Hartmut Porzig - Academia.edu (original) (raw)
Papers by Hartmut Porzig
Naunyn-schmiedebergs Archives of Pharmacology, Mar 2, 2022
Naunyn-schmiedebergs Archives of Pharmacology, 1968
Journal of Molecular and Cellular Cardiology, 1985
H. REUTER AND H. PORZIG. Regulation of8-Br-cAMP offl-adrenoceptors in Cultured Myocardial Cell's.... more H. REUTER AND H. PORZIG. Regulation of8-Br-cAMP offl-adrenoceptors in Cultured Myocardial Cell's. Journal of A4olecular and Celhdar C~rdiology (1985) 17, 307-316. Primary cultures of myocardial cells from neonatal rats were exposed for up to 5 days to the cyclic AMP derivative 8-Br-cAMP. After one day of exposure to the nucleotide, an increase in specific binding capacity of the hydrophilic fl-adrenoceptor antagonist 3H CGP 12177 was observed in intact cells. ( --)-Isoprenaline displaced the radioligand fi'om its binding site. Neither the K D of the antagonist, nor that of the agonist were significantly affected by 8-Br-cAMP. The loss of fladrenoceptors during desensitization by long term treatment of the cells with isoprenaline could be partially prevented by 8-Br-cAMP. Only in desensitized, but not in normal cells was isoprenaline-induced cAMP formation significantly enhanced by 8-Br-cAMP pretreatment. This may indicate that fl-adrenoceptors which appear during 8-Br-cAMP exposure are poorly coupled to the adenylate cyclase. Alternatively, the change in receptor density may be accompanied by alterations of other components in the fl-adrenergic system, e.g. an inhibition of the adenylate cyclase. We suggest that cAMP-dependent feed-back regulation of the fl-adrenergic system may play a role during postnatal myocardial differentiation.
The Journal of Membrane Biology, 1993
We have used a series of monoclonal antibodies (mAbs) to determine the degree of microscopic stru... more 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.
The Journal of Membrane Biology, 1973
Journal of Cellular Physiology, 1991
In intact reticulocytes, but not in fragmented membranes, the loss of adenylate cyclase activity ... more 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 (t,,2 = 2 h) during which the initial activity was reduced by 40-50% was followed by a slow phase with t , , , 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 G, and G i 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 p-adrenoceptors and MEL cell-derived adenylate cyclase and G proteins. Fusion
Journal of Biological Chemistry, 1997
To assess the role of G16, a trimeric G protein exclusively expressed in hematopoietic cells, Gal... more 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.
Journal of Biological Chemistry, 1999
Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiati... more Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiation. The G protein ␣-subunit G ␣16 , which is specifically expressed in hematopoietic cells, is highly regulated during differentiation of normal and leukemic cells. In human erythroleukemia cells, suppression of G ␣16 inhibited cellular growth rates. A reporter gene system was established to assess the role of G ␣16 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 -globin promoter. Expression of G ␣16 led to a significant increase in green fluorescent protein-positive cells, as did transfection with a G ␣16 antisense plasmid (154 and 156% of controls, respectively). The GTPase-deficient, constitutively active mutant of G ␣16 , G ␣16 R186C, further stimulated differentiation to 195% of control values. Because the effect of G ␣16 is triggered most efficiently by the GTP-bound protein, an indirect action through interference of overexpressed G ␣16 with G protein ␥-subunits can be excluded. The corresponding mutant of G ␣q (G ␣q R182C), the phylogenetically closest family member of G ␣16 , had no effect. The data define a specific role for G ␣16 -dependent signal transduction in cellular differentiation: deviations from optimal levels of G ␣16 functional activity lead to reduced growth rates and promote differentiation in hematopoietic cells.
