Biomedical subjects

L G Tertoolen

Publications and source records attributed to L G Tertoolen.

At least 37 records · Page 2Linked to original sources

PubMed1995

Evaluation of single-channel gating kinetics produced after amplitude-based separation of unitary currents.

Cell-attached patch-clamp recording has been established as a major technique for investigating ion channel behaviour in a physiological setting, despite the problems which arise in analysing records containing more than one type of unitary current. To circumvent these problems, single-channel amplitude-based assignment of discrete single-channel events to different channel types becomes increasingly necessary. Surprisingly, a systematic evaluation of the validity of this method in determining single-channel parameters has not been performed to date. Using computer-stimulated single-channel traces, and recordings from a biological preparation containing well-characterized ion channels (N1E-115 neuroblastoma cells), we have explored the accuracy by which amplitude-based separation recovers ion-channel parameters. Determination of gating kinetics after separation revealed that even a very small contamination in the selected population yields additional time constants in the probability density functions. Therefore, our results demonstrate that, whereas the use of amplitude-based separation is straightforward for determining slope conductance and reversal potential, it is prone to incorporate errors in establishing gating kinetics. Ways of identifying such errors are described.

Animals↗

PubMed1995

Intracellular acidification of gastrula ectoderm is important for posterior axial development in Xenopus.

There is evidence suggesting that pHi elevation can induce differentiation to cement gland, an extremely anterior structure, during the early development of Xenopus laevis (Picard, J. J. (1975) J. Embryol. exp. Morphol. 33, 957-967; Sive, H. L., Hattori, K. and Weintraub, H. (1989) Cell 58, 171-180). We wanted to investigate whether axial development or neural induction are mediated in Xenopus via regulation of pHi. Our interest was stimulated further because certain signal transduction pathways, which are thought to mediate anterior neural induction (Otte, A. P., Van Run, P., Heideveld, M., Van Driel, R. and Durston, A. J. (1989) Cell 58, 641-648; Durston and Otte (1991), Cell-Cell Interactions in Early Development, pp. 109-127), are also known to modify the activity of proton extruders (Mitsuka and Berk (1991) Am. J. Physiol. 260, C562-C569; Wakabayashi, S., Sardet, C., Fafournoux, P., Counillon, L., Meloche, S., Pages, G. and Pouysségur, J. (1993) Rev. Physiol. Biochem. Pharmacol. Vol. 119, pp. 157-186). We therefore measured pHi in explants of gastrula ectoderm and neurectoderm and identified ion exchangers that regulate pHi in these tissues. The measurements showed that pHi decreases in explants of both neurectoderm and uninduced ectoderm during the time course of gastrulation, this pHi decrease thus fails to correlate with neural induction. One important regulator of this cytoplasmic acidification is the Na+/H+ exchanger. The pHi set point, at which the acid extrusion activity of this alkalizing exchanger is shut off, shifts to more acidic values during the time course of gastrulation, thus permitting cytoplasmic acidification. We found also that preventing cytoplasmic acidification and thereby elevating pHi in late gastrula cells led to the specific suppression of posterior development. Neural induction and anterior development were unaffected by treatments leading either to an elevation of or a decrease in pHi. These findings indicate that the cellular processes mediating anterior development and neural induction are pHi tolerant, while the signals mediating posterior development require a sustained pHi decrease for their action, suggesting that downregulation of pHi is necessary for posterior axial development.

Animals↗

PubMed1994

Cytosolic calcium ions regulate lipid mobility in the plasma membrane of the human megakaryoblastic cell line MEG-01.

The fluidity of the plasma membrane is thought to play a role in the activation of blood platelets. We investigated the lateral diffusion of the lipophilic probe 1,1'-ditetradecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiIC14) and derivatives in the plasma membrane of the megakaryoblast MEG-01 by fluorescence recovery after photobleaching. The lateral diffusion coefficient (D) of DiIC14 in an unstimulated cell was (3.53 +/- 0.06) x 10(-9) cm2/s with a mobile fraction of 75%. Similar data were found with DiIC12 and DiIC18, but lipophilic probes specific for the outer leaflet showed a slower diffusion with a D value of (2.99 +/- 0.31) x 10(-9) cm2/s and a mobile fraction of 58%. Stimulation with platelet-activating agents decreased the diffusion of DiIC14 within 2 min, but left the mobile fraction unchanged. Signal processing was required for the decrease in D as D-Phenylalanyl-L-prolyl-L-arginyl-chloromethane-treated thrombin, which binds normally to the thrombin receptor but fails to activate the cell, had no effect. The decrease in D was accompanied by an increase in cytosolic Ca2+ content, [Ca2+]i, and studies using different concentrations of thrombin, the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester and the Ca2+ ionophore ionomycin revealed that lipid mobilty in the plasma membrane is regulated by Ca2+. In contrast, treatments thought to interfere with the mobility of membrane proteins had little effect. We conclude that the rigidification of the plasma membrane during cell activation is caused by an increase in [Ca2+]i and is therefore a late event and might only contribute to signal transduction at steps downstream of the mobilization/influx of Ca2+.

