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R Zippel

Publications and source records attributed to R Zippel.

At least 55 records · Page 3Linked to original sources
PubMed1989

Voltage-dependent calcium current in adherent mouse 3T3 fibroblasts.

Whole-cell recording was performed on adherent mouse Swiss 3T3 fibroblasts. Depolarizations from a holding potential of -100 mV gave rise to a transient inward current. The voltage dependence, kinetic properties, and ionic selectivity of this current are identical to those described in the same cells kept in suspension after detachment from the culture dish [A. Pandiella, A. Malgaroli, J. Meldolesi, and L.M. Vicentini (1987) Exp. Cell. Res. 170, 175-185; W.H. Moolenaar, L.G.J. Tertoolen, and S.W. de Laat (1984) J. Biol. Chem. 259, 8066-8069].

Action Potentials↗
PubMed1989

Inhibition of phosphotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase.

In normal fibroblasts stimulated by platelet derived growth factor (PDGF), PDGF receptors are transiently phosphorylated on tyrosine and represent the major phosphotyrosine containing protein. The phosphate of the phosphotyrosine groups turns over rapidly, and extensive evidence indicates a dynamic balance between phosphorylation and dephosphorylation reactions. Thus, the effect of an inhibitor of phosphatases, orthovanadate, on the pattern of the tyrosine phosphorylations induced by PDGF in Swiss 3T3 fibroblasts was investigated. Western blot analysis with antibodies against phosphotyrosine indicated that whereas in unstimulated cells no phosphotyrosine containing proteins were detected, treatment of cells with orthovanadate alone elicited the slow phosphorylation of several proteins including a 170 kDa component that was recognized to be the phosphorylated PDGF receptor. Addition of PDGF to cells shortly pretreated with vanadate highly increased the intensity of the 170 kDa band corresponding to the phosphorylated receptor and caused its stabilization during time. In addition, the phosphorylation on tyrosine of other proteins (molecular mass 116, 80, 73, 60, 50 and 39 kDa) was also induced. Both the receptor and the other tyrosine phosphorylated proteins appeared to be associated with the detergent insoluble matrix.

Animals↗
PubMed1988

Serum induces the immediate opening of Ca2+-activated channels in quiescent human fibroblasts.

Application of fetal calf serum to quiescent human fibroblasts produces an immediate (3-20 s delay) increase in membrane conductance which lasts about 20-30 s. This conductance is strongly outwardly-rectifying and has a reversal potential between -45 and -10 mV. The conductance increase may also be induced by application of the Ca2+ ionophore A23187 while it does not occur when intracellular K+ is replaced by Cs+. It is concluded that this early effect of serum is due to the opening of Ca2+-activated channels. This permeability change will alter the membrane potential and thus possibly interact with other voltage-sensitive processes induced by serum growth factors.

Blood↗
PubMed1988

Effect of the growth conditions on the expression of cell-surface-associated platelet-derived growth factor receptors in mouse fibroblasts.

The conditions affecting the appearance and disappearance of platelet-derived growth factor (PDGF) receptors from the pool of active cell surface-associated receptors were studied. Receptor molecules were revealed in intact Swiss 3T3 fibroblasts by their ability to bind 125I-labeled PDGF and, due to their property to become phosphorylated in tyrosine following ligand binding, by antibodies to phosphotyrosine. PDGF receptor molecules were found to be quite scarce in exponentially growing fibroblasts as compared to quiescent cells. When growing cells were either shifted to a medium containing plasma or received suramin in the culture medium, cell surface-associated PDGF receptors largely increased. This process required about 12 h. Incubation of quiescent cells in serum, but not in plasma, induced a slow decrement of ligand-activatable receptors. In the presence of PDGF the rate of receptor removal from the cell surface was very rapid and was a function of the PDGF concentration. Quiescent cells deprived of cell-surface receptors by incubation with PDGF reexpressed PDGF receptors in about 14 h.

Animals↗
PubMed1988

Dissociation of the ligand and dephosphorylation of the platelet-derived growth factor receptor.

The ligand-induced phosphorylation of the platelet-derived growth factor (PDGF) receptor was followed at 37 degrees C by a rapid dephosphorylation which was roughly parallel to the down regulation of the 125I-PDGF binding sites. At 4 degrees C, when the ligand-receptor complexes remain associated with the cell surface, the phosphorylated form of the receptor was more stable. However if the ligand was dissociated from the receptor by means of a mild acid wash or a treatment with suramin, the dephosphorylation of the receptor also occurred at a low temperature. These data suggest that, due to the dissociation of the ligand, the kinase activity of the receptor is switched off so that the phosphotyrosine-containing receptors remain exposed to the action of phosphatases that rapidly dephosphorylate them.

Animals↗
PubMed1988

Epidermal growth factor-induced phosphoinositide hydrolysis. Modulation by protein kinase C.

