P2X receptors-mediated cytosolic phospholipase A2 activation in primary afferent sensory neurons contributes to neuropathic pain

Activation of P2X₃ and P2X_2/3 receptors (P2X₃R/P2X_2/3R), ionotropic ATP receptor subtypes, in primary sensory neurons is involved in neuropathic pain, a debilitating chronic pain that occurs after peripheral nerve injury. However, the underlying mechanisms remain unknown. We investigated the role of cytosolic phospholipase A₂ (cPLA₂) as a downstream molecule that mediates the P2X₃R/P2X_2/3R-dependent neuropathic pain. We found that applying ATP to cultured dorsal root ganglion (DRG) neurons increased the level of Ser505-phosphorylated cPLA₂ and caused translocation of Ser505-phosphorylated cPLA₂ to the plasma membrane. The ATP-induced cPLA₂ activation was inhibited by a selective antagonist of P2X₃R/P2X_2/3R and by a selective inhibitor of cPLA₂. In the DRG in vivo , the number of cPLA₂-activated neurons was strikingly increased after peripheral nerve injury but not after peripheral inflammation produced by complete Freund's adjuvant. Pharmacological blockade of P2X₃R/P2X_2/3R reversed the nerve injury-induced cPLA₂ activation in DRG neurons. Moreover, administering the cPLA₂ inhibitor near the DRG suppressed nerve injury-induced tactile allodynia, a hallmark of neuropathic pain. Our results suggest that P2X₃R/P2X_2/3R-dependent cPLA₂ activity in primary sensory neurons is a key event in neuropathic pain and that cPLA₂ might be a potential target for treating neuropathic pain.     

P2X7 pre-synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP-induced glutamate release

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X₇ receptors, it is still unclear whether neuronal functions can be attributed to P2X₇ receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X₇ receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non-neuronal cells and allows exposure of 'nude' release-regulating pre-synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X₇ receptors. Together, our findings suggest that (i) P2X₇ receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X₇ receptors are localized on glutamatergic nerve terminals; (iii) P2X₇ receptors play a significant role in ATP-evoked glutamate efflux, which involves Ca²⁺-dependent vesicular release; and (iv) the P2X₇ receptor itself constitutes a significant Ca²⁺-independent mode of exit for glutamate.     

ATP scavenging by the intracellular pathogen Porphyromonas gingivalis inhibits P2X7-mediated host-cell apoptosis

The purinergic receptor P2X₇ is involved in cell death, inhibition of intracellular infection and secretion of inflammatory cytokines. The role of the P2X₇ receptor in bacterial infection has been primarily established in macrophages. Here we show that primary gingival epithelial cells, an important component of the oral innate immune response, also express functional P2X₇ and are sensitive to ATP-induced apoptosis. Porphyromonas gingivalis, an intracellular bacterium and successful colonizer of oral tissues, can inhibit gingival epithelial cell apoptosis induced by ATP ligation of P2X₇ receptors. A P. gingivalis homologue of nucleoside diphosphate kinase (NDK), an ATP-consuming enzyme, is secreted extracellularly and is required for maximal suppression of apoptosis. An ndk -deficient mutant was unable to prevent ATP-induced host-cell death nor plasma membrane permeabilization in the epithelial cells. Treatment with purified recombinant NDK inhibited ATP-mediated host-cell plasma membrane permeabilization in a dose-dependent manner. Therefore, NDK promotes survival of host cells by hydrolysing extracellular ATP and preventing apoptosis-mediated through P2X₇.     

Neurosteroids differentially modulate P2X4 ATP-gated channels through non-genomic interactions

As neuroactive steroids modulate several ionotropic receptors, we assessed whether the ATP-gated currents elicited by P2X₄ receptors are modulated by these compounds. We transfected HEK293 cells or injected Xenopus laevis oocytes with the cDNA coding for rat P2X₄ receptor. Application of 0.1–10 μM alfaxolone potentiated within 60-s the 1 μM ATP-evoked currents with a maximal potentiation of 1.8 and 2.6-fold in HEK293 or oocytes cells respectively. Allopregnalolone or 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) also potentiated the ATP-gated currents but with a maximal effect only averaging 1.25 and 1.35-fold respectively. In contrast, 0.3–10 μM pregnanolone, but not its sulfated derivative, inhibited the ATP-gated currents; the maximal inhibition reached 40% in both cell types. THDOC, but not other neurosteroids increased significantly the τ_off of the ATP-evoked currents, revealing another mode of neurosteroid modulation. Sexual steroids such as 17β-estradiol or progesterone were inactive revealing explicit structural requirements. Alfaxolone or THDOC at concentrations 30- to 100-fold larger than required to modulate the receptor, gated the P2X₄ receptor eliciting ATP-like currents that were reduced with suramin or brilliant blue G, but potentiated the P2X₄ receptor more than 10-fold by 10 μM zinc. In conclusion, neurosteroids rapidly modulate via non-genomic mechanisms and with nanomolar potencies, the P2X4 receptor interacting likely at distinct modulator sites.     

