Ca2+ signalling in K562 human erythroleukaemia cells: effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways

The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway.

Download Full-text

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646319 ◽

1992 ◽

Vol 68 (05) ◽

pp. 570-576 ◽

Cited By ~ 20

Author(s):

Mary A Selak

Keyword(s):

Neutrophil Elastase ◽

Cell Activation ◽

Thromboxane A2 ◽

Creatine Phosphate ◽

Dense Granule ◽

Cathepsin G ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Low Concentrations ◽

High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

Download Full-text

Synthesis and anticancer evaluation of sulfur containing 9-anilinoacridines

Recent Patents on Anti-Cancer Drug Discovery ◽

10.2174/1574892816666210728122910 ◽

2021 ◽

Vol 16 ◽

Author(s):

Pranav Gupta ◽

Radhika V. Kumar ◽

Chul-Hoon Kwon ◽

Zhe-Sheng Chen

Keyword(s):

Cell Cycle ◽

Anticancer Activity ◽

Topoisomerase Ii ◽

Critical Role ◽

K562 Cells ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

In Vivo Models ◽

Topo Ii ◽

Sulfur Containing

Background: DNA topoisomerases are a class of enzymes that play a critical role in fundamental biological processes of replication, transcription, recombination, repair and chromatin remodeling. Amsacrine (m-AMSA), the best-known compound of 9-anilinoacridines series was one of the first DNA-intercalating agents to be considered as a Topoisomerase II inhibitor. Objective: A series of sulfur containing 9-anilinoacridines related to amsacrine were synthesized and evaluated for their anticancer activity. Methods: Cell viability was assessed by the MTT assay. The topoisomerase II inhibitory assay was performed using the Human topoisomerase II Assay kit and flow cytometry was used to evaluate the effects on cell cycle of K562 cells. Molecular docking was performed using Schrödinger Maestro program. Results: Compound 36 was found to be the most cytotoxic of the sulfide series against SW620, K562, and MCF-7. The limited SAR suggested the importance of the methansulfonamidoacetamide side chain functionality, the lipophilicity and relative metabolic stability of 36 in contributing to the cytotoxicity. Topoisomerase II α inhibitory activity appeared to be involved in the cytotoxicity of 36 through inhibition of decatenation of kinetoplast DNA (kDNA) in a concentration dependent manner. Cell cycle analysis further showed the Topo II inhibition through accumulation of K562 cells in G2/M phase of cell cycle. Docking of 36 into the Topo II α-DNA complex suggested that it may be an allosteric inhibitor of Topo II α. Conclusion: Compound 36 exhibits anticancer activity by inhibiting topoisomerase II and it could further be evaluated in in vivo models.

Download Full-text

Endothelium and mechanical responses of isolated monkey pulmonary veins to histamine

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1990.259.4.h1032 ◽

1990 ◽

Vol 259 (4) ◽

pp. H1032-H1037 ◽

Cited By ~ 2

Author(s):

T. Matsuki ◽

T. Ohhashi

Keyword(s):

Pulmonary Veins ◽

Dependent Manner ◽

High Concentration ◽

Concentration Dependent Manner ◽

Mechanical Responses ◽

Relaxing Factor ◽

Prostaglandin F2 Alpha ◽

H2 Receptors ◽

Endothelium Derived Relaxing Factor ◽

Stimulation Of

Ring strips of monkey pulmonary veins precontracted with a high concentration of prostaglandin F2 alpha (PGF2 alpha) relaxed in a concentration-dependent manner in response to histamine. Treatment with mepyramine and/or famotidine attenuated the relaxation. 2-Pyridylethylamine (2PEA) and dimaprit caused relaxations in the precontracted preparations, which were inhibited by pretreatment with mepyramine and famotidine, respectively. Removal of endothelium reversed the histamine- and 2PEA-induced relaxations to dose-related contractions. On the other hand, the removal had no effect on the dimaprit-induced relaxations, which were significantly reduced by pretreatment with famotidine. Histamine-induced relaxations in the precontracted strips with endothelium in the presence and absence of famotidine were suppressed or abolished by treatment with methylene blue or hemoglobin but were unaffected by aspirin. It may be concluded that histamine-induced relaxation in monkey pulmonary veins precontracted with PGF2 alpha is mediated by H2-receptors in smooth muscle and H1-receptors in endothelium. Also, stimulation of the endothelial H1-receptors liberates an endothelium-derived relaxing factor.