Adenosine Diphosphate↗

PubMed1994

Rapid alterations in lateral mobility of lipids in the plasma membrane of activated human megakaryocytes.

In the present study we measured membrane fluidity as the lateral mobility of the lipid probe 1,1'-ditetradecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by fluorescence recovery after photobleaching in the plasma membrane of a single megakaryocyte, the progenitor cell of platelets. Megakaryocytes after 13 days in culture (maturation stage III) had a lateral diffusion coefficient (D) of (4.56 +/- 0.10) x 10(-9) cm2/s and a mobile fraction of 65 +/- 2% (means +/- SEM, n = 140). Megakaryocytes isolated from rib had a similar D and mobile fraction. Stimulation with alpha-thrombin (1-10 U/ml) induced a dose-dependent decrease in D to (3.40 +/- 0.22) x 10(-9) cm2/s between 1-5 min after stimulation (P < 0.001). The mobile fraction did not change. A similar decrease in D was found following stimulation with ADP (20 microM) and ionomycin (100 nM). Modulation of calpain I activity with calpain I inhibitor or tetracain had no effect. Pretreatment with cytochalasin B or colchicine decreased D to (3.64 +/- 0.29) x 10(-9) cm2/s (P < 0.003) and (3.96 +/- 0.18) x 10(-9) cm2/s (P < 0.013) respectively. After stimulation D decreased further in cytochalasin-treated cells (3.37 +/- 0.16) x 10(-9) cm2/s (P < 0.020) but remained at the same level in colchicine-treated cells. Both treatments increased the mobile fraction to 73-75% in stimulated megakaryocytes (P < 0.03). These data indicate that the diffusion velocity of lipids in megakaryocytes is low and decreases further after stimulation. These changes are independent of calpain I. Treatments that decrease the cytoskeletal mass and thereby increase the mobility of proteins in the plasma membrane increase the number of lipids that participate in this process.

Adenosine Diphosphate↗

PubMed1993

Extracellular ATP elevates cytoplasmatic free Ca2+ in HeLa cells by the interaction with a 5'-nucleotide receptor.

In the present study we have characterized the effects of ATP and several other nucleotides on the intracellular Ca2+ levels of HeLa cells. Using fura-2 microscopy fluorescence measurements, the ATP-mediated increase in intracellular Ca2+ was shown to consist of a rapid rise which decreased after a few seconds to a sustained elevated level. In the absence of extracellular Ca2+ or in the presence of 10 microM La3+, only a transient elevation of the Ca2+ concentration was observed. The ATP responses were not altered after treatment of HeLa cells with cholera toxin or pertussis toxin. Pharmacological analysis of this calcium response revealed that this effect was not mediated by the classical P2y purinoceptor but by a 5'-nucleotide receptor.

Adenosine Triphosphate↗

PubMed1993

Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances.

Using the human Intestine 407 cell line as a model, we investigated a possible role for tyrosine kinase(s) in regulating the ion efflux pathways induced by hyposmotic stimulation (regulatory volume decrease, RVD). Pretreatment of 125I(-)-and 86Rb(+)-loaded cells with the phosphotyrosine phosphatase inhibitor sodium orthovanadate (200 microM) potentiated isotope efflux triggered by mild hypotonicity (10-20%) but did not further increase the efflux in response to more vigorous osmotic stimulation (30% hypotonicity). The tyrosine kinase inhibitors herbimycin A and genistein largely reduced the osmoshock-induced efflux in both control and vanadate-pretreated cells, while not affecting calcium-activated 86Rb+ efflux. Potentiation of the RVD response by vanadate was confirmed by direct measurements of hypotonicity-induced changes in cell volume. Hypotonic shock alone triggered a rapid and transient increase in tyrosine phosphorylation of several proteins as well as phosphorylation of mitogen-activated protein kinase. Furthermore, the potentiating effects of vanadate on hypotonicity-induced ion efflux and mitogen-activated protein (MAP) kinase phosphorylation were mimicked by epidermal growth factor. Neither vanadate nor epidermal growth factor provoked a RVD-like ionic response under isotonic conditions. These results indicate that tyrosine phosphorylation is an essential step in the RVD response and suggest a novel role of growth factors in the cellular defense against osmotic stress.