P2Y receptor mediated inhibitory modulation of noradrenaline release in response to electrical field stimulation and ischemic conditions in superfused rat hippocampus slices

In this study, the inhibitory regulation of the release of noradrenaline (NA) by P2 receptors was investigated in hippocampus slices pre-incubated with [³H]NA. Electrical field stimulation (EFS; 2 Hz, 240 shocks, and 1 ms) released NA in an outside [Ca²⁺]-dependent manner, and agonists of P2Y receptors inhibited the EFS-evoked [³H]NA release with pharmacological profile similar to that of the P2Y₁ and P2Y₁₃ receptor subtypes. This inhibitory modulation was counteracted by bicuculline and 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline + 2-amino-5-phosphonovalerate and 2-amino-4-phosphonobutyrate. In contrast, the excess release in response to 30 min combined oxygen and glucose deprivation was outside [Ca²⁺] independent, but still sensitive to the inhibition of both facilitatory P2X₁ and inhibitory P2Y₁ receptors. Whereas mRNA encoding P2Y₁₂ and P2Y₁₃ receptor subunits were expressed in the brainstem, P2Y₁ receptor immunoreactivity was localized to neuronal somata and dendrites innervated by the mossy fiber terminals in the CA3 region of the hippocampus, as well as somata of granule cells and interneurons in the dentate gyrus. In summary, in addition to the known facilitatory modulation via P2X receptors, EFS-evoked [³H]NA outflow in the hippocampus is subject to inhibitory modulation by P2Y₁/P2Y₁₃ receptors. Furthermore, endogenous activation of both facilitatory and inhibitory P2 receptors may participate in the modulation of pathological NA release under ischemic-like conditions.     

The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells

The effects of 17β-estradiol (E₂) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E₂ at physiological concentrations on DA efflux in nerve growth factor-differentiated rat pheochromocytoma cells that express estrogen receptors (ER) α, ERβ, and G-protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of ³H-DA from pre-loaded cells; a 9–15 min 10⁻⁹ M E₂ treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non-genomic response, and an E₂-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 min. Efflux dose–responses for E₂ were non-monotonic, also characteristic of non-genomic estrogenic actions. ERα siRNA knockdown abolished E₂-mediated DA efflux, while ERβ knockdown did not, and GPR30 knockdown increased E₂-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ERα is the predominant mediator of E₂-mediated DA efflux, with inhibitory contributions from GPR30 and ERβ. E₂ also caused trafficking of ERα to the plasma membrane, trafficking of ERβ away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERα is largely responsible for non-genomic estrogenic effects on DAT activity.     

P2X7 and P2Y13 purinergic receptors mediate intracellular calcium responses to BzATP in rat cerebellar astrocytes

Previous work has established the presence of functional P2X₇ subunits in rat cerebellar astrocytes, which after stimulation with 3'- O -(4-benzoyl)benzoyl ATP (BzATP) evoked morphological changes that were not reproduced by any other nucleotide. To further characterize the receptor(s) and signaling mechanisms involved in the action of BzATP, we have employed fura-2 microfluorometry and the patch-clamp technique. BzATP elicited intracellular calcium responses that typically exhibited two components: the first one was transient and metabotropic in nature – sensitive to phospholipase C inhibition and pertussis toxin treatment –, whereas the second one was sustained and depended on the presence of extracellular calcium. The ionotropic nature of this latter component was corroborated by measurements of Mn²⁺ entry and macroscopic non-selective cation currents evoked by either BzATP (100 μM) or ATP (1 mM). The two components of the calcium response to BzATP differed in their pharmacological sensitivity. The metabotropic component was partially sensitive to pyridoxalphosphate-5'-phosphate-6-azo-(-2-chloro-5-nitrophenyl)-2,4-disulfonate, a selective antagonist of P2Y₁₃ receptors, while the ionotropic component was modulated by external magnesium and markedly reduced by brilliant blue G and 3-(5-(2,3-dichlorophenyl)-1 H -tetrazol-1-yl)methyl pyridine (A438079), thus implying the involvement of P2X₇ purinergic receptors. It is concluded that P2Y₁₃ and P2X₇ purinergic receptors are functionally expressed in rat cerebellar astrocytes and mediate the increase in intracellular calcium elicited by BzATP in these cells.     