Download Full-text

In Vivo Targets of Pasteurella Multocida Toxin

International Journal of Molecular Sciences ◽

10.3390/ijms21082739 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2739

Author(s):

Arshiya Banu ◽

Alistair J. Lax ◽

Agamemnon E. Grigoriadis

Keyword(s):

G Proteins ◽

Protein Modification ◽

Pasteurella Multocida ◽

Term Treatment ◽

Atrophic Rhinitis ◽

Short Term Treatment ◽

Experimental Conditions ◽

Proliferation Markers ◽

Pasteurella Multocida Toxin

Many Pasteurella multocida strains are carried as commensals, while some cause disease in animals and humans. Some type D strains cause atrophic rhinitis in pigs, where the causative agent is known to be the Pasteurella multocida toxin (PMT). PMT activates three families of G-proteins—Gq/11, G12/13, and Gi/o—leading to cellular mitogenesis and other sequelae. The effects of PMT on whole animals in vivo have been investigated previously, but only at the level of organ-specific pathogenesis. We report here the first study to screen all the organs targeted by the toxin by using the QE antibody that recognizes only PMT-modified G-proteins. Under our experimental conditions, short-term treatment of PMT is shown to have multiple in vivo targets, demonstrating G-alpha protein modification, stimulation of proliferation markers and expression of active β-catenin in a tissue- and cell-specific manner. This highlights the usefulness of PMT as an important tool for dissecting the specific roles of different G-alpha proteins in vivo.

Download Full-text

Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.1.f129 ◽

1997 ◽

Vol 273 (1) ◽

pp. F129-F135 ◽

Cited By ~ 9

Author(s):

J. M. Arthur ◽

G. P. Collinsworth ◽

T. W. Gettys ◽

L. D. Quarles ◽

J. R. Raymond

Keyword(s):

G Protein ◽

G Proteins ◽

Plasma Membranes ◽

Mdck Cells ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cellular Activation ◽

Protein Activation ◽

A Cell ◽

Cation Sensing

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

Download Full-text

Adrenergic and cholinergic responsiveness of isolated canine bronchial arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1990.259.1.h156 ◽

1990 ◽

Vol 259 (1) ◽

pp. H156-H161 ◽

Cited By ~ 1

Author(s):

S. T. O'Rourke ◽

P. M. Vanhoutte

Keyword(s):

Electrical Stimulation ◽

Bronchial Artery ◽

Cholinergic Receptor ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bronchial Arteries ◽

Prostaglandin F2 Alpha ◽

The Right ◽

Endothelium Dependent Relaxation ◽

Concentration Response Curve

The purpose of this study was to characterize the adrenergic and cholinergic responsiveness of isolated rings of canine bronchial arteries. Electrical stimulation of the vessels produced frequency-dependent contractions that were inhibited by phenoxybenzamine, tetrodotoxin, and phentolamine but not by atropine. Norepinephrine caused concentration-dependent contractions of the rings; the concentration-response curve to norepinephrine was shifted to the right by phentolamine and prazosin, whereas rauwolscine had no effect. Isoproterenol, norepinephrine, tyramine, and electrical stimulation (in the presence of phentolamine) failed to elicit relaxation in rings contracted with prostaglandin F2 alpha. Contracted rings relaxed to acetylcholine in an endothelium- and concentration-dependent manner. At concentrations up to 10(-4) M, acetylcholine did not evoke contractions in these tissues. Endothelium-dependent relaxation to acetylcholine was inhibited by atropine and 4-diphenyl-acetoxy-N-methyl piperidine methiodine but not by pirenzepine. These results suggest that alpha 1-adrenoceptors mediate contraction to norepinephrine, and M3-muscarinic receptors mediate endothelium-dependent relaxation to acetylcholine in the canine bronchial artery. Moreover, the smooth muscle of these vessels does not respond directly to beta-adrenergic- or cholinergic-receptor stimulation.