Benzoquinones↗

PubMed1993

Epidermal growth factor-induced actin remodeling is regulated by 5-lipoxygenase and cyclooxygenase products.

In a number of cell types, epidermal growth factor (EGF) evokes dramatic morphological changes, cortical actin polymerization, and stress fiber breakdown. The molecular processes by which increased EGF receptor tyrosine kinase activity results in actin reorganization and morphological changes are unresolved. Recently, we demonstrated that arachidonic acid metabolites function in EGF signal transduction. We now report that in A431 cells, HeLa cells, and rat-1 fibroblasts, the EGF-induced cortical actin polymerization is produced by lipoxygenase metabolism, whereas in these cells stress fiber breakdown is mediated by cyclooxygenase metabolites. Also, the EGF-provoked rounding up in A431 cells is dependent on arachidonic acid metabolism. We conclude that leukotrienes and prostaglandins act in concert, as second messengers, to produce morphological effects and actin reorganization, providing a novel mechanism for directing growth factor-induced cytoskeletal changes.

Actins↗

PubMed1993

Mouse P19 embryonal carcinoma cells express functional histamine H1-receptors.

In the present study, we have investigated the response in P19 embryonal carcinoma (EC) cells to histamine. We show that these cells, that resemble the pluripotent cells of an early mouse embryo, respond to histamine addition by a transient increase in intracellular Ca2+. The response is stereoselectively inhibited by the enantiomers of the H1-receptor antagonists chlorpheniramine and cicletanine. [3H]-mepyramine was found to bind with high affinity (Kd 4 nM) to a membrane preparation of P19 EC cells. The profile of these binding sites corresponded well with the results of the Ca2+ measurements. A high affinity [3H]-mepyramine binding site was also identified on intact cells. These data demonstrate that embryonal carcinoma cells express functional histamine H1-receptors and suggest that histamine might act as a regulatory factor in the early development of the mouse embryo.

Animals↗

PubMed1993

Receptor protein tyrosine phosphatase alpha activates pp60c-src and is involved in neuronal differentiation.

Here we report that protein tyrosine phosphatases (PTPases), like their enzymatic counterpart the protein tyrosine kinases, can play an important role in cell differentiation. Expression of the transmembrane PTPase receptor protein tyrosine phosphatase alpha (RPTP alpha) is transiently enhanced during neuronal differentiation of embryonal carcinoma (EC) and neuroblastoma cells. Retinoic acid induces wild type P19 cells to differentiate into endoderm- and mesoderm-like cells. By contrast, retinoic acid treatment leads to neuronal differentiation of P19 cells, ectopically expressing functional RPTP alpha, as illustrated by their ability to generate action potentials. Endogenous pp60c-src kinase activity is enhanced in the RPTP alpha-transfected cells, which may be due to direct dephosphorylation of the regulatory Tyr residue at position 527 in pp60c-src by RPTP alpha. Our results demonstrate that RPTP alpha is involved in neuronal differentiation and imply a role for pp60c-src in the differentiation process.

Animals↗

PubMed1993

Epidermal growth factor induces phosphorylation of extracellular signal-regulated kinase 2 via multiple pathways.

Expression of p21rasAsn-17, a dominant negative mutant of p21ras that blocks p21ras activation by growth factors, inhibits activation of extracellular signal-regulated kinase 2 (ERK2) by insulin and platelet-derived growth factor in rat-1 cells [A. M. M. de Vries-Smits, B. M. T. Burgering, S. J. Leevers, C. J. Marshall, and J. L. Bos, Nature (London) 357:602-604, 1992]. Here we report that expression of p21rasAsn-17 does not abolish epidermal growth factor (EGF)-induced phosphorylation of ERK2 in fibroblasts. Since EGF activates p21ras in these cells, this indicates that EGF induces a p21ras-independent pathway for the phosphorylation of ERK2 as well. We investigated whether activation of protein kinase C (PKC) or increase in intracellular calcium could be involved in p21ras-independent signaling. In rat-1 cells, inhibition of either PKC, by prolonged 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment, or calcium influx, by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) pretreatment, did not abolish EGF-induced ERK2 phosphorylation. However, a combined inhibition of both p21ras and calcium influx, but not PKC, resulted in a complete inhibition of EGF-induced ERK2 phosphorylation. In contrast, in Swiss 3T3 cells, inhibition of both p21ras activation and TPA-sensitive PKC, but not calcium influx, inhibited EGF-induced ERK2 phosphorylation. These results demonstrate that in fibroblasts, EGF induces alternative pathways of ERK2 phosphorylation in a cell-type-specific manner.