Experimental Hematology, 2000
The lyl-1 gene encodes a protein that belongs to the basic helixloop-helix (bHLH) family of trans... more The lyl-1 gene encodes a protein that belongs to the basic helixloop-helix (bHLH) family of transcription factors. In humans lyl-1 was first identified as a fusion gene due to a chromosomal translocation found in a subset of T-ALL. Expression of lyl-1 in humans is restricted to hematopoietic cells where it parallels the expression of two related bHLH proteins: SCL/tal-1 and tal-2, both involved in leukemogenesis as well. Expression of lyl-1 in mice and humans is similar. To investigate the role of lyl-1 in mice we first performed a refined analysis of lyl-1 expression by RT-PCR. We examined the following cell lines: two embryonic (NIH-3T3, ES) and one adult (MEL), and the following tissues: lung, kidney, liver, spleen, brain, muscle, heart, ovary, and testis, normal whole embryo and peripheral blood lymphocytes. A 192-bp lyl-1 fragment was amplified in all analyzed samples, suggesting that lyl-1 is ubiquitously expressed in mice. Northern blot analysis confirmed this pattern of expression and evidenced a larger amount of lyl-1 mRNA in the embryo. Furthermore, we performed an antisense oligomer treatment against the starting codon of the translation region of lyl-1 mRNA in MEL cells and embryonic fibroblasts. After 35/48 hours of treatment the cells underwent growth arrest in a dose dependent manner. Taken together these findings show for the first time that lyl-1 is ubiquitously expressed in the mouse, and participates in the control of cell proliferation, suggesting a new role for this transcription factor.
Experimental Cell Research, 1990
European Journal of Pharmacology, 1990
Mouse erythroblastoma cells were used as a model system to study sensitivity and regulation of th... more Mouse erythroblastoma cells were used as a model system to study sensitivity and regulation of the beta-adrenergic system during DMSO-induced differentiation from the proerythroblast to the normoblast stage. Differentiation was characterized by a initial marked increase in hormonal sensitivity lasting 24 to 40 h followed by a gradual loss of beta-adrenergic responsiveness. These changes are mainly due to a rapid but transient increase in receptor density (4 fold) and a marked shift of the membrane concentrations of the transmembrane signalling proteins Gs and Gi. The Gs to Gi ratio changed from 1:7 in native MEL cells to 2:1 in differentiated cells. By contrast, fusion experiments between rat reticulocytes and and MEL cells indicated that cytosolic factors, while not prominently involved in the regulation of the beta-adrenergic system of differentiating MEL cells, may be responsible for the rapid loss of cyclase activity during reticulocyte maturation, the last step in red cell formation.
European Journal of Applied Physiology and Occupational Physiology, 1984
Six male non-endurance trained subjects (S) and six marathon runners (M) underwent graded treadmi... more Six male non-endurance trained subjects (S) and six marathon runners (M) underwent graded treadmill exercise (T) and isoproterenol stimulation (I; 2 and 4 ~tg 9 rain-l), fl-adrenergic receptor density was additionally determined as the amount of 3H-Dihydroalprenolol (DHA) specifically bound on intact polymorphonuclear leucocytes. Heart rate, Vo2 uptake, lactate, plasma noradrenaline, and adrenaline were estimated during T. Heart rate, stroke volume, cardiac output, as well as lactate, glucose, free fatty acids (FFA), and glycerol levels in the blood were determined during I. M showed the known training-dependent responses during T, such as lower heart rates, lactate levels, and plasma catecholamines at identical work loads, as well as higher Vo2ma x than S. I-induced cardiac output increase was quite similar in both groups. Stroke volume, however, increased significantly in M and stayed constant in S. Lactate decreased (S), glucose increased significantly (M), glycerol increased similarly in both groups, FFA rise was less marked in S. I-induced stroke volume response (I) may be indicative of a more economic regulation of heart work in M than S. Lactate decrease and less marked FFA increase, as observed in S, may be the result of a somewhat higher cardiac energy demand, dependent on less economic heart work. Higher DHA-binding as observed in M, as well as stroke volume response and glucose increase, may be indicators of a training-dependent rise in sensitivity to catecholamines. The unsolved question is, however, to what extent fl-receptor responses in intact blood cells are significant for receptor behavior in other organs.