Regions of the amino terminus of the P2X1 receptor required for modification by phorbol ester and mGluR1α receptors

The potentiation of P2X₁ receptor currents by phorbol ester (PMA) treatment and stimulation of mGluR1α receptors was sensitive to inhibition of novel forms of protein kinase C. Potentiation was also reduced by co-expression of an amino terminal P2X₁ receptor minigene. Cysteine point mutants of residues Tyr¹⁶-Gly³⁰ were expressed in Xenopus oocytes. Peak current amplitudes to ATP for Y16C, T18C and R20C mutants were reduced, however this did not result from a decrease in surface expression of the channels. The majority of the mutants showed changes in the time-course of desensitization of ATP evoked currents indicating the important role of this region in regulation of channel properties. PMA and mGluR1α potentiation was abolished for the mutants Y16C, T18C, R20C, K27C and G30C. Minigenes incorporating either Y16C, K27C, V29C or G30C still inhibited PMA responses. However D17C, T18C or R20C mutant minigenes were no longer effective suggesting that these residues are important for interaction with regulatory factors. These results demonstrate that the conserved YXTXK/R sequence and a region with a conserved glycine residue close to the first transmembrane segment contribute to PMA and GPCR regulation of P2X₁ receptors.     

Demonstration of the presence of alditols and galacturonic acid in Vibrio parahaemolyticus O10 lipopolysaccharide

Abstract Identification of 4 unidentified neutral substances (X₁, X₂, X₃ and X₄) in lipopolysaccharides of Vibrio parahaemolyticus (Miyano et al. (1980) FEMS Microbiol. Lett. 8, 23–28, and 14, 145–148) was attempted. X₁ (1,4-anhydroribitol) was found to be formed from ribitol-5-phosphate during hydrolysis. X₂ was identified to be 2- O -methylribitol. X₃ and X₄ were found to be formed during hydrolysis of galacturonic acid and D-glycero-L-mannoheptose (or L-glycero-D-mannoheptose), respectively. The chemical structures of X₃ and X₄ remain to be determined.     

Microglial phagocytosis attenuated by short-term exposure to exogenous ATP through P2X7 receptor action

Microglia, the CNS resident macrophages responsible for the clearance of degenerating cellular fragments, are essential to tissue remodeling and repair after CNS injury. ATP can be released in large amounts after CNS injury and may mediate microglial activity through the ionotropic P2X and the metabotropic P2Y receptors. This study indicates that exposure to a high concentration of ATP for 30 min rapidly induces changes of the microglial cytoskeleton, and significantly attenuates microglial phagocytosis. A pharmacological approach showed that ATP-induced inhibition of microglial phagocytotic activity was due to P2X₇R activation, rather than that of P2YR. Activation of P2X₇R by its agonist, 2'-3'- O -(4-benzoyl)benzoyl-ATP (BzATP), produced a Ca²⁺-independent reduction in microglial phagocytotic activity. In addition, the knockdown of P2X₇R expression by lentiviral-mediated shRNA interference or the blockade of P2X₇R activation by the specific antagonists, oxidized ATP (oxATP) and brilliant blue G, has efficiently restored the phagocytotic activity of ATP and BzATP-treated microglia. Our results reveal that P2X₇R activation may induce the formation of a Ca²⁺-independent signaling complex, which results in the reduction of microglial phagocytosis. This suggests that exposure to ATP for a short-term period may cause insufficient clearance of tissue debris by microglia through P2X₇R activation after CNS injury, and that blockade of this receptor may preserve the phagocytosis of microglia and facilitate CNS tissue repair.     

Contribution of extracellular negatively charged residues to ATP action and zinc modulation of rat P2X2 receptors

Two histidines are known to be essential for zinc potentiation of rat P2X₂ receptors, but the chemistry of zinc coordination would suggest that other residues also participate in this zinc-binding site. There is also a second lower affinity zinc-binding site in P2X₂ receptors whose constituents are unknown. To assess whether the extracellular acidic residues of the P2X₂ receptor contribute to zinc potentiation or inhibition, site-directed mutagenesis was used to produce alanine substitutions at each extracellular glutamate or aspartate. Two electrode voltage clamp recordings from Xenopus oocytes indicated that 7 of the 34 mutants (D82A, E85A, E91A, E115A, D136A, D209A, and D281A) were deficient in zinc potentiation and one mutant (E84A) was deficient in zinc inhibition. Additional tests on cysteine mutants at these eight positions indicated that D136 is the only residue that is a strong candidate to be at the potentiating zinc-binding site, and that E84 is unlikely to be at the inhibitory zinc-binding site.     

