kn-62 and Leukemia--Lymphocytic--Chronic--B-Cell

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Amino Acid Sequence; Animals; Anti-Bacterial Agents; Calcium; Cell Line; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Ethidium; Humans; Intracellular Fluid; K562 Cells; Leukemia, Lymphocytic, Chronic, B-Cell; Macrophages; Mice; Molecular Sequence Data; Oxidation-Reduction; Polymyxin B; Purinergic P2 Receptor Antagonists; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Transfection

The effects of extracellular ATP, ADP, AMP and adenosine on cAMP accumulation have been studied in freshly isolated B-lymphocytes from patients with chronic lymphocytic leukemia. Extracellular ATP and several nucleotide analogs stimulated cAMP accumulation with the following order of potency: ATP (EC(50)=120+/-20 microM)>ADP>>AMP. ADP was less effective than ATP and may be a partial agonist. AMP exhibited variable but generally weak activity. The stable analog of ATP, alpha,beta-methylene ATP (EC(50)=110+/-15 microM) also stimulated cAMP accumulation and exhibited similar efficacy to ATP. The P2Y(2) receptor agonist, UTP had no effect on intracellular cAMP levels. Adenosine and the A(2A)/A(2B) receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated cAMP accumulation in CLL lymphocytes. Adenosine deaminase inhibited the cAMP response to adenosine but had no effect on the ATP-induced cAMP response. On the other hand, the AMP analog, adenosine 5'-thiomonophosphate, (AMPS; 1.0 mM) inhibited ATP-induced and alpha,beta-methylene ATP-induced cAMP production but had no effect on adenosine-induced cAMP production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of P2Y(11) receptor as well as A(2A) and A(2B) receptor mRNA in chronic lymphocytic leukemia lymphocytes. However, A(2B) receptors would appear to be relatively ineffective because the A(2A) selective agonist, CGS-21680 exhibited comparable efficacy to NECA. Furthermore, the A(2A)-selective antagonist 8-(3-chlorostyryl)-caffeine (CSC) right-shifted the concentration-response curve for NECA. Taken together, the data indicate that ATP induces cAMP accumulation via the activation of P2Y(11) receptors whereas adenosine induces cAMP accumulation via the activation of A(2A) receptors. Coordinate activation of P2Y(11) and A(2A) receptors may influence the developmental fate of normal B-lymphocytes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Cyclic AMP; Dose-Response Relationship, Drug; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocytes; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X7; RNA, Messenger

1. Although extracellular adenosine 5'-triphosphate (ATP) is the natural ligand for the P2Z receptor of human lymphocytes it is less potent than 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) in opening the associated ion channel, which conducts a range of permeants including Ba2+ and ethidium+. We have quantified the influx of ethidium+ into lymphocytes produced by BzATP, ATP, 2-methylthio-ATP (2MeSATP) and ATPgammaS, studied competition between ATP and BzATP and investigated the effects of KN-62, a new and potent inhibitor of the P2Z receptor. 2. BzATP and ATP stimulated ethidium+ influx with EC50 values of 15.4+/-1.4 microM (n=5) and 85.6+/-8.8 microM (n=5), respectively. The maximal response to ATP was only 69.8+/-1.9% of that for BzATP. Hill analysis gave nH of 3.17+/-0.24 (n=3) and 2.09+/-0.45 (n=4) for BzATP and ATP, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z receptor-operated ion channel. 3. A rank order of agonist potency of BzATP>ATP=2MeSATP>ATPgammaS was observed for agonist-stimulated ethidium+ influx, while maximal influxes followed a rank order of BzATP>ATP>2MeSATP>ATPgammaS. 4. Preincubation with 30-50 microM oxidized ATP (ox-ATP), an irreversible P2Z inhibitor, reduced the maximal response but did not change the steepness of the Ba2+ influx-response curve produced by BzATP (nH 3.2 and 2.9 for 30 and 50 microM ox-ATP, respectively (n=2)). 5. ATP (300-1000 microM) added simultaneously with 30 microM BzATP (EC90) inhibited both ethidium+ and Ba2+ fluxes to a maximum of 30-40% relative to the values observed with BzATP alone. Moreover, ATP (300 microM) shifted the concentration-response curve to the right for BzATP-stimulated Ba2+ influx, confirming competition between ATP and BzATP. 6. KN-62, a new and powerful inhibitor of the lymphocyte P2Z receptor, showed less potency in antagonizing BzATP-mediated fluxes than ATP-induced fluxes when maximal concentrations of both agonists (BzATP, 50 microM; ATP, 500 microM) were used. 7. These data suggest that the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a more efficacious agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Affinity Labels; Barium; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethidium; Flow Cytometry; Humans; In Vitro Techniques; Ion Channels; Leukemia, Lymphocytic, Chronic, B-Cell; Linear Models; Lymphocytes; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Structure-Activity Relationship; Thionucleotides