Clinical Chemistry and Laboratory Medicine, 2000
Zusammenfassung: Zur Bestimmung der ß-Rezeptoren-Dichte wurde die spezifische Bindung von [ 3 H]D... more Zusammenfassung: Zur Bestimmung der ß-Rezeptoren-Dichte wurde die spezifische Bindung von [ 3 H]Dihydroalprenolol an intakten polymorphkernigen Leukocyten bei 6 ausdauertrainierten (VO 2 max. 65,7 ±2,0 ml/kg · min) und 9 nicht ausdauertrainierten männlichen Probanden (VOz max. 52,0 ± 4,0 ml/kg · min) untersucht. Die spezifische Bindung ist definiert als Differenz zwischen Gesamtbindung und nicht verdrängbarer unspezifischer Bindung. Für die Bindungsstudien wurden die Leukocyten im autologen Plasma reinkubiert. Die spezifische Bindung zeigt bei trainierten und untrainierten Probanden ein Sättigungsverhalten bei ungefähr 2 nmol/l Dihydroalprenolol. Sie beträgt 85% (0,1 nmol/1 Dihydroalprenolol) bis 51% (2,0 nmol/1 Dihydroalprenolol) der Gesamtbindung. B max . beträgt, basierend auf der Scatchard-Analyse, 41,2 fmol/10 7 Zellen (Trainierte) und 21,6 fmol/10 7 Zellen (Untrainierte). Die Dissonanzkonstante (Ko) wurde zu 0,44 nmol/l Dihydroalprenolol (Untrainierte) und 0,49 nmol/1 Dihydroalprenolol (Trainierte) berechnet, die Zahl der ß-adrenergen Bindungsstellen beträgt ungefähr 1300 (Untrainierte) und 2500 pro Zelle (Trainierte). Die spezifische Bindung von Dihydroalprenolol an intakte polymorphkernige Leukocyten von ausdauertrainierten ist signifikant höher als bei untrainierten Probanden. Die trainings-abhängige Änderung der ß-Rezeptoren-Dichte kann ein Indikator einer gesteigerten Empfindlichkeit gegenüber Katecholaminen sein.
Circulation Research, 2002
Hypertrophied and failing cardiac myocytes generally show alterations in intracellular Ca 2ϩ hand... more Hypertrophied and failing cardiac myocytes generally show alterations in intracellular Ca 2ϩ handling associated with changes in the contractile function and arrhythmogenicity. The cardiac Na ϩ -Ca 2ϩ exchange (NCX) is an important mechanism for Ca 2ϩ extrusion and cell relaxation. Its possible involvement in changes of excitation-contraction coupling (EC-coupling) with disease remains uncertain. We analyzed the NCX function in rat ventricular myocytes 5 to 6 months after experimental myocardial infarction (PMI) produced by left coronary artery ligation and from sham-operated (SO) hearts. Caged Ca 2ϩ was dialyzed into the cytoplasm via a patch-clamp pipette and Ca 2ϩ was released by flash photolysis to activate NCX and measure the associated currents (I NaCa ), whereas [Ca 2ϩ ] i changes were simultaneously recorded with a confocal microscope. I NaCa density normalized to the [Ca 2ϩ ] i jumps was 2.6-fold higher in myocytes from PMI rats. The level of total NCX protein expression in PMI myocytes was also increased. Interestingly, although the I NaCa density in PMI cells was larger, PMI and SO myocytes presented virtually identical Ca 2ϩ transport via the NCX. This discrepancy was explained by a reduced surface/volume ratio (34.8%) observed in PMI cells. We conclude that the increase in NCX density may be a mechanism to maintain the required Ca 2ϩ extrusion from a larger cell to allow adequate relaxation. (Circ Res. 2002;91:323-330.)
Cellular Signalling, 2005
The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite ... more The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells. CXCL12 promoted growth in multipotent cells by activating the RhoA-Rho kinase pathway. Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins. Rho activation required a G i -mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively. By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCh activity. The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCh. Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by Gprotein-coupled receptor agonists. D
Cellular Signalling, 2005
Hematopoietic cells uniquely express G a16 , a G protein a-subunit of the G q -type. G a16 is obl... more Hematopoietic cells uniquely express G a16 , a G protein a-subunit of the G q -type. G a16 is obligatory for P2Y 2 receptor-dependent Ca 2+mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y 2 receptors physically interact with G a16 . Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca 2+ -signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G a16 and P2Y 2 receptors form constitutive complexes, and that the P2Y 2 receptor is present as an oligomer. D
Experientia, 1973
ABSTRACT Zusammenfassung In Herzmuskelpräparaten sind zwei Systeme für den Ca-Durchtritt durch di... more ABSTRACT Zusammenfassung In Herzmuskelpräparaten sind zwei Systeme für den Ca-Durchtritt durch die Plasmamembranen zu unterscheiden: 1. Ein spannungs- und zeitabhängiger Ca-Einwärtsstrom, der während der Plateauphase des Aktionspotentials fliesst und durch La3+ gehemmt wird; 2. ein Na−Ca-Austauschsystem, das vor allem für den Auswärtstransport von Ca aus der Zelle verantwortlich ist, und durch La3+ nicht beeinflusst wird.
