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.     

Ca2+-Dependent and Ca2+-Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

Abstract: We examined protein kinase C (PKC) activity in Ca²⁺-dependent PKC (Ca²⁺-dependent PKC activities) and Ca²⁺-independent PKC (Ca²⁺-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca²⁺-dependent and Ca²⁺-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca²⁺-independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-ε, was used in the assay; whereas Ca²⁺-independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-δ, was not significantly reduced in AD. Further studies on the protein levels of Ca²⁺-independent PKC-δ, PKC-ε, and PKC-ζ in AD brain revealed reduction of the PKC-ε level in both cytosolic and membranous fractions, although PKC-δ and PKC-ζ levels were not changed. These findings indicated that Ca²⁺-dependent and Ca²⁺-independent PKC are changed in AD, and that among Ca²⁺-independent PKC isozymes, the alteration of PKC-ε is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.     

Inhibitory effects of aripiprazole on interferon-gamma-induced microglial activation via intracellular Ca2+ regulation in vitro

The activation of the inflammatory/immunological response system is suggested to be related to the pathophysiology of schizophrenia. Aripiprazole is a novel atypical antipsychotic, which is a high-affinity dopamine D₂ receptor partial agonist. Atypical antipsychotics, all of which have dopamine D₂ receptor antagonism, have recently reported to have significantly inhibitory effects on interferon (IFN)-γ-induced microglial activation in vitro . In the present study, we investigated whether or not aripiprazole also has anti-inflammatory effect on IFN-γ-induced microglial activation. Not quinpirole, dopamine D₂ full agonist, but aripiprazole significantly inhibited the generation of nitric oxide (NO) and tumor necrosis factor (TNF)-α from IFN-γ-activated microglia and suppressed the IFN-γ-induced elevation of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in murine microglial cells. Increased [Ca²⁺]_i has been reported to be required, but by itself not sufficient, for the release of NO and certain cytokines. As a result, we can speculate that aripiprazole may inhibit IFN-γ-induced microglial activation through the suppression of IFN-γ-induced elevation of [Ca²⁺]_i in microglia. Our results demonstrated that not only antipsychotics which have dopamine D₂ receptor antagonism but also aripiprazole have anti-inflammatory effects via the inhibition of microglial activation. Antipsychotics may therefore have a potentially useful therapeutic effect on patients with schizophrenia by reducing the microglial inflammatory reactions.     

Stimulation of an Insulin Receptor Activates and Down-Regulates the Ca2+-Independent Protein Kinase C, Apl II, Through a Wortmannin-Sensitive Signaling Pathway in Aplysia

Abstract: Activation of tyrosine kinase-linked receptors has been shown to stimulate Ca²⁺-independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase-linked receptor in these cells persistently activated and down-regulated the Ca²⁺-independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down-regulate the Ca²⁺-activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3-kinase because (a) diC₁₆phosphatidylinositol 3,4,5-trisphosphate, a synthetic phosphoinositide 3-kinase product, stimulated autophosphorylation of baculovirus-expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the activation and down-regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase-linked receptors may mediate some of their effects in neurons through activation of Ca²⁺-independent protein kinase C isoforms.     

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.     

Role of Ciliary Neurotrophic Factor in Microglial Phagocytosis

Microglia, CNS-resident macrophages, serve as scavengers to remove cellular debris and facilitate tissue remodeling in the developing and injured CNS. Little is known as what and how microenvironmental factors mediate the phagocytotic ability of microglia. Our previous study has indicated that treatment with glial cell line-derived neurotrophic factor (GDNF) increased the phagocytotic activity of primary rat microglia possibly through the upregulation of α5 integrin. In the present study, ciliary neurotrophic factor (CNTF), which has been reported to be produced by glia, was shown to have stimulatory effect on the phagocytosis of primary rat microglia and mouse microglial cell line BV2. Ca²⁺ imaging analysis and the application of intracellular calcium chelator BAPTA-AM revealed that CNTF-induced increase in microglial phagocytosis was mediated by a calcium signaling pathway. Furthermore, treatment with CNTF led to an increase in the expression of αv integrin, which has been reported to be involved in the phagocytosis of the apoptotic cells. In summary, we have provided evidence that CNTF can increase microglial phagocytosis through a calcium-mediated pathway. Our results also suggest that the upregulation of αv integrin by CNTF could be involved in the increased phagocytotic activity of microglia. Special issue article in honor of Dr. George DeVries.     

