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J Boonstra

Publications and source records attributed to J Boonstra.

At least 127 records · Page 7Linked to original sources
PubMed1985

Immunocytochemical demonstrations of cytoplasmic and cell-surface EGF receptors in A431 cells using cryo-ultramicrotomy, surface replication, freeze-etching and label fracture.

In this paper we describe the use of a number of complimentary methods to visualize cytoplasmic and cell-surface located epidermal growth factor (EGF) receptors in cultured A431 cells. Cryo-ultramicrotomy in combination with immuno-gold labelling will be shown to provide an excellent method in visualizing cytoplasmic located EGF receptors in addition to cell-surface located EGF receptors. An important aspect in this method involves the possible effects of the fixatives on antigenicity. Using radioactive labelled anti EGF receptor antibodies, it was shown that formaldehyde as a fixative had no significant effect on label-efficiency. The density and lateral distribution of EGF receptors at the cell surface has been studied by three methods, i.e. surface replication, freeze etching and label fracture, all methods in conjunction with immuno-gold labelling. These methods allow in principle a quantitation of the surface distribution of the EGF receptors. The surface-replication method involves, however, dehydration and critical-point drying steps, and using radioactive labelled anti EGF receptor antibodies it was shown that in particular OsO4 fixation and dehydration caused a significant loss of cell-associated antibodies. This disadvantage is overcome by freeze etching and the label-fracture method, and as such these techniques provide the best methods for quantitative analysis of the planar distribution of cell-surface located EGF-receptors.

Antigen-Antibody Reactions↗
PubMed1985

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling.

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.

Carcinoma, Squamous Cell↗
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

Analysis of K+ and Na+ transport and intracellular contents during and after heat shock and their role in protein synthesis in rat hepatoma cells.

Heat shock at 42 degrees caused a rapid inhibition of protein synthesis in Reuber H35 hepatoma cells. Inhibition was maximal within 5 min after the temperature was increased. After heat shock at 42 degrees for 30 min, protein synthesis was restored in 4 to 5 hr. Heat shock did not inhibit amino acid transport or cause a decrease of cellular amino acid pools, excluding a direct effect of these parameters on the inhibition of protein synthesis. The same heat shock caused a stimulation of Na+-K+ pump activity, as monitored by ouabain-sensitive Rb+ influx, but the activity returned rapidly to pretreated levels after heat shock. Similar effects were observed in the passive K+ efflux. Furthermore, heating did not affect the intracellular K+ and Na+ contents. A clear difference in the effect of temperature on protein synthesis and active K+ and Na+ influx was observed. In an Arrhenius plot, a sharp break for protein synthesis was observed at 40 degrees (D. H. J. Schamhart et al., Radiat. Res., in press, 1984), while no discontinuity was observed in the Arrhenius plot for active K+ and Na+ influxes. The results demonstrate that, during and after heat shock and at various temperatures, the K+ and Na+ balances are in a continuous steady state. Experimental modification of the intracellular K+ and Na+ contents by using ouabain or the Na+ ionophore monensin revealed that, within large limits of intracellular cation contents, protein synthesis is unimpaired. These results exclude any direct involvement of K+ and Na+ in the effects of heat shock on protein synthesis in Reuber H35 hepatoma cells.

Animals↗
PubMed1983

Membrane regulation of the Na+,K+-ATPase during the neuroblastoma cell cycle: correlation with protein lateral mobility.

The pumping activity of the plasma membrane-bound Na+,K+-ATPase shows considerable variation during the cell cycle of mouse neuroblastoma Neuro-2A cells. Addition of external ATP at millimolar concentrations, which selectively enhances the plasma membrane permeability of Neuro-2A cells for sodium ions, stimulates the Na+,K+-ATPase pumping activity at all phases of the cell cycle from a factor of 1.05 in mitosis up to 2.2 in G1 phase. Determination of the number of Na+,K+-ATPase copies per cell by direct 3H-ouabain binding studies in the presence of external ATP shows a gradual increase in the number of pump sites on passing from mitosis to the late S/G2-phase by approximately a factor of 2. From these data the pumping activity per copy of Na+,K+-ATPase, optimally stimulated with respect to its various substrate ions, has been determined during the various phases of the cell cycle. This optimally stimulated pumping activity per enzyme copy, which is a reflection of the physicochemical state of the plasma membrane, is high in mitosis, almost twofold lower in early G1 phase, and increases gradually again during the other phases of the cell cycle. This shows that the observed regulation of Na+,K+-ATPase activity during the cell cycle is caused by a combination of three independent factors--namely variation in intracellular substrate availability (Na+), changes in number of enzyme copies per cell, and modulation of the plasma membrane environment of the protein molecules. The modulation of the optimal pumping activity per enzyme copy shows a good correlation (rho = 0.96) with the known modulation of protein lateral mobility during the cell cycle, such that a high protein lateral mobility correlates with a low enzyme activity. It is concluded that changes in plasma membrane properties take place during the Neuro-2A cell cycle that result in changes in the rate of protein lateral diffusion and Na+,K+-ATPase activity in directly correlated way.

Animals↗
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

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells.

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3'-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from -51 mV to -36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using 86Rb+ as a radioactive tracer for K+, demonstrated that the depolarization was not caused by changes in (Na+ + K+)-ATPase-mediated K+ influx or in the transmembrane K+ gradient. The permeability ratio (PNa/PK), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of PNa/PK was shown by 24Na+ and 86Rb+ flux measurements to be due to both an increase of the Na+ permeability and a decrease of the K+ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.

