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H Porzig

Publications and source records attributed to H Porzig.

At least 19 recordsLinked to original sources
PubMed2002

Immunohistochemical detection of the sodium-calcium exchanger in rat hippocampus cultures using subtype-specific antibodies.

All of the known Na+/Ca2+ exchanger subtypes, NCX1-3, are expressed in the brain, albeit with marked regional differences. On the mRNA level, overall expression seems most prominent for NCX2, intermediate for NCX1, and, except for a few regions, low for NCX3. Using three subtype-specific antibodies, we have now studied the cellular expression of the NCX subtypes in rat hippocampus cultures by immunohistochemical techniques. Our results provide evidence for a highly cell-specific expression pattern of NCX subtypes and show surprisingly little colocalization. NCX1 and NCX3 are both primarily expressed in neuronal cells. While NCX1 is found in the large majority of neurons, NCX3 expression was restricted to a small minority of cells. By contrast, NCX2 was almost exclusively present in glial cells. The NCX2 antibody, a IgM, stained glial cell membranes as well as an intermediate fibrillar system. In spite of extensive screening, the nature of this fiber system has not yet been identified.

Animals↗
PubMed1999

Erythropoietin- and stem cell factor-induced DNA synthesis in normal human erythroid progenitor cells requires activation of protein kinase Calpha and is strongly inhibited by thrombin.

Proliferation, differentiation, and survival of erythroid progenitor cells are mainly regulated by stem cell factor (SCF) and erythropoietin (Epo). Using normal human progenitors, we analyzed the role of Ca2+-sensitive protein kinase C (PKC) subtypes and of G-protein-coupled receptor ligands on growth factor-dependent DNA synthesis. We show that stimulation of DNA synthesis by the two growth factors requires activation of PKCalpha. Inhibitors of Ca2+-activated PKC subtypes blocked the growth factor-induced 3H-thymidine incorporation. SCF and Epo caused no significant translocation of PKCalpha into the membrane, but treatment of intact cells with either of the two cytokines resulted in enhanced activity of immunoprecipitated cytosolic PKCalpha. Stimulation of PKC with the phorbol ester PMA mimicked the cytokine effect on DNA synthesis. Epo-, SCF-, and PMA-induced thymidine incorporation was potently inhibited by thrombin (half-maximal inhibition with 0.1 U/mL). This effect was mediated via the G-protein-coupled thrombin receptor and the Rho guanosine triphosphatase. Adenosine diphosphate caused a modest Ca2+-dependent stimulation of DNA synthesis in the absence of cytokines and specifically enhanced the effect of SCF. Cyclic 3', 5'-adenosine monophosphate exerted a selective inhibitory effect on Epo-stimulated thymidine incorporation. Our results define PKCalpha as major intermediate effector of cytokine signaling and suggest a role for thrombin in controlling erythroid progenitor proliferation.

Cells, Cultured↗
PubMed1999

Induction of erythroid differentiation by altered Galpha16 activity as detected by a reporter gene assay in MB-02 cells.

Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiation. The G protein alpha-subunit Galpha16, which is specifically expressed in hematopoietic cells, is highly regulated during differentiation of normal and leukemic cells. In human erythroleukemia cells, suppression of Galpha16 inhibited cellular growth rates. A reporter gene system was established to assess the role of Galpha16 on erythroid differentiation of MB-02 erythroleukemia cells. It is based on transient transfection with a plasmid that expresses green fluorescent protein under the control of the beta-globin promoter. Expression of Galpha16 led to a significant increase in green fluorescent protein-positive cells, as did transfection with a Galpha16 antisense plasmid (154 and 156% of controls, respectively). The GTPase-deficient, constitutively active mutant of Galpha16, Galpha16R186C, further stimulated differentiation to 195% of control values. Because the effect of Galpha16 is triggered most efficiently by the GTP-bound protein, an indirect action through interference of overexpressed Galpha16 with G protein betagamma-subunits can be excluded. The corresponding mutant of Galphaq (GalphaqR182C), the phylogenetically closest family member of Galpha16, had no effect. The data define a specific role for Galpha16-dependent signal transduction in cellular differentiation: deviations from optimal levels of Galpha16 functional activity lead to reduced growth rates and promote differentiation in hematopoietic cells.