Comparison of Type 2 Inositol 1,4,5-Trisphosphate Receptor Distribution and Subcellular Ca2+ Release Sites that Support Ca2+ Waves in Cultured Astrocytes

Abstract: We have examined the mechanisms that underlie Ca²⁺ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca²⁺ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca²⁺ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca²⁺ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP₃R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP₃Rs with Ca²⁺ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP₃Rs (InsP₃R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca²⁺ responses and InsP₃R2 immunofluorescence were compared in the same cell, domains of elevated Ca²⁺ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP₃R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca²⁺ release sites that support regenerative wave propagation include increased levels of InsP₃R2 expression.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

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.     

Differential Inactivation of Postsynaptic Density-Associated and Soluble Ca2+/Calmodulin-Dependent Protein Kinase II by Protein Phosphatases 1 and 2A

Abstract: Autophosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) at Thr²⁸⁶ generates Ca²⁺-independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr²⁸⁶ in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A-like enzymes account for >70% of activity toward exogenous soluble Thr²⁸⁶-autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs); each protein phosphatase is also present in isolated PSDs, but only PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr²⁸⁶-autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (³²P autophosphorylated in the presence of Ca²⁺/calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD-associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca²⁺-independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments.     

Activation of the histaminergic H3 receptor induces phosphorylation of the Akt/GSK-3β pathway in cultured cortical neurons and protects against neurotoxic insults

Stimulation of histamine H₃ receptors (H₃R) activates G_i/o-proteins that inhibit adenylyl cyclase and triggers MAPK and phospholipase A₂. In a previous study, we showed that H₃R-mediated phosphorylation of Akt at Ser473 occurs in primary cultures of rat cortical neurons, but neither the downstream targets nor the function of such activation were explored. In this report we address these questions. Western blotting experiments showed that H₃R-mediated activation of Akt in cultured rat cortical neurons was inhibited by LY 294004 and U0126, suggesting that it depends on phosphoinositide-3-kinase and mitogen-activated protein kinase kinase. H₃R activation phosphorylated, hence inactivated, the Akt downstream effector glycogen synthase kinase-3β, increased the expression of the antiapoptotic protein Bcl-2 and protected cultured rat and mouse cortical neurons from neurotoxic insults in a dose-dependent manner. All these effects were inhibited by the H₃R antagonist inverse/agonist thioperamide. Mouse cortical cells expressed H₃R as revealed by immunostaining experiments, and stimulation of H₃R phoshorylated Akt and decreased caspase 3 activity. Hence, we uncovered a yet unexplored action of the H₃R that may help understand the impact of H₃R signaling in the CNS.     

Characterization of the Exocytotic Release of Glutamate from Guinea-Pig Cerebral Cortical Synaptosomes

Abstract: A continuous enzyme-linked fluorometric assay was used for determining the characteristics for glutamate exocytosis from guinea-pig cerebrocortical synaptosomes. Ca²⁺-dependent release can be induced not only by K⁺, but also by the Na⁺ channel activator veratridine and the Ca²⁺ ionophore ionomycin. K⁺-induced release can be inhibited by the Ca²⁺ channel inhibitor verapamil. Sr²⁺ and Ba²⁺ substitute for Ca²⁺ in promoting K⁺-induced release. Agents that would be predicted to transform the transvesicular pH gradient into a membrane potential are without effect on glutamate release. However, the protonophore carbonylcy-anide p -trifluoromethoxyphenylhydrazone causes a time-dependent loss of exocytosis that is oligomycin insensitive and may be due to depletion of vesicular glutamate. The Ca²⁺-independent release of glutamate from the cytosol on depolarization is unchanged or promoted by metabolic inhibitors that lower the ATP/ADP ratio. In contrast, Ca²⁺-dependent release is ATP dependent and is blocked by the combined inhibition of oxidative phosphorylation and glycolysis.     

Hydrolysis-Resistant GTP Analogs Stimulate Catecholamine Release from Digitonin-Permeabilized PC12 Cells