Cell Calcium, 2006
We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control... more We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca 2+ release and store-operated Ca 2+ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 M) and the Src inhibitor PP2 (10 M). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca 2+ transients were reduced by imatinib and/or PP2. Ca 2+ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca 2+ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKC␣ catalytic activity and PKC␣ co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca 2+ influx was reduced by complexing extracellular Ca 2+ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca 2+ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.
Cell Calcium, 2005
Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expr... more Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expression of the cardiac Na + -Ca 2+ exchanger (NCX1) to correlate suppression of the Na + -Ca 2+ exchange function with down-regulation of NCX1 protein expression. In a denovo expression system (Sf9 cells), a decrease in both, NCX1 mRNA and protein after AS-ODN application was paralleled by diminished NCX1 activity, a typical hallmark of a true "antisense effect". Although AS-ODN uptake was also efficient in rat neonatal cardiac myocytes, in wholecell extracts of these cells treated with AS-ODNs, the amount of NCX1 protein determined in a quantitative binding assay remained almost unchanged, despite a prompt loss of NCX1 function. Immunocytochemical staining of myocytes revealed that most of the immunoreactivity was not localized in the plasma membrane, but in intracellular compartments and was barely affected by AS-ODN treatment. These results indicate that the "functional half-life" of the NCX1 protein in the plasma membrane of neonatal cardiac myocytes is surprisingly short, much shorter than reported half-lifes of about 30 h for other membrane proteins.
Naunyn-schmiedebergs Archives of Pharmacology, Mar 2, 2022
Naunyn-schmiedebergs Archives of Pharmacology, 1968
Journal of Molecular and Cellular Cardiology, 1985
H. REUTER AND H. PORZIG. Regulation of8-Br-cAMP offl-adrenoceptors in Cultured Myocardial Cell's.... more H. REUTER AND H. PORZIG. Regulation of8-Br-cAMP offl-adrenoceptors in Cultured Myocardial Cell's. Journal of A4olecular and Celhdar C~rdiology (1985) 17, 307-316. Primary cultures of myocardial cells from neonatal rats were exposed for up to 5 days to the cyclic AMP derivative 8-Br-cAMP. After one day of exposure to the nucleotide, an increase in specific binding capacity of the hydrophilic fl-adrenoceptor antagonist 3H CGP 12177 was observed in intact cells. ( --)-Isoprenaline displaced the radioligand fi'om its binding site. Neither the K D of the antagonist, nor that of the agonist were significantly affected by 8-Br-cAMP. The loss of fladrenoceptors during desensitization by long term treatment of the cells with isoprenaline could be partially prevented by 8-Br-cAMP. Only in desensitized, but not in normal cells was isoprenaline-induced cAMP formation significantly enhanced by 8-Br-cAMP pretreatment. This may indicate that fl-adrenoceptors which appear during 8-Br-cAMP exposure are poorly coupled to the adenylate cyclase. Alternatively, the change in receptor density may be accompanied by alterations of other components in the fl-adrenergic system, e.g. an inhibition of the adenylate cyclase. We suggest that cAMP-dependent feed-back regulation of the fl-adrenergic system may play a role during postnatal myocardial differentiation.
The Journal of Membrane Biology, 1993
We have used a series of monoclonal antibodies (mAbs) to determine the degree of microscopic stru... more 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.
