Chemosensory Adaptation to Lysozyme and GTP Involves Independently Regulated Receptors in Tetrahymena thermophila

ABSTRACT. Chemosensory adaptation is seen in Tetrahymena thermophila following prolonged exposure (ten minutes) to micromolar concentrations of the chemorepellents lysozyme or guanosine triphosphate (GTP). Since these cells initially show repeated backward swimming episodes (avoidance reactions) in these repellents, behavioral adaptation is seen as a decrease in this repellent-induced behavior. The time course of this behavioral adaptation is paralleled by decreases in the extents of surface binding of either [³²P]GTP or [³H]lysozyme in vivo. Scatchard plot analyses of repellent binding in adapted cells suggests the behavioral adaptation is due to a dramatic decrease in the number of surface binding sites, as represented by decreased Bmax values. The estimated K_D values for nonadapted cells are 6.6 μM and 8.4 μM for lysozyme and GTP binding, respectively. Behavioral adaptation and decreased surface receptor binding are specific for each repellent. The GTP adapted cells (20 μM for ten minutes) still respond behaviorally to 50 μM lysozyme and bind [³H]lysozyme normally. Lysozyme adapted cells (50 μM for ten minutes) still bind [³²P]GTP and respond behaviorally to GTP. All the behavioral and binding changes seen are also reversible (deadaptation). Neomycin was shown to be a competitive inhibitor of [³H]lysozyme binding and lysozyme-induced avoidance reactions, but it had no effect on either [³²P]GTP binding or GTP-induced or avoidance reactions. These results are consistent with the hypothesis that there are two separate repellent receptors, one for GTP and the other for lysozyme, that are independently downregulated during adaptation to cause specific receptor desensitization and consequent behavioral adaptation.     

Enhanced lipid peroxidation by extracellular ATP in PC12 cells

Recently we have demonstrated that extracellular ATP acts as an excitatory neurotransmitter and enhances cell death in the presence of ferrous ions. By using a newly developed cis-parinaric acid fluorescence technique, we demonstrated that ATP, in a dose dependent manner, enhanced the increased membrane lipid peroxidation in PC12 cells when cells were incubated with micromolar FeCl₂/DTP. P₂ purinoceptor agonists, α,β-methylene ATP and 2-methylthio-ATP, induced PC12 cell lipid peroxidation, but to a lesser extent than ATP. ATP-induced Ca²⁺ influx via P₂ purinoceptor activation significantly increased the intracellular Ca²⁺ concentration, which may have triggered a free radical generating cascade(s), and led to membrane lipid peroxidation and cell death. Since oxidative stress has been implicated in certain neurodegenerative diseases such as aging, extracellular ATP may contribute to neuronal cell death by an oxidative mechanism involving lipid peroxidation.     

Characterization of ATP‐induced cell death in the GL261 mouse glioma

Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP, a P2X7 agonist, induced cell death at concentrations that are described to activate the P2X7 receptor in mouse. oATP, an antagonist of P2X7, blocked the ATP‐induced cell death. Agonists of purinergic receptors expressed in GL261 such as adenosine, ADP, UTP did not cause any cell death, even at mM concentrations. A sub‐population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP‐induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP‐induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. These data show the importance of P2X7 in ATP‐induced cell death and shed light on the importance of ATP‐induced cell death in glioma development. J. Cell. Biochem. 109: 983–991, 2010. © 2010 Wiley‐Liss, Inc.     

Agonists of the P2Y(AC)-receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma

We have previously shown that an ecto-NPPase modulates the ATP- and ADP-mediated P2Y(AC)-receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap(3)A induced no detectable PI turnover and were identified as specific agonists of the P2Y(AC)-receptor with EC(50) values of 250 +/- 37 pM and 1 +/- 0.5 microM, respectively. P2Y(AC)-receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2Y(AC)-purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP(3)-independent Ca(2+)-influx was observed after P2Y(AC)-receptor activation. Inhibition of this influx by Ca(2+)-chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a specific MEK-inhibitor inhibited the 2MeSADP- and Ap(3)A-induced MAP kinase activation. In addition, transfection with dominant negative RhoA(Asn19) rendered C6 cells insensitive to P2Y(AC)-receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf-1 after P2Y(AC)-receptor activation. We may conclude that P2Y(AC)-purinoceptor agonists activate MAP kinase through a G(i)-RhoA-PKC-raf-MEK-dependent, but ras- and Ca(2+)-independent cascade.     

An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons

Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA(A) receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X(3) or P2X(2+3) and GABA(A) receptors does not depend on chloride and calcium ions. We identified an intracellular QST(386-388) motif in P2X(3) subunits which is required for the functional coupling with GABA(A) receptors. Moreover the cross-inhibition between native P2X(3) and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA(A)beta3 subunits. We provide evidence that P2X(3) and GABA(A) receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X(3)-GABA(A) subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.     

P2Y1嘌呤受体在心血管系统的研究进展

P2Y1嘌呤受体是最早被发现并克隆的P2Y受体家族成员,属G蛋白偶联受体,其内源性激动剂主要是ADP及ATP。随着研究深入,人们发现该受体在多个方面有着重要作用,参与机体多种生理病理过程。本文对该受体近年来在心血管系统方面的研究进行综述,提示P2Y1受体有可能成为疾病治疗的潜在靶点。     

Plasticity of purine release during cerebral ischemia: clinical implications?

Adenosine is a powerful modulator of neuronal function in the mammalian central nervous system. During a variety of insults to the brain, adenosine is released in large quantities and exerts a neuroprotective influence largely via the A₁ receptor, which inhibits glutamate release and neuronal activity. Using novel enzyme-based adenosine sensors, which allow high spatial and temporal resolution recordings of adenosine release in real time, we have investigated the release of adenosine during hypoxia/ischemia in the in vitro hippocampus. Our data reveal that during the early stages of hypoxia adenosine is likely released per se and not as a precursor such as cAMP or an adenine nucleotide. In addition, repeated hypoxia results in reduced production of extracellular adenosine and this may underlie the increased vulnerability of the mammalian brain to repetitive or secondary hypoxia/ischemia.     

Control of P2X(2) channel permeability by the cytosolic domain

ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na(+)-selective I(1) state to the I(2) state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X(2) (rP2X(2)) channels does not occur at mouse P2X(2) (mP2X(2)) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X(2) and mP2X(2) channels have a pore diameter of approximately 11 A in the I(1) state, but the transition to the I(2) state increases the rP2X(2) diameter by at least 3 A. The I(1) to I(2) transition occurs with a rate constant of approximately 0.5 s(-1). The data focus attention on specific residues of P2X(2) channel cytoplasmic domains as determinants of permeation in a state-specific manner.