris is followed by a prolonged after-hyperpolarization (a.h.p.) lasting 0.5-4 sec. The a.h.p. reverses sign between -75 and -85 mV. 3. Externally applied tetraethylammonium (TEA, 15 mM) increases the Ca2+ spike overshoot, prolongs the falling phase and enhances the a.h.p. duration. The a.h.p. is inhibited by Ca2+ antagonists such as La3+, Co2+ and Mn2+. 4. After replacement of Ca2+ by Ba+ or Sr2+ (20mM) action potentials can still be elicited in Na+-free solution, but no a.h.p. is observed. 5. Increasing [Ca2+] from 1.8 up to 20 mM results in an increased capability of neuroblastoma cells to fire repetitively and in a consistent reduction of the firing rate from about 4-10 sec-1 to 0.5-1.8 sec-1. 6. It is concluded that Ca2+ entry during the action potential activates a TEA-resistant K+ conductance which gives rise to the prolonged a.h.p. Data from repetitively firing cells are consistent with the view that the a.h.p. plays a role in the regulation of low-frequency firing.

Action Potentials↗