The Journal of Membrane Biology, 1973
Journal of Cellular Physiology, 1991
In intact reticulocytes, but not in fragmented membranes, the loss of adenylate cyclase activity ... more 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 (t,,2 = 2 h) during which the initial activity was reduced by 40-50% was followed by a slow phase with t , , , 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 G, and G i 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 p-adrenoceptors and MEL cell-derived adenylate cyclase and G proteins. Fusion
Journal of Biological Chemistry, 1997
To assess the role of G16, a trimeric G protein exclusively expressed in hematopoietic cells, Gal... more 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.
Journal of Biological Chemistry, 1999
Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiati... more Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiation. The G protein ␣-subunit G ␣16 , which is specifically expressed in hematopoietic cells, is highly regulated during differentiation of normal and leukemic cells. In human erythroleukemia cells, suppression of G ␣16 inhibited cellular growth rates. A reporter gene system was established to assess the role of G ␣16 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 -globin promoter. Expression of G ␣16 led to a significant increase in green fluorescent protein-positive cells, as did transfection with a G ␣16 antisense plasmid (154 and 156% of controls, respectively). The GTPase-deficient, constitutively active mutant of G ␣16 , G ␣16 R186C, further stimulated differentiation to 195% of control values. Because the effect of G ␣16 is triggered most efficiently by the GTP-bound protein, an indirect action through interference of overexpressed G ␣16 with G protein ␥-subunits can be excluded. The corresponding mutant of G ␣q (G ␣q R182C), the phylogenetically closest family member of G ␣16 , had no effect. The data define a specific role for G ␣16 -dependent signal transduction in cellular differentiation: deviations from optimal levels of G ␣16 functional activity lead to reduced growth rates and promote differentiation in hematopoietic cells.
Experimental Hematology, 2000
The lyl-1 gene encodes a protein that belongs to the basic helixloop-helix (bHLH) family of trans... more The lyl-1 gene encodes a protein that belongs to the basic helixloop-helix (bHLH) family of transcription factors. In humans lyl-1 was first identified as a fusion gene due to a chromosomal translocation found in a subset of T-ALL. Expression of lyl-1 in humans is restricted to hematopoietic cells where it parallels the expression of two related bHLH proteins: SCL/tal-1 and tal-2, both involved in leukemogenesis as well. Expression of lyl-1 in mice and humans is similar. To investigate the role of lyl-1 in mice we first performed a refined analysis of lyl-1 expression by RT-PCR. We examined the following cell lines: two embryonic (NIH-3T3, ES) and one adult (MEL), and the following tissues: lung, kidney, liver, spleen, brain, muscle, heart, ovary, and testis, normal whole embryo and peripheral blood lymphocytes. A 192-bp lyl-1 fragment was amplified in all analyzed samples, suggesting that lyl-1 is ubiquitously expressed in mice. Northern blot analysis confirmed this pattern of expression and evidenced a larger amount of lyl-1 mRNA in the embryo. Furthermore, we performed an antisense oligomer treatment against the starting codon of the translation region of lyl-1 mRNA in MEL cells and embryonic fibroblasts. After 35/48 hours of treatment the cells underwent growth arrest in a dose dependent manner. Taken together these findings show for the first time that lyl-1 is ubiquitously expressed in the mouse, and participates in the control of cell proliferation, suggesting a new role for this transcription factor.
Experimental Cell Research, 1990
European Journal of Pharmacology, 1990
Mouse erythroblastoma cells were used as a model system to study sensitivity and regulation of th... more Mouse erythroblastoma cells were used as a model system to study sensitivity and regulation of the beta-adrenergic system during DMSO-induced differentiation from the proerythroblast to the normoblast stage. Differentiation was characterized by a initial marked increase in hormonal sensitivity lasting 24 to 40 h followed by a gradual loss of beta-adrenergic responsiveness. These changes are mainly due to a rapid but transient increase in receptor density (4 fold) and a marked shift of the membrane concentrations of the transmembrane signalling proteins Gs and Gi. The Gs to Gi ratio changed from 1:7 in native MEL cells to 2:1 in differentiated cells. By contrast, fusion experiments between rat reticulocytes and and MEL cells indicated that cytosolic factors, while not prominently involved in the regulation of the beta-adrenergic system of differentiating MEL cells, may be responsible for the rapid loss of cyclase activity during reticulocyte maturation, the last step in red cell formation.