Animals↗
PubMed1999

Functional expression of voltage-gated Na+ and Ca2+ channels during neuronal differentiation of PC12 cells with nerve growth factor or forskolin.

Voltage-gated ion channels and morphological differentiation were studied in rat PC12 pheochromocytoma cells after treatment with nerve growth factor (NGF) or forskolin. Ca2+ and Na+ channels were analyzed by electrophysiological techniques (using Ba2+ as charge carrier through Ca2+ channels) and by binding studies with specific ligands. With NGF, Na+ current (I(Na)) density increased in parallel with neurite extension. Ba2+ current (I(Ba)) density and Ca2+ channel numbers were both enhanced after a 2-day latency period. The tyrosine kinase inhibitor genistein blocked NGF-induced neurite extension but not the increase in I(Na) density. With forskolin, neurite outgrowth was linked to an apparent increase in I(Ba) density similar to the one induced by NGF, while no change in I(Na) was observed. Dihydropyridine-sensitive (L-type) as well as omega-conotoxin-sensitive (N-type) currents contributed to this effect. In spite of its stimulating effect on I(Ba), binding studies with radiolabeled ligands in forskolin-treated cells showed no change in N-type and an apparent loss of high affinity L-type Ca2+ channel binding. Our results suggest that induction of individual voltage-dependent channel types as well as morphological differentiation each require the activation of different signaling pathways. NGF and forskolin both enhanced current flow through voltage-dependent Ca2+ channels. However, only NGF increased channel expression while forskolin appeared to modulate channel kinetics.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Thrombin and phorbol esters potentiate Gs-mediated cAMP formation in intact human erythroid progenitors via two synergistic signaling pathways converging on adenylyl cyclase type VII.

In intact, but not in permeabilized, human erythroid progenitor cells, thrombin and phorbol esters potentiate cellular cAMP formation in response to Gs-coupled receptor agonists such as prostaglandin E1 (PGE1). We show here that the two agonists achieve their phenotypically similar effects by using distinctly different signaling pathways, both of which require protein kinase C (PKC) activation. After short term exposure (11 min), phorbol esters caused an alkaline shift of cellular pH by approximately 0.1 unit, resulting in a 1.5-2-fold increase in PGE1-induced cAMP formation. The effect of phorbol esters was inhibited by 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the Na+/H+ exchanger, and by the PKC inhibitors GF 109203X, Gö 6976, and staurosporine. Thrombin increased cellular pH by only 0.02-0.05 unit but seemed to potentiate PGE1-stimulated cAMP formation by an effect on the Gs-activated adenylyl cyclase involving a Ca2+-independent (novel) PKC. This effect was inhibited by GF 109203X and staurosporine but was resistant to 5-(N-ethyl-N-isopropyl)amiloride or Gö 6976. Inactivation of PKC by incubation of the cells in the presence of 10 nM phorbol-12-myristate-13-acetate for 18 hr completely abolished the potentiating effect of thrombin on cyclase activity, whereas the pH-dependent stimulation was fully retained. Northern blots with specific cDNA probes and a lack of Ca2+ sensitivity indicate that progenitor cells predominantly express adenylyl cyclase type VII. Our results suggest that in normal human erythroid progenitors, thrombin can activate pH-dependent and -independent, PKC-linked pathways converging on adenylyl cyclase type VII to potentiate cAMP formation in response to Gs-coupled receptor agonists.

Adenylyl Cyclases↗
PubMed1997

The P2U purinoceptor obligatorily engages the heterotrimeric G protein G16 to mobilize intracellular Ca2+ in human erythroleukemia cells.