A short-term treatment with phorbol 12,13-dibutyrate (PDBu) was found to inhibit totally the epidermal growth factor (EGF)-stimulated phosphoinositide hydrolysis in A431 cells, whereas long-term pretreatment with PDBu, which is known to down regulate protein kinase C, induced a greater accumulation of the EGF-triggered inositol phosphate accumulation, particularly of Ins(1,3,4,5)P4. The increased Ins(1,4,5)P3/Ins(1,3,4,5)P4 formation in the PDBu long-term pretreated cells was coincident with the increased Ca2+ influx stimulated by EGF in the same cells. Since long-term pretreatment with PDBu was found to enhance the EGF signals, an explanation for the synergism between EGF and phorbol esters in the induction of DNA synthesis is provided.

Chlorides↗
PubMed1988

A voltage-dependent calcium current in mouse Swiss 3T3 fibroblasts.

Patch-clamp experiments in the whole-cell mode have been performed in Swiss 3T3 mouse fibroblasts. Depolarizations from negative holding potential (Vh less than -60 mV) gave rise to a rapidly activating, fully inactivating, inward current of few tenths of nA in physiological saline at 35 degrees C. The current persisted when external Na+ was replaced by impermeant TMA+ and disappeared in 0 Ca2+, 1 mM EGTA. The current was reversible blocked by Co2+ and it was slightly reduced when external Ca2+ was substituted by Ba2+. Finally its reversal potential changed with Nernstian slope with increasing external Ca2+ concentrations. It is concluded that these cells possess a voltage-dependent Ca2+ channel.

Animals↗
PubMed1988

Properties of the voltage-dependent calcium channel of mouse Swiss 3T3 fibroblasts.

1. Suspended Swiss 3T3 fibroblasts were voltage clamped using the whole-cell technique. 2. Passage from the cell-attached to the whole-cell mode was accompanied by only a minor decrease in input resistance. Direct measurement of resting potential gave values between O and -15 mV. 3. In order to account for the effects of leak on the membrane potential measurements, I-V curves were obtained immediately before and after patch rupture by applying voltage ramps. After subtraction of the cell-attached current from the whole-cell current, the true membrane potential was estimated as the zero-current potential in the I-V curve. An average value of -8.2 +/- 0.9 mV in 8 mM-Ca2+ was obtained in this way. 4. In 2 mM-Ca2+, step depolarizations 100 ms long from holding potentials (Vh) more negative than -60 mV caused a transient inward current to appear. From Vh greater than -60 mV only a linear leakage component was apparent. 5. In 2 mM-Ca2+ depolarizations to potentials greater than +40 mV (from Vh = -100 mV) generated transient, outwardly directed currents. 6. Increasing extracellular Ca2+ up to 32 mM shifted the peak current vs. voltage curve and the reversal potential (Erev) towards more positive potentials, and caused an increase of the peak current. 7. The steady-state inactivation curve was the same for both inward and outward currents, indicating that they flow through the same channels. The currents are completely inactivated at V = -60 mV. 8. Recovery of the fully inactivated current upon hyperpolarization had an exponential time course with tau = 0.22 s at V = -80 mV and tau = 0.18 s at V = -100 mV. 9. In the absence of Ca2+ (but with Mg2+ present) the inward current disappeared but a large, inactivating outward current appeared when V greater than 0 mV. The current was strongly reduced by Cd2+ (1 mM) or Co2+ (10 mM). 10. Complete removal of divalent cations from the external solution caused the channel to become highly permeable to monovalent cations. 11. Nitrendipine (10 microM) and verapamil (5 microM) were unable to block the current. 12. On the whole the present results indicate that voltage-dependent Ca2+ channels are present in these cells. Their sensitivity to divalent cations, to organic blockers and to potential is similar to that of the low-voltage-activated, or 'T' type, Ca2+ channels described in other cells.

Action Potentials↗
PubMed1988

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.

Carcinoma, Squamous Cell↗
PubMed1986

PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse Swiss 3T3 and human skin fibroblasts.

Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts we demonstrate that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA. Feed-back inhibition of surface receptors by protein kinase C-mediated phosphorylation is therefore not general, and cannot be the only process responsible for the attenuation of receptor-mediated responses in eukaryotic cells.

Animals↗
PubMed1986

In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies.