European Journal of Applied Physiology and Occupational Physiology, 1984
Six male non-endurance trained subjects (S) and six marathon runners (M) underwent graded treadmi... more Six male non-endurance trained subjects (S) and six marathon runners (M) underwent graded treadmill exercise (T) and isoproterenol stimulation (I; 2 and 4 ~tg 9 rain-l), fl-adrenergic receptor density was additionally determined as the amount of 3H-Dihydroalprenolol (DHA) specifically bound on intact polymorphonuclear leucocytes. Heart rate, Vo2 uptake, lactate, plasma noradrenaline, and adrenaline were estimated during T. Heart rate, stroke volume, cardiac output, as well as lactate, glucose, free fatty acids (FFA), and glycerol levels in the blood were determined during I. M showed the known training-dependent responses during T, such as lower heart rates, lactate levels, and plasma catecholamines at identical work loads, as well as higher Vo2ma x than S. I-induced cardiac output increase was quite similar in both groups. Stroke volume, however, increased significantly in M and stayed constant in S. Lactate decreased (S), glucose increased significantly (M), glycerol increased similarly in both groups, FFA rise was less marked in S. I-induced stroke volume response (I) may be indicative of a more economic regulation of heart work in M than S. Lactate decrease and less marked FFA increase, as observed in S, may be the result of a somewhat higher cardiac energy demand, dependent on less economic heart work. Higher DHA-binding as observed in M, as well as stroke volume response and glucose increase, may be indicators of a training-dependent rise in sensitivity to catecholamines. The unsolved question is, however, to what extent fl-receptor responses in intact blood cells are significant for receptor behavior in other organs.
Clinical Chemistry and Laboratory Medicine, 2000
Zusammenfassung: Zur Bestimmung der ß-Rezeptoren-Dichte wurde die spezifische Bindung von [ 3 H]D... more Zusammenfassung: Zur Bestimmung der ß-Rezeptoren-Dichte wurde die spezifische Bindung von [ 3 H]Dihydroalprenolol an intakten polymorphkernigen Leukocyten bei 6 ausdauertrainierten (VO 2 max. 65,7 ±2,0 ml/kg · min) und 9 nicht ausdauertrainierten männlichen Probanden (VOz max. 52,0 ± 4,0 ml/kg · min) untersucht. Die spezifische Bindung ist definiert als Differenz zwischen Gesamtbindung und nicht verdrängbarer unspezifischer Bindung. Für die Bindungsstudien wurden die Leukocyten im autologen Plasma reinkubiert. Die spezifische Bindung zeigt bei trainierten und untrainierten Probanden ein Sättigungsverhalten bei ungefähr 2 nmol/l Dihydroalprenolol. Sie beträgt 85% (0,1 nmol/1 Dihydroalprenolol) bis 51% (2,0 nmol/1 Dihydroalprenolol) der Gesamtbindung. B max . beträgt, basierend auf der Scatchard-Analyse, 41,2 fmol/10 7 Zellen (Trainierte) und 21,6 fmol/10 7 Zellen (Untrainierte). Die Dissonanzkonstante (Ko) wurde zu 0,44 nmol/l Dihydroalprenolol (Untrainierte) und 0,49 nmol/1 Dihydroalprenolol (Trainierte) berechnet, die Zahl der ß-adrenergen Bindungsstellen beträgt ungefähr 1300 (Untrainierte) und 2500 pro Zelle (Trainierte). Die spezifische Bindung von Dihydroalprenolol an intakte polymorphkernige Leukocyten von ausdauertrainierten ist signifikant höher als bei untrainierten Probanden. Die trainings-abhängige Änderung der ß-Rezeptoren-Dichte kann ein Indikator einer gesteigerten Empfindlichkeit gegenüber Katecholaminen sein.