Download Full-text

Pre- and postjunctional effects of some prostanoids in human isolated vas deferens

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1991.260.4.r792 ◽

1991 ◽

Vol 260 (4) ◽

pp. R792-R797 ◽

Cited By ~ 1

Author(s):

F. Holmquist ◽

H. Hedlund ◽

K. E. Andersson

Keyword(s):

Vas Deferens ◽

Thromboxane A2 ◽

Electrical Field Stimulation ◽

Inhibitory Effect ◽

Dependent Manner ◽

Inhibitory Effects ◽

Concentration Dependent Manner ◽

Thromboxane A2 Receptor ◽

Thromboxane Receptor ◽

Thromboxane A2 Analogue

The effects of prostaglandin (PG) E1, PGE2, the thromboxane A2 analogue U-44069, and the prostacyclin derivative iloprost were studied on isometric contractions induced by norepinephrine (NE) and by electrical field stimulation of nerves in isolated preparations of the human vas deferens. The effects of these agents on the electrically induced release of 3H from preparations preincubated with [3H]NE were also investigated. PGE1 and PGE2 inhibited the electrically induced contractions concentration dependently. U-44069 augmented the contractions without affecting baseline tension, and in preparations where the contractions had been inhibited by PGE1 or PGE2, U-44069 restored the contractions almost to starting levels. The thromboxane A2-receptor antagonist BM 13505, having no effect or inhibitory effects on electrically induced contractions, abolished the stimulatory effect of U-44069. Contractions induced by exogenous NE were augmented by U-44069, whereas PGE1 and BM 13505 were without effects. The electrically induced release of 3H was inhibited by PGE1 and PGE2 in a concentration-dependent manner, whereas U-44069 and BM 13505 increased the release of 3H. Furthermore, the inhibitory effect of PGE1 on 3H release was partly counteracted by U-44069. Iloprost had no significant effect on electrically induced contractions or on 3H release. These results suggest that, in the human vas deferens, thromboxane A2 augments contractions predominantly through a postjunctional site of action, whereas PGs of the E type have a prejunctional inhibitory effect. In addition, the pre- and post-junctional effect profiles of U-44069 and BM 13505 suggest that there may be more than one thromboxane receptor.

Download Full-text

Functionally Selective Inhibition of the Oxytocin Receptor by Retosiban in Human Myometrial Smooth Muscle

Endocrinology ◽

10.1210/endocr/bqz043 ◽

2020 ◽

Vol 161 (2) ◽

Author(s):

Paul J Brighton ◽

Michael J Fossler ◽

Siobhan Quenby ◽

Andrew M Blanks

Keyword(s):

Smooth Muscle ◽

G Proteins ◽

Small Molecule ◽

Selective Inhibition ◽

Oxytocin Receptor ◽

Human Myometrium ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Prostaglandin Production ◽

Downstream Signaling

Abstract Novel small molecule inhibitors of the oxytocin receptor (OTR) may have distinct pharmacology and mode of action when compared with first-generation oxytocin antagonists when used for the prevention of preterm birth. The aim was to determine the mechanism of action of small molecule OTR antagonists retosiban and epelsiban compared with the currently used peptide-based compound atosiban. Human myometrial samples were obtained at cesarean section and subjected to pharmacological manipulations to establish the effect of antagonist binding to OTR on downstream signaling. Retosiban antagonism of oxytocin action in human myometrium was potent, rapid, and reversible. Inhibition of inositol 1,4,5-trisphosphate (IP3) production followed single-site competitive binding kinetics for epelsiban, retosiban, and atosiban. Retosiban inhibited basal production of IP3 in the absence of oxytocin. Oxytocin and atosiban but not retosiban inhibited forskolin, and calcitonin stimulated 3′,5′-cyclic adenosine 5′-mono-phosphate (cAMP) production. Inhibition of cAMP was reversed by pertussis toxin. Oxytocin and atosiban, but not retosiban and epelsiban, stimulated extracellular regulated kinase (ERK)1/2 activity in a time- and concentration-dependent manner. Oxytocin and atosiban stimulated cyclo-oxygenase 2 activity and subsequent production of prostaglandin E2 and F2α. Prostaglandin production was inhibited by rofecoxib, pertussin toxin, and ERK inhibitor U0126. Oxytocin but not retosiban or atosiban stimulated coupling of the OTR to Gα q G-proteins. Oxytocin and atosiban but not retosiban stimulated coupling of the OTR to Gα i G-proteins. Retosiban and epelsiban demonstrate distinct pharmacology when compared with atosiban in human myometrial smooth muscle. Atosiban displays agonist activity at micromolar concentrations leading to stimulation of prostaglandin production.