Antibodies against the synthetic hapten azobenzyl phosphonate which specifically crossreact with phosphotyrosine have been produced and used to detect the proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to the platelet-derived growth factor (PDGF). Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated proteins followed by decoration with phosphotyrosine antibodies and 125I-labeled protein A have been used. The major tyrosine-phosphorylated component was a 170 kDa protein. The following lines of evidence suggest that this protein is the PDGF receptor in its tyrosine-phosphorylated form: (a) both proteins have the same (170 kDa) molecular weight; (b) the phosphorylated 170 kDa protein was detectable only in cell lines bearing the PDGF receptor; (c) the phosphorylation of the 170 kDa protein required PDGF and was dose-dependent. Kinetic studies showed that the phosphorylation of the receptor was maximal after 5-10 min at 37 degrees C and was followed by a rapid decrement of the band. The loss of the 170 kDa component was not prevented by inhibitors of membrane internalization and of lysosomal proteinases, while it was inhibited by lowering the temperature to 5 degrees C. In PDGF-stimulated cells, phosphotyrosine antibodies detected also a minor 36 kDa component phosphorylated at tyrosine.

Adenosine Triphosphate↗
PubMed1986

The interaction with PDGF as a source of G1 phase heterogeneity in normal mouse fibroblasts.

In normal mouse fibroblasts exponentially growing at suboptimal serum concentration the population of cells having a presynthetic (G1) DNA content is heterogeneous, since there are present both competent and non-competent cells (Sturani et al, 1984). We show here that SV40 transformed 3T3 fibroblasts, which have lost growth control and serum requirement, lack the G1 non-competent phase. These findings are confirmed by the different kinetics of exit from the G1 compartment of transformed and normal cells. In addition a positive correlation is reported between the fraction of non-competent cells and the average abundance of the phosphorylable PDGF receptor on the cell surface of normal cells.

Animals↗
PubMed1984

G1 phase heterogeneity in exponentially growing Swiss 3T3 mouse fibroblasts.

The growth rate of normal cultured Swiss 3T3 fibroblasts is function of serum concentration and the fraction of G1 cells, and hence the average residence time in G1, increases with the generation time. Serum contains two sets of factors: competence factors, essentially platelet-derived growth factor (PDGF), which induces competence in quiescent fibroblasts and prevents replicating cells from entering G0, and plasma, which allows progression. The increase in the duplication time and the duration of G1 at low serum concentration could hence be due to the fact that competence factors become limiting. The fraction of non-competent cells, operationally defined as those G1 cells unable to leave G1 in the presence of plasma alone, was determined in populations exponentially growing at serum concentrations between 5 and 20%. To do so exponentially growing cultures were shifted to plasma plus colcemid: one part of the cell population progressed through the cycle and accumulated with a G2 DNA content, whereas non-competent cells remained in G1. Analysis of the DNA distributions performed 24 h after the shift showed that as serum concentration was lowered more cells were found in the non-competent state: they were less than 5% in 20% serum and almost 50% in 5% serum. The non-competent cells constitute a dynamic fraction of the population, since in the presence of serum they can leave G1 and progress in the cycle. These data indicate that one of the steps limiting exponential growth is the acquisition of competence and that this event gives rise to heterogeneity within the G1 population.

Animals↗
PubMed1983

[Determination of aerosol deposition in the maxillary sinus].

The penetration of aerosol into the maxillary sinus depends on the function of the maxillary ostium. Exact findings regarding the velocity of the air flow in the maxillary sinus can be gained by anemometry under optical control of the position of the thermistor. The penetration of aerosol (Patent Blue) can be observed even with a partially closed ostium, and deposit can be documented by photographs. The resorptive process on the mucosa - using fluorescent aerosols - can be visualized by direct excision under UV microscopy. Finally, it is pointed out that the aerosol has a therapeutic value in chronic sinusitis maxillaris even with only partial patency of the ostium.

Aerosols↗
PubMed1983

[Functional aspects of endoscopic surgery of the fundus of the frontal sinus].

Obstruction of the ostium frontale leads to hyperplasia of the mucosa in the cranial part of the sinus frontalis and caudal of the recessus frontalis. The velocity of air flow in the ostium frontale can be recorded by means of thermistor anemometry after elimination by endoscopy, of the obstruction to drainage and to ventilation in the ostium frontale. The method of functional examination by contrast yields information on the postoperative flow of secretion. Mucociliar transportation, which shows that normal functions have been resumed, can be demonstrated accurately by endoscopy of the nose after the application of colouring matter. The exact indication for radical operation can be determined in this manner.

Endoscopy↗
PubMed1982

Cell cycle analysis in a human cell line (EUE cells).

A new procedure to determine the durations of cell cycle phases is based on th autoradiography of labeled nuclei and a standard chemical determination of DNA content per cell. It has been used to study the cell cycle timing of a human established cell line, EUE cells, so far poorly characterized in its kinetic aspects. The cycle phase durations obtained with this method are compared with the DNA frequency profiles determined by flow cytometry and found to agree closely. The relevance of the growth conditions of the exponentially growing cultures for a correct estimation of the cycle phase duration is indicated. Cell size distribution has been shown to vary according to growth condition.

Autoradiography↗