Circulation Research, 2002
Hypertrophied and failing cardiac myocytes generally show alterations in intracellular Ca 2ϩ hand... more Hypertrophied and failing cardiac myocytes generally show alterations in intracellular Ca 2ϩ handling associated with changes in the contractile function and arrhythmogenicity. The cardiac Na ϩ -Ca 2ϩ exchange (NCX) is an important mechanism for Ca 2ϩ extrusion and cell relaxation. Its possible involvement in changes of excitation-contraction coupling (EC-coupling) with disease remains uncertain. We analyzed the NCX function in rat ventricular myocytes 5 to 6 months after experimental myocardial infarction (PMI) produced by left coronary artery ligation and from sham-operated (SO) hearts. Caged Ca 2ϩ was dialyzed into the cytoplasm via a patch-clamp pipette and Ca 2ϩ was released by flash photolysis to activate NCX and measure the associated currents (I NaCa ), whereas [Ca 2ϩ ] i changes were simultaneously recorded with a confocal microscope. I NaCa density normalized to the [Ca 2ϩ ] i jumps was 2.6-fold higher in myocytes from PMI rats. The level of total NCX protein expression in PMI myocytes was also increased. Interestingly, although the I NaCa density in PMI cells was larger, PMI and SO myocytes presented virtually identical Ca 2ϩ transport via the NCX. This discrepancy was explained by a reduced surface/volume ratio (34.8%) observed in PMI cells. We conclude that the increase in NCX density may be a mechanism to maintain the required Ca 2ϩ extrusion from a larger cell to allow adequate relaxation. (Circ Res. 2002;91:323-330.)
Cellular Signalling, 2005
The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite ... more The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells. CXCL12 promoted growth in multipotent cells by activating the RhoA-Rho kinase pathway. Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins. Rho activation required a G i -mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively. By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCh activity. The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCh. Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by Gprotein-coupled receptor agonists. D
Cellular Signalling, 2005
Hematopoietic cells uniquely express G a16 , a G protein a-subunit of the G q -type. G a16 is obl... more Hematopoietic cells uniquely express G a16 , a G protein a-subunit of the G q -type. G a16 is obligatory for P2Y 2 receptor-dependent Ca 2+mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y 2 receptors physically interact with G a16 . Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca 2+ -signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G a16 and P2Y 2 receptors form constitutive complexes, and that the P2Y 2 receptor is present as an oligomer. D
Experientia, 1973
ABSTRACT Zusammenfassung In Herzmuskelpräparaten sind zwei Systeme für den Ca-Durchtritt durch di... more ABSTRACT Zusammenfassung In Herzmuskelpräparaten sind zwei Systeme für den Ca-Durchtritt durch die Plasmamembranen zu unterscheiden: 1. Ein spannungs- und zeitabhängiger Ca-Einwärtsstrom, der während der Plateauphase des Aktionspotentials fliesst und durch La3+ gehemmt wird; 2. ein Na−Ca-Austauschsystem, das vor allem für den Auswärtstransport von Ca aus der Zelle verantwortlich ist, und durch La3+ nicht beeinflusst wird.
Cell Calcium, 2006
We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control... more We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca 2+ release and store-operated Ca 2+ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 M) and the Src inhibitor PP2 (10 M). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca 2+ transients were reduced by imatinib and/or PP2. Ca 2+ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca 2+ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKC␣ catalytic activity and PKC␣ co-immunoprecipitated with Bcr/Abl. Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca 2+ influx was reduced by complexing extracellular Ca 2+ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca 2+ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.
Cell Calcium, 2005
Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expr... more Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expression of the cardiac Na + -Ca 2+ exchanger (NCX1) to correlate suppression of the Na + -Ca 2+ exchange function with down-regulation of NCX1 protein expression. In a denovo expression system (Sf9 cells), a decrease in both, NCX1 mRNA and protein after AS-ODN application was paralleled by diminished NCX1 activity, a typical hallmark of a true "antisense effect". Although AS-ODN uptake was also efficient in rat neonatal cardiac myocytes, in wholecell extracts of these cells treated with AS-ODNs, the amount of NCX1 protein determined in a quantitative binding assay remained almost unchanged, despite a prompt loss of NCX1 function. Immunocytochemical staining of myocytes revealed that most of the immunoreactivity was not localized in the plasma membrane, but in intracellular compartments and was barely affected by AS-ODN treatment. These results indicate that the "functional half-life" of the NCX1 protein in the plasma membrane of neonatal cardiac myocytes is surprisingly short, much shorter than reported half-lifes of about 30 h for other membrane proteins.