To assess the role of G16, a trimeric G protein exclusively expressed in hematopoietic cells, Galpha16 antisense RNA was stably expressed in human erythroleukemia (HEL) cells. Western blot analysis showed that in transfected cell lines, the expression of endogenous Galpha16 protein was suppressed, but the expression of Galphaq/11, Galphai2, and Galphai3 remained unaffected. Suppression of Galpha16 in transfected HEL cells did not interfere with transient elevations of intracellular free Ca2+ concentrations induced by prostaglandin E1 (PGE1), platelet-activating factor, or thrombin. In parental HEL cells, UTP and ATP mobilized Ca2+ from intracellular stores with half-maximum effective concentrations of 3. 6 +/- 0.7 and 4.7 +/- 1.6 microM, respectively, apparently by stimulating P2U purinoceptors. By contrast, Ca2+ mobilization by UTP or ATP was completely abrogated in Galpha16-suppressed cells, indicating specific coupling of G16 to P2U purinoceptors. Pertussis toxin inhibited the effect of UTP in parental HEL cells by 57.6 +/- 4.9%. These data indicate that signaling by the P2U purinoceptor obligatorily requires G16 but may be modulated further by activation of Gi. Priming of HEL cells with UTP or ATP prior to stimulation with PGE1 markedly enhanced the PGE1-induced intracellular Ca2+ release. This indirect, potentiating effect of UTP and ATP was not impaired in Galpha16-suppressed cells but was inhibited by pertussis toxin, indicating that functional P2U purinoceptors are present on these cells and that the potentiating effect primarily depends on Gi. The data demonstrate (i) that Galpha16 antisense RNA selectively inhibits endogenous Galpha16 protein expression in HEL cells; (ii) that stimulation of endogenous P2U (P2Y2) purinoceptors leads to the mobilization of intracellular Ca2+ by a mechanism that strictly depends on Galpha16; and (iii) that P2U purinoceptors in HEL cells can communicate with two distinct signaling pathways diverging at the G protein level.

Adenosine Triphosphate↗
PubMed1997

Suppression of polarity, locomotion and F-actin levels of Walker carcinosarcoma cells by the inhibitor CI-959.

Locomotor activity of tumor cells is an important factor for the capacity for invasion and metastasis. Therefore, inhibitors interfering with cellular mechanisms regulating spontaneous cell locomotion are of particular interest for cancer therapy. CI-959, a new benzothiophene cell activation inhibitor, has the capacity to suppress spontaneous polarity and locomotion of Walker carcinosarcoma cells. Suppression of polarity and locomotion was closely associated with a reduction in the relative amount of F-actin. The mechanisms involved in suppression of motility are Ca2(+)-independent and not related to cell-substratum adhesion. Walker carcinosarcoma cells appear to be able to locomote at very low (nM) levels of free [Ca2+]i.

Actins↗
PubMed1995

G-protein-coupled receptors in normal human erythroid progenitor cells.

Human erythroid progenitor cells were isolated from peripheral blood of healthy donors and amplified in a suspension culture system using recombinant growth factors (stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin) as well as conditioned medium from a human bone marrow stroma cell line to support cell proliferation. After 6-8 days of culture, the cell population consisted mainly of erythroid colony-forming cells (burst-forming units, BFU-Es and colony-forming units, CFU-Es). In these cells, we studied ligand-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and cAMP formation as the primary effector systems of guanine nucleotide-binding protein (G protein)-coupled receptors. The results confirmed the functional expression of receptors for adenosine (type A2B), prostaglandin E1 and isoprenaline (beta-adrenoceptor), all of which stimulated adenylyl cyclase, as well as for ADP (purinoceptor types P2T and P2U), platelet-activating factor and thrombin all of which caused a transient increase in [Ca2+]i. The efficacy of adenosine and prostaglandin E1 in stimulating cAMP formation was more than 5 times higher than that of isoprenaline, suggesting a low beta-adrenoceptor density. The response to adenosine and isoprenaline decreased by 80 and 55% respectively during maturation into the proerythroblast stage. Similarly, thapsigargin-sensitive intracellular Ca2+ stores and ligand-induced Ca2+ release declined by about 60% during the CFU-E-to-erythroblast transition. The overall functional expression pattern of G protein-coupled receptors differed from that in human erythroleukaemia cell lines or from that in platelets. Primary culture systems for nontransformed cells, such as the one presented here, thus will be indispensable for the study of the functional role of G protein-dependent signalling during haematopoiesis.

Adenylyl Cyclases↗
PubMed1995

Crosstalk between thrombin and adenylyl cyclase-stimulating agonists in proliferating human erythroid progenitor cells.