Identification of GABAA/Benzodiazepine Receptors on Clathrin-Coated Vesicles from Rat Brain

Abstract: To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABA_A/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [³H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin-coated vesicles. Homogeneity of the coated-vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light-chain kinase. Vesicles exhibiting specific binding of [³H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [³H]flunitrazepam to coated vesicles yielded a K_D value of 21 ± 4.7 nM and a B_max value of 184 ± 28 fmol/mg. The K_D value for coated vesicles was 12-19-fold that found with microsomal or crude synaptic membranes. This low-affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The B_maxvalue for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the removal of GABA_A/benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down-regulation that is produced by exposure to GABA and benzodiazepine agonists.     

Tachykinins Potentiate N-Methyl-D-Aspartate Responses in Acutely Isolated Neurons from the Dorsal Horn

Abstract: Substance P and neurokinin A both potentiated N -methyl- d -aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca²⁺]_i in these cells. Substance P, but not neurokinin A, increased [Ca²⁺]_i in a subpopulation of neurons. The increase in [Ca²⁺]_i was found to be due to Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Substance P and neurokinin A also potentiated the increase in [Ca²⁺]_i produced by NMDA, but not by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 m M K⁺. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca²⁺]_i can be dissociated from their effects on NMDA receptors.     

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

Chronic Dopamine D2 Receptor Activation Does Not Affect Survival and Differentiation of Cultured Dopaminergic Neurons: Morphological and Neurochemical Observations

Abstract: Primary cultures of rat ventral mesencephalon were used to elucidate the role of chronic stimulation of dopamine (DA) D₂ autoreceptors in the development of fetal dopaminergic neurons in vitro. Cultured dopaminergic neurons, as visualized by tyrosine hydroxylase immunocytochemistry, became more differentiated in the course of cultivation time and exhibited specific high-affinity uptake for [³H]DA. In rat striatal tissue, activation of D₂ receptors has been shown to inhibit the release of DA. Previously accumulated [³H]DA was released from the cultures upon depolarization in a Ca²⁺-dependent manner. K⁺-evoked [³H]DA release could be inhibited by the selective D₂ receptor agonists LY 171555 and N0437 in a concentration-dependent manner. The inhibitory effects of LY 171555 and N0437 were antagonized by the selective DA D₂ receptor antagonist sulpiride. These observations are indicative for the expression of functional D₂ receptors in the cultures. Daily treatment of these cultures for 7 days with LY 171555 or sulpiride did not lead to any change in protein content, the number of tyrosine hydroxylase-immunoreactive neurons, or the uptake capacity for [³_H]DA. Our data demonstrate that chronic stimulation of DA D₂ receptors does not impair survival or differentiation of cultured fetal dopaminergic neurons.     

P2Y1 receptor-mediated glutamate release from cultured dorsal spinal cord astrocytes

P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5'-O-2-thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y₁ receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS-induced [Ca²⁺]_i increase and glutamate release are mediated by P2Y₁ receptor. Ca²⁺ release from IP₃-sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y₁ receptor-evoked glutamate release is regulated by volume-sensitive Cl⁻ channels and anion co-transporter, which open up the possibility that P2Y₁ receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5-nitro-2 (3-phenyl propy lamino)–benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y₁ receptor-evoked glutamate may be mediated via volume-sensitive Cl⁻ channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y₁ receptors-evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.     

The Kv2.1 channels mediate neuronal apoptosis induced by excitotoxicity

Chronic loss of intracellular K⁺ can induce neuronal apoptosis in pathological conditions. However, the mechanism by which the K⁺ channels are regulated in this process remains largely unknown. Here, we report that the increased membrane expression of Kv2.1 proteins in cortical neurons deprived of serum, a condition known to induce K⁺ loss, promotes neuronal apoptosis. The increase in I _K current density and apoptosis in the neurons deprived of serum were inhibited by a dominant negative form of Kv2.1 and MK801, an antagonist to NMDA receptors. The membrane level of Kv2.1 and its interaction with SNAP25 were increased, whereas the Kv2.1 phosphorylation was inhibited in the neurons deprived of serum. Botulinum neurotoxin, an agent known to prevent formation of soluble N -ethylmaleimide-sensitive factor attachment protein receptor complex, suppressed the increase in I _K current density. Together, these results suggest that NMDA receptor-dependent Kv2.1 membrane translocation is regulated by a soluble N -ethylmaleimide-sensitive factor attachment protein receptor-dependent vesicular trafficking mechanism and is responsible for neuronal cell death induced by chronic loss of K⁺.