Download Full-text

Differential cholera-toxin- and pertussis-toxin-catalysed ADP-ribosylation of G-proteins coupled to formyl-peptide and leukotriene B4 receptors

Biochemical Journal ◽

10.1042/bj2890469 ◽

1993 ◽

Vol 289 (2) ◽

pp. 469-473 ◽

Cited By ~ 8

Author(s):

T M Schepers ◽

K R McLeish

Keyword(s):

Cholera Toxin ◽

G Proteins ◽

Pertussis Toxin ◽

Leukotriene B4 ◽

Guanine Nucleotides ◽

Dependent Manner ◽

Functional Responses ◽

Concentration Dependent Manner ◽

Adp Ribosylation ◽

Formyl Peptide

N-Formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and leukotriene B4 (LTB4) induce disparate second-messenger generation and functional responses in neutrophils and HL-60 granulocytes. Receptors for these chemoattractants couple to a common pool of G-proteins which are substrates for both pertussis-toxin- and cholera-toxin-catalysed ADP-ribosylation. The hypothesis that formyl-peptide and LTB4 receptors induce different receptor-specific conformations of activated G-proteins was tested. The ability of pertussis toxin and cholera toxin to ADP-ribosylate G(i) proteins coupled to formyl-peptide or LTB4 receptors in membranes isolated from HL-60 granulocytes was used to assess the conformational state of the alpha subunits. Cholera-toxin-catalysed ADP-ribosylation of alpha 40 (40 kDa alpha subunit) was inhibited by guanosine 5′-[beta gamma-imido]triphosphate and GDP in a concentration-dependent manner. Addition of fMet-Leu-Phe, but not LTB4, re-established cholera-toxin labelling of alpha 40 in the presence of either guanine nucleotide. In the absence of guanine nucleotides, fMet-Leu-Phe and C5a enhanced cholera-toxin-catalysed labelling of alpha 40, whereas LTB4 and platelet-activating factor had no effect. Preincubation with fMet-Leu-Phe, but not LTB4, inhibited pertussis-toxin labelling of alpha 40 in the presence of guanosine 5′-[gamma-thio]triphosphate and in the absence of guanine nucleotides. Preincubation with fMet-Leu-Phe or LTB4 enhanced pertussis-toxin labelling of alpha 40 in the presence of GDP. These data suggest that activated G(i) proteins coupled to formyl-peptide and LTB4 receptors exist in different conformations determined by the receptor with which they interact.

Download Full-text

Decitabine Downregulates TIGAR to Induce Apoptosis and Autophagy in Myeloid Leukemia Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/8877460 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Lanzhu Li ◽

Wenjie Liu ◽

Qian Sun ◽

Han Zhu ◽

Ming Hong ◽

...

Keyword(s):

Myeloid Leukemia ◽

Dna Methyltransferase ◽

Negative Impact ◽

Cell Metabolism ◽

Leukemia Cell ◽

K562 Cells ◽

Glycolytic Pathway ◽

Dependent Manner ◽

Ros Production ◽

Concentration Dependent Manner

Decitabine (DAC) is a well-known DNA methyltransferase inhibitor, which has been widely used for the treatment of acute myeloid leukemia (AML). However, in addition to hypomethylation, DAC in AML is also involved in cell metabolism, apoptosis, and immunity. The TP53-induced glycolysis and apoptosis regulator (TIGAR) functions to inhabit glycolysis and protect cancer cells from reactive oxygen species- (ROS-) associated apoptosis. Our previous study revealed that TIGAR is highly expressed in myeloid leukemia cell lines and AML primary cells and associated with poor prognosis in adult patients with cytogenetically normal AML. In the present study, it was found that in a time- and concentration-dependent manner, DAC downregulates the TIGAR expression, induces ROS production, and promotes apoptosis in HL-60 and K562 cells. However, blocking the glycolytic pathway partially reversed the combined effects of DAC and TIGAR knockdown on apoptosis, ROS production, and cell cycle arrest, indicating that DAC induced apoptosis through the glycolytic pathway. Furthermore, TIGAR also has a negative impact on autophagy, while DAC treatment upregulates autophagy-related proteins LC3, Beclin-1, ATG3, and ATG-5, downregulates p62, and promotes the formation of autophagosomes, indicating that DAC may activate autophagy by downregulating TIGAR. Taken together, DAC plays an unmethylated role in inducing apoptosis and activating autophagy in myeloid leukemia by downregulating TIGAR.

Download Full-text