Animals↗

PubMed1992

Epidermal growth factor activates calcium channels by phospholipase A2/5-lipoxygenase-mediated leukotriene C4 production.

Epidermal growth factor (EGF) induces a Ca2+ influx in many cell types, but the underlying mechanisms are so far unresolved. We report that: EGF-induced Ca2+ channel activity is eliminated by lipoxygenase inhibition and is mimicked by artificial induction of lipoxygenase activity; addition of leukotriene C4 can fully mimic EGF in its ability to activate Ca2+ channels; and EGF induces a rapid accumulation of intracellular leukotriene C4. In addition, we show that EGF-induced, Ca(2+)-dependent membrane hyperpolarization and junB proto-oncogene expression are dependent on lipoxygenase activity, whereas EGF-induced cytoplasmic alkalinization is not. We conclude that PLA2/5-lipoxygenase-mediated leukotriene C4 production constitutes a novel and specific signal transduction pathway in growth factor action.

Acetophenones↗

PubMed1992

Short-term desensitization of the histamine H1 receptor in human HeLa cells: involvement of protein kinase C dependent and independent pathways.

1. In this study we have investigated the effects of short-term exposure of cells to histamine on the subsequent H1 receptor responsiveness in HeLa cells, using Ca2+ fluorescence microscopy and video digital imaging. 2. In HeLa cells, histamine (100 microM) induces an immediate H1 receptor-mediated biphasic elevation of the intracellular Ca2+ concentration ([Ca2+]i) (basal [Ca2+]i: 81 +/- 30 nM, histamine-induced Ca2+ response: first phase: 1135 +/- 79 nM; second phase: 601 +/- 52 nM, n = 11). 3. The histamine H1 receptors on HeLa cells are readily susceptible to desensitization since repetitive exposure of the same group of cells to histamine (100 microM) markedly affected the release and influx component of the induced Ca2+ response (second application of histamine: first phase: 590 +/- 92 nM, second phase: 279 +/- 47 nM; third application of histamine: first phase: 454 +/- 127 nM, second phase: 240 +/- 45 nM, n = 6). Video digital imaging revealed an increase in the lag time between stimulation and monitoring of the Ca2+ response and a reduced increase in [Ca2+]i after desensitization with histamine. 4. Neither the release component of the ATP response (50 microM) nor the caffeine (3 mM)-induced Ca2+ release were found to be affected by desensitization with 100 microM histamine. However, the second phase of the ATP response was significantly reduced after desensitization with histamine (control cells: 516 +/- 33 nM; desensitized cells: 331 +/- 96 nM, n = 4, P < 0.05).5. Activation of protein kinase C (PKC) by phorbol-12-myristate-1 3-acetate was found to inhibit the histamine as well as ATP-induced Ca2" response in a dose-dependent manner.6. In PKC downregulated cells the second phase of the histamine-induced Ca2+ response was significantly elevated, indicating the involvement of PKC in the negative feedback on the Ca2+ influx(control cells: second phase: 601 +/- 52 nM (n = 11); PKC downregulated cells: second phase:890 +/- 90nM, n = I0, P<0.05).7. Homologous desensitization of H, receptor responsiveness was still observed in PKC downregulated cells, implying the rapid activation of a regulatory mechanism other than PKC.8. Based on our experimental data we suggest that short-term desensitization of the histamine H,receptor evolves from two different processes: a selective reduction of the histamine-induced Ca2+ release, mediated by a PKC-independent pathway, and a non-selective inhibition of the receptormediated Ca2+ influx activated by a PKC-dependent pathway.

Adenosine Triphosphate↗

PubMed1991

Epidermal growth factor-activated calcium and potassium channels.