Human erythroid progenitor cells grown in a suspension culture system were used to study possible interactions between different guanine nucleotide-binding protein (G-protein)-coupled receptor-effector systems during normal cell differentiation. Agonist-stimulated adenylyl cyclase was not inhibited by any one of a panel of ligands (ADP, UTP, platelet-activating factor, thrombin, alpha2-adrenoceptor agonists, interleukin 8, lysophosphatidic acid) most of which are known, in other cells, to reduce cAMP formation by a Gi-mediated, pertussis toxin-sensitive mechanism. The first four of these ligands are also known to cause transient changes in intracellular [Ca2+] in erythroid cells. Rather than inhibiting, thrombin (but not ADP, UTP or PAF) specifically caused a fivefold increase in the maximum adenosine- or prostaglandin E1-stimulated cAMP formation, without any shift of the concentration/response curves. Thrombin did not enhance forskolin- and AlF4-stimulated cyclase activity and had only a marginal effect on isoprenaline-dependent stimulation. The effect of thrombin seemed to be unrelated to intracellular Ca2+ release but could be partially mimicked by phorbol ester (PMA)-induced stimulation of protein kinase C (PKC) and was inhibited by staurosporin or by inactivation of PKC after long-term incubation with PMA. The activity of thrombin was restricted to proliferating, colony-forming progenitor cells while proerythroblasts were completely unresponsive. Our results suggest that the interaction of thrombin with Gs-linked receptors requires phosphorylation of a target protein that is different from adenylyl cyclase, Gs or Gi but may be involved in the regulation of receptor desensitization.

Adenosine↗
PubMed1995

Localization and functional significance of the Na+/Ca2+ exchanger in presynaptic boutons of hippocampal cells in culture.

Immunocytochemical evidence for localized distribution of the Na+/Ca2+ exchange protein in nerve terminals of cultured hippocampal cells is presented together with results on the functional relevance of the exchanger in the control of [Ca2+]i and of synaptic vesicle recycling. The monoclonal antibody R3F1, directed against an epitope on the intracellular loop of the protein, revealed higher densities of expression in synaptic regions than in other parts of the neurons. Removal of extracellular Na+ produced enhanced and prolonged elevation of [Ca2+]i in nerve terminals during and after electrical stimulation of the cells. Correspondingly, initial rates of exocytosis, measured by fluorescence changes of FM 1-43 during stimulation, were faster in LiCl-containing solution than in NaCl-containing solution. By contrast, endocytosis at 20 s was the same in both solutions.

Animals↗
PubMed1995

Phorbol esters downregulate expression of the sodium/calcium exchanger in renal epithelial cells.

The Na+/Ca2+ exchanger (NCE) contributes to Ca2+ reabsorption by connecting tubules of the nephron. A line of renal epithelial cells from monkey kidney (LLC-MK2) was used to investigate the regulation of NCE expression. After the activation of protein kinase C (PKC) by phorbol myristate acetate (PMA), NCE activity decreased exponentially by 75% in 48 h (half time approximately 19 h). PMA decreased NCE mRNA by 85% in 24 h. The decrease in NCE transcript preceded the downregulation of NCE activity. NCE protein was quantified with a monoclonal antibody to cardiac NCE. PMA decreased the binding of 3H-labeled antibody to cell sonicates by 40% in 24 h. Immunoblots show that PMA produced a marked and extended increase in membrane-associated PKC-alpha, although PMA depleted total PKC-alpha by 65% in 24 h. In vivo 32P labeling of myristolated alanine-rich C kinase substrate, a specific PKC substrate, confirmed that PMA produced a rapid and extended activation of PKC. 4 alpha-PMA, a stereoisomer of PMA that neither binds nor activates PKC, had no effect on NCE activity or transcript. These findings indicate that activation of PKC with phorbol esters downregulates NCE mRNA, protein, and activity in renal epithelial cells.

Animals↗
PubMed1994

Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells.

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an erythropoetin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein alpha subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.

Adenosine Diphosphate↗
PubMed1993

Immunological crossreactivity between the retinal Na(+)-Ca2+,K+ and the cardiac Na(+)-Ca2+ exchanger proteins.

