Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today. 16:524–528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R.A. North and G. Buell. 1996. Science (Wash. DC). 272:735–737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1β. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1β release triggered by LPS. Our data suggest that LPS-dependent IL-1β release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1β secretion.
We investigated the effect of pharmacologic modulation of the ATP receptor on intracellular ion changes and proliferative response of human peripheral blood lymphocytes (PBLs) and purified T lymphocytes. Extracellular ATP (ATPe) triggered in these cells an increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization. Whereas both Ca2+ release from intracellular stores and influx across the plasma membrane were detected in the whole PBL population, only Ca2+ influx was observed in T cells. In the presence of near physiologic extracellular Na+ concentrations (125 mmol/L), Ca2+ permeability through the ATPe-gated channel was very low, suggesting a higher selectivity for monovalent over divalent cations. The selective P2Z agonist benzoylbenzoic ATP (BzATP) increased [Ca2+]i in the presence but not the absence of extracellular Ca2+ and also caused plasma membrane depolarization. The covalent blocker oxidized ATP (oATP), an inhibitor of P2X and P2Z receptors, prevented Ca2+ influx and plasma membrane depolarization, but had no effect on Ca2+ release from stores. Stimulation with ATPe alone had no significant effects on PBL 3H-thymidine incorporation. On the contrary, ATPe or BzATP had a synergistic effect on DNA synthesis stimulated by selective T-cell mitogens such as phytohemagglutinin, anti-CD3 monoclonal antibody, or allogenic PBLs (mixed lymphocyte cultures). Treatment with oATP inhibited mitogenic stimulation by these receptor-directed agents but not by the combined application of the Ca2+ ionophore ionomycin and phorbol myristate acetate. Interleukin-2 partially relieved inhibition by oATP. These results suggest that human T lymphocytes express a plasma membrane channel gated by ATPe that is involved in mitogenic stimulation.
We have observed a striking difference in the response to extracellular ATP in lymphoblastoid cell lines established from Duchenne muscular dystrophy patients and normal subjects. Duchenne muscular dystrophy cells stimulated by extracellular ATP underwent a large increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization, while normal cell lines were little or not at all responsive. These changes in intracellular ion homeostasis were due to activation of an ATP-gated membrane channel permeable to Na+ and Ca2+, with little or no contribution of Ca2+ release from intracellular stores. The channel was selectively activated by ATP, since other purine/pyrimidine nucleotides were ineffective, and it was inhibited by pretreatment with oxidized ATP, a compound previously reported to irreversibly inhibit P2 purinergic receptors. In the presence of extracellular ATP, lymphoblastoid cells established from Duchenne muscular dystrophy patients, but not from healthy controls, underwent rounding and swelling and eventually lysed. The results of this study suggest that lymphoblastoid cells isolated from Duchenne muscular dystrophy patients are eminently sensitive to stimulation by extracellular ATP.
Two series of compounds were designed to mimic the transition state and high-energy intermediates (HEI) of the enzymatic reaction of 6-phosphogluconate dehydrogenase (6PGDH). Sulfoxide analogues (7-11) were designed to mimic the transition state during the oxidation of the substrate to 3-keto-6-phosphogluconate, an enzyme-bound intermediate of the enzyme. Hydroxamate and amide derivatives of d-erythronic acid were designed to mimic the 1,2-cis-enediol HEI of the 6PGDH reaction. These two series of compounds were assayed as competitive inhibitors of the Trypanosoma brucei and sheep liver enzymes, and their selectivity value (ratio sheep/parasite) was calculated. The sulfoxide transition-state analogues showed weak and selective inhibition of the T. brucei enzyme. The hydroxamic derivatives showed potent and selective inhibition of the T. brucei 6PGDH with a Ki in the nanomolar range.
New drugs are urgently required for Human African Trypanosomiasis (sleeping sickness), a disease which has re-emerged as a major health threat in Sub-Saharan Africa. The third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, has been shown to be a good target for drugs. The enzyme is essential to the trypanosomes that causes sleeping sickness and structural differences when compared to its mammalian counterpart allow for selective inhibition. Three series of inhibitors have been designed, these include phosphorylated carbohydrate substrate and transition state analogues, non-carbohydrate substrate analogues and also triphenylmethane-based compounds. All have shown selective inhibition of the trypanosomal 6-phosphogluconate dehydrogenase and representatives of each have trypanocidal activity.
The kinetics of 6-phosphogluconate dehydrogenase from Trypanosoma brucei was examined and compared to those of the same enzyme from lamb's liver. Variation of kinetic parameters as a function of pH suggests a chemical mechanism similar to other 6-phosphogluconate dehydrogenases. The comparison extended to a detailed analysis of the effect on enzyme activity by several inhibitors including the trypanocidal drugs suramin, melarsoprol and analogues of these compounds. The 7: brucei enzyme differs significantly from its mammalian counterpart with respect to several inhibitors, particularly the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate which have respectively 170-fold and 40-fold higher affinity for the parasite enzyme.
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