The earliest responses to activation of the epidermal growth factor (EGF) receptor include a transient increase in calcium influx and a transient membrane hyperpolarization. The underlying mechanisms are, however, not well understood as yet. In the present study, we have applied patch clamp recording in the cell-attached and the outside-out mode, and fluorimetric cytosolic Ca2+ determinations, to identify the nature of the ion channels involved, to characterize their properties at the level of single channels, and to unravel their mechanism of activation. We provide evidence that activation of the EGF receptor results initially in the activation of voltage-independent Ca2+ channels that can be defined as direct receptor-operated channels. This in turn causes the activation of Ca(2+)-dependent K+ channels, which results in a (delayed) membrane hyperpolarization and then leads to the activation of a second class of Ca2+ channels that are sensitive to hyperpolarization. An autocatalytic generation of further hyperpolarization and Ca2+ influx is the predicted outcome of this ionic cascade. Based on the observed inhibitory effects of protein kinase C activation on the activity of Ca(2+)-dependent K+ channels, we propose that protein kinase C is involved in the negative regulation of this cascade, which explains the transient nature of these responses.

Calcium Channels↗

PubMed1991

Transforming growth factor-beta enhances the extent of intercellular communication between normal rat kidney cells.

Normal rat kidney (NRK) cells cultured in the presence of epidermal growth factor are contact-inhibited at confluent densities. In the additional presence of transforming growth factor (TGF)-beta, however, cells undergo phenotypic transformation which is accompanied by a loss of contact inhibition. In this study, we show by means of the fluorescence photobleaching recovery technique and a scrape-loading dye transfer technique that quiescent confluent cultures of NRK cells do not show extensive gap junction-mediated intercellular communication. Cells contact-inhibited in the presence of epidermal growth factor also show only limited intercellular communication, although with an enhanced permeability coefficient. Cells phenotypically transformed upon addition of TGF beta, however, show extensive intercellular communication, with a similarly enhanced permeability coefficient. This enhanced intercellular communication induced by TGF beta is paralleled by an increase in intracellular pH. It is concluded that in contrast to what has been observed during tumorigenic transformation, phenotypic transformation of NRK cells induced by TGF beta results in an enhancement of the extent of gap junction-mediated intercellular communication.

Animals↗

PubMed1991

Characterization of a tyrosine kinase signaling pathway in undifferentiated P19 embryonal carcinoma cells.

Embryonal carcinoma (EC) cells are the malignant stem cells of teratocarcinoma and have the capacity to proliferate in the absence of serum growth factors. As yet no receptor protein tyrosine kinases have been identified on undifferentiated EC cells and as a consequence tyrosine kinase signaling pathways could not be studied in these cells. We have used stably transfected P19 embryonal carcinoma cells expressing a well-characterized receptor protein tyrosine kinase, the human epidermal growth factor receptor (hEGF-R) to study protein tyrosine kinase signaling mechanisms in undifferentiated EC cells. Here we report that the ectopically expressed hEGF-R contains EGF-inducible autophosphorylation activity and is rapidly internalized and degraded upon ligand binding. In addition, the exogenous hEGF-R confers EGF-responsiveness to these cells in that inositol phosphate formation and cytoplasmic-free Ca2+ concentration are enhanced in response to EGF. Furthermore, the Na+/H+ exchanger is activated in response to EGF, leading to a sustained rise in intracellular pH. Our results show that undifferentiated P19 EC cells contain the necessary components of protein tyrosine kinase signal transduction machinery.

Adenosine Triphosphate↗

PubMed1990

Regulation of phosphoinositide hydrolysis induced by histamine and guanine nucleotides in human HeLa carcinoma cells. Calcium and pH dependence and inhibitory role of protein kinase C.

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.

Calcium↗

PubMed1990

Histamine-H1-receptor-mediated phosphoinositide hydrolysis, Ca2+ signalling and membrane-potential oscillations in human HeLa carcinoma cells.

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+.

Aluminum↗

PubMed1990

Intercellular communication is cell cycle modulated during early Xenopus laevis development.

We investigated intercellular communication during the seventh and tenth cell cycles of Xenopus laevis development using microinjection of Lucifer yellow and FITC-dextran as well as freeze-fracture electron microscopy. We found that gap junction-mediated dye coupling visualized using Lucifer yellow was strongly cell cycle modulated in the tenth cell cycle. Cytoplasmic bridge-mediated dye coupling visualized via FITC-dextran was also, of course, cell cycle modulated. The basis of cell cycle-modulated gap junctional coupling was investigated by measuring the abundance of morphologically detectable gap junctions through the tenth cell cycle. These proved to be six times more abundant at the beginning than at the end of this cell cycle.

Animals↗