Abstract: The effect of the hydrolysis-resistant GTP analogs, guanosine 5'- O -(3-thiotriphosphate) (GTPγS) and guanylyl imidodiphosphate (GMPPNP), on norepinephrine (NE) secretion from digitonin-permeabilized rat pheochromocytoma cells, PC12, was examined. Although secretion in the presence of saturating Ca²⁺ (10 μ M ) was not affected by GTP7S or GMPPNP, secretion in the absence of Ca²⁺ was stimulated by these GTP analogs. Secretion induced by saturating concentrations of GTPγS or GMPPNP was approximately 80% of that induced by 10 μ M Ca²⁺. Half-maximum stimulation was induced by 30 μ M GTPγS or GMPPNP. Both Ca²⁺-stimulated and GTPγS-stimulated secretion were ATP dependent and inhibited by N -ethylmaleimide. The GTPγS-stimulated secretion of NE from permeabilized PC12 cells does not appear to result from either the release of Ca²⁺ or the activation of protein kinase C. Activation of protein kinase C by pretreatment of intact cells with 12- O -tetradecanoyl-phorbol 13-acetate caused a 50% increase in both Ca²⁺-stimulated and GTP7S-stimulated secretion. Cholera and pertussis toxins did not affect Ca²⁺-stimulated or GTPγS-stim-ulated NE secretion. Guanosine 5'- O -(2-thiodiphosphate) (GDPβS) and GTP inhibited GTPγS-stimulated secretion but not Ca²⁺-stimulated secretion. The inability of GDPβS to inhibit Ca²⁺-stimulated secretion indicates that the process affected by GTPγS is not an essential step in the Ca²⁺-stimulated pathway.     

Effect of Ca2+ on In Vitro Astrocyte Injury

Abstract: Current literature suggests that a massive influx of Ca²⁺ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch-induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca²⁺ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca²⁺ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca²⁺ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca²⁺ content to twofold above physiological levels (2 m M ) produced a reduction in cell injury. The reduction in injury afforded by Ca²⁺ could not be mimicked with Ba²⁺, Mn²⁺, Zn²⁺, or Mg²⁺, suggesting that a Ca²⁺-specific mechanism is involved. Using ⁴⁵Ca²⁺, we demonstrate that injury induces a rapid influx of extracellular Ca²⁺ into the astrocyte, achieving an elevation in total cell-associated Ca²⁺ content two- to threefold above basal levels. Pharmacological elevation of intracellular Ca²⁺ levels with the Ca²⁺ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell-associated Ca²⁺ reduces astrocyte injury produced by a traumatic insult.     

CELL ASSOCIATION PATTERN IN AGGREGATES CONTROLLED BY MULTIPLE CELL-CELL ADHESION MECHANISMS

We have previously assumed the presence of two mechanisms for the aggregation of Chinese hamster V79 cells, the Ca²⁺-dependent one and the Ca²⁺-independent one. In order to examine if each of these mechanisms contributed differently to the various aspects of cell aggregation, the morphology of V79 cell aggregates, pretreated so that they were provided with only one of the two adhesion mechanisms, was compared by light and electron microscopy. The adhesion among cells with only the Ca²⁺-dependent mechanism was very tight, with the formation of gap and intermediate junctions. Cells were arranged in a rod or dendric shape in aggregates. In aggregates of cells with only the Ca²⁺-independent mechanism, cells were loosely attached to each other without the formation of specialized junctions and the aggregates were of globular shape. In aggregates of cells with both mechanisms, both characteristics of the above two aggregates were found. Four clones of V79 cells, which formed colonies with different morphology when they were grown in soft agar, were isolated. It was found that such differences were due to the different activity of the Ca²⁺-independent mechanism among these clones. These results suggested that the two adhesion mechanisms play different roles in the cell arrangement in aggregates.     

A2A Adenosine Receptors Inhibit ATP-Induced Ca2+ Influx in PC12 Cells by Involving Protein Kinase A

Abstract: The regulatory role of A_2A adenosine receptors in P₂ purinoceptor-mediated calcium signaling was investigated in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with 2- p -(2-carboxyethyl)-phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680), a specific agonist of the A_2A adenosine receptor, the extracellular ATP-evoked rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was inhibited by 20%. Both intracellular calcium release and inositol 1,4,5-trisphosphate production evoked by ATP were not affected by CGS-21680 treatment. However, ATP-evoked Ca²⁺ influx was inhibited following CGS-21680 stimulation. The CGS-21680-mediated inhibition occurred independently of nifedipine-induced inhibition of the [Ca²⁺]_i rise. The CGS-21680-induced inhibition was completely blocked by reactive blue 2. The CGS-21680 effect was mimicked by forskolin and dibutyryl-cyclic AMP and blocked by Rp -adenosine 3',5'-cyclic monophosphothioate, a protein kinase A inhibitor, or by staurosporine, a general kinase inhibitor. The data suggest that in PC12 cells activation of A_2A adenosine receptors leads to inhibition of P₂ purinoceptor-mediated Ca²⁺ influx through ATP-gated cation channels and involves protein kinase A.