Animals↗
PubMed1982

Effect of external ATP on the plasma membrane permeability and (Na+ +K+)-ATPase activity of mouse neuroblastoma cells.

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na+ relative to K+, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K+ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na+. Under these conditions the pumping activity of the Neuro-2A (Na+ +K+)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na+ +K+)-ATPase for ouabain, as measured by direct [3H]ouabain-binding studies and by inhibition studies of active K+ uptake. In the presence of 3.5 mM ATP and the absence of external K+ both techniques indicate an apparent dissociation constant for ouabain of 2 X 10(-6)M. Neuro-2A cells contain (3.5 +/- 0.7) X 10(5) ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7 +/- 0.4) X 10(-20) mol K+/min per copy of (Na+ +K+)-ATPase at room temperature.

Adenosine Triphosphate↗
PubMed1982

Regulation of Na+,K+ pump activity by nerve growth factor in chick embryo dorsal root ganglion cells.

Nerve growth factor (NGF) is required for the growth and development of sensory and sympathetic neurons. Incubation of chick dorsal root ganglionic cells without NGF resulted in a decrease of active (Na+,K+-pump-mediated) K+ influx over a period of several hours. Addition of NGF to NGF-deprived cells caused 1) a return of the active K+ influx to the values occurring in cells continuously exposed to NGF, preceded by 2) a very rapid, but transient overstimulation of the Na+,K+-pump-mediated K+ influx. Restoration of normal Na+,K+-pump activity occurred at NGF concentrations of 1 biological unit/ml or greater, whereas the NGF concentration in the 1-100 biological unit/ml range affected the rapidity with which the pump restoration took place. The transient pump behavior was only observed in NGF-deprived cells and could not be elicited in NGF-supported steady-state cells or in cells having already received delayed NGF once. This transient Na+,K+-pump behavior was exclusively displayed in conjunction with a high intracellular Na+ concentration. Decreasing the external Na+ concentration below 70 mM reduced the hyperstimulation response to NGF, until at 10 mM Na+ the delayed presentation of NGF caused no overshoot at all. The effect of NGF on the Na+,K+-pump was specific for the NGF molecule and could not be mimicked by other proteins.

Animals↗
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

Characterization of 42K+ and 86Rb+ transport and electrical membrane properties in exponentially growing neuroblastoma cells.

For measuring K+ efflux from exponentially growing neuroblastoma cells (clone Neuro-2A), two methods were used, a sampling method and a washing method. Both methods indicated that K+ efflux kinetics were as from a two-compartment system, but the two compartments could only be resolved completely using the washing method. A fast compartment, containing 143 +/- 16 nmol K+/10(6) cells, was found to be associated to the cell surface, and a slow compartment, containing 151 +/- 7 nmol K+/10(6) cells, was found to represent the intracellular K+. The rate constant of the slow compartment was 0.0164 +/-0.0005 min-1, and the K+ efflux rate was 2.46 +/- 0.14 nmol K+/10(6) cells per min. Using the appropriate conditions to measure K+ influx, the kinetics of influx were equal to the kinetics of efflux, indicating steady-state conditions. In addition a comparison was made between 42K+ and 86Rb+ as radioactive tracers for K+ flux. It was found that 86Rb+ was specifically bound on both the inside and the outside of the cells, and for this reason was not a suitable tracer for studying K+ flux kinetics in neuro-2A cells. A membrane potential of -42.9 +/- 1.3 mV and intracellular K+ activity of 108.1 +/- 3.0 mM were measured using conventional and ion-selective microelectrodes. A correlation was made between the K+ flux and electrophysiological data, using the equations of electrodiffusion theory. Thus, the permeabilities of K+ and Na+ were calculated as (3.9 +/- 0.4) . 10(-8) cm/s and (0.6 and 0.1) . 10(-8) cm/s respectively, together with K+ conductance of (2.8 +/- 0.3) . 10(-6) omega-1/cm2.

Animals↗
PubMed1981

Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells.

Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle.

Animals↗
PubMed1981

Cation transport and growth regulation in neuroblastoma cells. Modulations of K+ transport and electrical membrane properties during the cell cycle.

Cation transport and membrane potential were studied during the cell cycle of neuroblastoma cells (clone Neuro-2A) to investigate the role of these parameters in growth regulation. The cells were synchronized by selective detachment of mitotic cells. The membrane potential and intracellular K+ activity were measured with conventional and K+-selective microelectrodes respectively. Both the membrane potential and K+ activity were high in mitosis, decreased to half maximal in G1 phase, and rose again during S phase. K+ efflux across the plasma membrane was studied with 42K+ as a radioactive tracer using a washing method for cells grown in monolayer and a continuous efflux method for mitotic cells in suspension. The intracellular K+ content and unidirectional K+ efflux rate obtained from these measurements showed modulations during the cell cycle similar to those of the membrane potential. Using equations of electrodiffusion theory the membrane permeabilities to K+ and Na+ were calculated. These permeabilities were high in mitosis, decreased rapidly in G1 phase and increased during S phase, followed by a transient decrease in G2 phase. A rapid increase was observed between G2 phase and the next mitosis. A similar pattern was obtained for the K+ conductance. K+ resistance changes during the cell cycle were similar to changes in the specific membrane resistance, measured by microelectrodes, except for the early cell cycle phases (mitosis and G1). These studies clearly demonstrate large modulations of the passive membrane permeability properties during the cell cycle. These modulations can be correlated with physicochemical membrane variations during the cell cycle, such as membrane fluidity and lateral mobility of lipids.

Animals↗