We have used a series of monoclonal antibodies (mAbs) to determine the degree of microscopic structural homology between the retinal Na(+)-Ca2+,K+ and the cardiac Na(+)-Ca2+ exchange proteins. Sets of mAbs were raised separately to partially purified preparations of either the retinal or the recombinant myocardial exchanger. Each panel of mAbs was then screened for crossreactivity with the respective heterologous exchanger using enzyme-linked immunoassay and immunoblotting techniques. Out of 43 anti-retinal exchanger mAbs, we found 3 detecting the cardiac exchanger on immunoblots, while 4 out of 36 anti-cardiac exchanger mAbs reacted with the retinal exchanger. The strength of the crossreactions was generally weak and suggested that only low affinity epitopes were available on the heterologous proteins. For two crossreacting anti-retinal mAbs the apparent binding affinities to the cardiac exchanger were lower by more than two orders of magnitude. The overall low degree of epitope sharing among the two sets of mAbs confirms that in spite of their obvious functional and topological similarities, microscopic structural homologies between the two proteins are scarce.

Animals↗
PubMed1993

Mapping of the cardiac sodium-calcium exchanger with monoclonal antibodies.

We used a panel of monoclonal antibodies raised against the canine cardiac Na(+)-Ca2+ exchanger expressed in Sf9 insect cells to analyze the immunoreactive domains and the topological organization of this membrane protein. Antibodies, which reacted strongly on Western blots of the recombinant protein, were used to screen an expression sublibrary composed of exchanger cDNA fragments. Positive clones thus indicated the expression of antibody binding sites. Linear epitopes, 16-155 amino acids in length, could be identified for four antibodies. One antibody recognized two neighboring, but nonoverlapping, sequences. All epitopes were localized to the large hydrophilic region of the exchanger connecting the putative transmembrane segments 5 and 6. The immunodominant region of the protein is a highly charged domain in the carboxy-terminal half of the hydrophilic region. Binding studies with the 3H-labeled high-affinity antibody R3F1 establish that the immunodominant region is located on the intracellular surface of the membrane. The same antibody was used to directly determine the membrane concentration of the exchanger in different cell types. Newborn rat heart cells contain approximately 6 x 10(5) exchanger molecules per cell. Exchanger densities in different cells seem to correlate with the Na(+)-dependent Ca2+ transport activity in the corresponding membrane vesicles.

Amino Acid Sequence↗
PubMed1991

Analysis by cell hybridization of mechanisms that regulate beta-adrenergic responses in reticulocytes and in differentiating erythroid cells.

In intact reticulocytes, but not in fragmented membranes, the loss of adenylate cyclase activity during cell maturation followed a biphasic time course. A rapid phase (t1/2 approximately 2 h) during which the initial activity was reduced by 40-50% was followed by a slow phase with t1/2 close to 3 days. The fast decay seemed to occur on the adenylate cyclase level since (-)isoprenaline- or forskolin-stimulated activities behaved similarly and bacterial toxin-monitored Gs and Gi proteins remained stable. The mechanism of the initial decrease in hormonal responsiveness was further analysed in hybrid cells prepared by fusing reticulocytes with Friend erythroleukemia (MEL) cells. The hybrids contained reticulocyte-derived beta-adrenoceptors and MEL cell-derived adenylate cyclase and G proteins. Fusion of reticulocytes to native MEL cells caused adenylate cyclase activity to drop by 30% at 2 h and 45% at 18 h after fusion. By contrast, hybrids prepared after dimethylsulfoxide-induced differentiation of MEL cells showed stable or increasing rates of receptor-coupled cAMP formation between 2 and 18 h after fusion, concomitant with the enhanced activity of the Gs protein in these cells. A cyclase-stimulating factor present in the cytosol of MEL cells and of reticulocytes appeared not to be involved in short-term regulation of hormonal responsiveness. We conclude that the strength of beta-adrenergic responses in erythroid progenitor cells is primarily regulated by modulating G protein-mediated receptor cyclase coupling while reticulocytes, during early maturation, seem to rely on direct inactivation of adenylate cyclase, probably via a cytosolic proteolytic pathway.

Adenylyl Cyclases↗