Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM-CSF): priming of human neutrophils in vitro with GM-CSF is associated with accumulation of phosphatidic acid and diradylglycerol.

The generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF). Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked approximately 100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production. GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)P2-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance of PdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence of ethanol. The formation of PEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism of action of GM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.

Intracellular localization of phospholipase D1 in mammalian cells.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.

Tumor necrosis factor-alpha induces stress fiber formation through ceramide production: role of sphingosine kinase.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.

Differential innervation of superficial versus deep laminae of the dorsal horn by bulbo-spinal serotonergic pathways in the rat.

Convergent data showed that bulbo-spinal serotonergic projections exert complex modulatory influences on nociceptive signaling within the dorsal horn. These neurons are located in the B3 area which comprises the median raphe magnus (RMg) and the lateral paragigantocellular reticular (LPGi) nuclei. Because LPGi 5-HT neurons differ from RMg 5-HT neurons regarding both their respective electrophysiological properties and responses to noxious stimuli, we used anatomical approaches for further characterization of the respective spinal projections of LPGi versus RMg 5-HT neuron subgroups. Adult Sprague-Dawley rats were stereotaxically injected into the RMg or the LPGi with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L). The precise location of injection sites and RMg vs LPGi spinal projections into the different dorsal horn laminae were visualized by PHA-L immunolabeling. Double immunofluorescent labeling of PHA-L and the serotonin transporter (5-HTT) allowed detection of serotonergic fibers among bulbo-spinal projections. Anterograde tracing showed that RMg neurons project preferentially into the deep laminae V-VI whereas LPGi neuron projections are confined to the superficial laminae I-II of the ipsilateral dorsal horn. All along the spinal cord, double-labeled PHA-L/5-HTT immunoreactive fibers, which represent only 5-15% of all PHA-L-immunoreactive projections, exhibit the same differential locations depending on their origin in the RMg versus the LPGi. The clear-cut distinction between dorsal horn laminae receiving bulbo-spinal serotonergic projections from the RMg versus the LPGi provides further anatomical support to the idea that the descending serotonergic pathways issued from these two bulbar nuclei might exert different modulatory influences on the spinal relay of pain signaling neuronal pathways.

Hedgehog Pathway-Mediated Vascular Alterations Following Trigeminal Nerve Injury.

Whereas neurovascular interactions in spinal neuropathic pain models have been well characterized, little attention has been given to such neurovascular interactions in orofacial neuropathic pain models. This study investigated in male Sprague-Dawley rats the vascular changes following chronic constriction injury (CCI) of the infraorbital nerve (IoN), a broadly validated preclinical model of orofacial neuropathic pain. Following IoN-CCI, an early downregulation of tight junction proteins Claudin-1 and Claudin-5 was observed within the endoneurium and perineurium, associated with increased local accumulation of sodium fluorescein (NaFlu) within the IoN parenchyma, as compared with sham animals. These events were evidence of local blood-nerve barrier disruption and increased vascular permeability. A significant upregulation of immunocytes (CD3, CD11b) and innate immunity (TLR2, TLR4) mRNA markers was also observed, suggestive of increased local inflammation. Finally, a significant downregulation of Hedgehog pathway readouts Patched-1 and Gli-1 was observed within the IoN after CCI and local injections of cyclopamine, a Hedgehog pathway inhibitor, replicated in naïve rats the molecular, vascular, and behavioral changes observed following IoN-CCI. These results suggest a major role of Hedgehog pathway inhibition in mediating local increased endoneurial and perineurial vascular permeability following trigeminal nerve injury, thus facilitating immunocytes infiltration, neuroinflammation development, and neuropathic pain-like aversive behavior.

Effects of pyrrophenone, an inhibitor of group IVA phospholipase A2, on eicosanoid and PAF biosynthesis in human neutrophils.

BACKGROUND AND PURPOSE: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA2 (cPLA2alpha). This paper aims at characterizing the inhibitory properties of the cPLA2alpha inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). EXPERIMENTAL APPROACH: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA2alpha inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE2 was measured. KEY RESULTS: Pyrrophenone potently inhibited LT, PGE2 and PAF biosynthesis in PMN with IC50s in the range of 1-20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluoro-phosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF3) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenone-treated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 microM pyrrophenone, suggesting that the cPLA2alpha inhibitor does not directly inhibit PLD. CONCLUSIONS AND IMPLICATIONS: Pyrrophenone is a more potent and specific cPLA2alpha inhibitor than MAFP and AACOCF3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF.

Regulation of phospholipase D by phosphorylation-dependent mechanisms.

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4, 5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Calpha as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.

C2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils.

Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C2-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O2-) production. The preincubation of PMN for 15 min with C2-ceramide increased by up to almost 3-fold the amounts of O2- generated in response to 0.1 and 1 microM fMLP. Priming was detected at C2-ceramide concentrations of 2 microM to 4 microM per million PMN. Though less potent than C2-ceramide, C6-ceramide (N-hexanoylsphingosine) could prime for O2- generated in response to 0.1 microM fMLP, with maximal effects obtained at 10-20 microM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O2- generation. As expected, TNF-alpha (1000 U/ml), also primed for fMLP-induced O2- production; however, the combination of TNF-alpha and C2-ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C2-ceramide and TNF-alpha. C2-ceramide at 2 microM did not enhance the production of O2- induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B4 (LTB4), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C2-ceramide (2 microM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A2 or D (PLC, PLA2, PLD) were involved in the priming effect by C2-ceramide. However, C2-ceramide inhibited in a dose-related manner the production of O2- induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C2-ceramide. The priming of O2- production by C2-ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-alpha have different mechanisms than C2-ceramide.

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: evidence for an involvement of phospholipase D-derived phosphatidic acid.

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.

Granulocyte-macrophage colony-stimulating factor primes phospholipase D activity in human neutrophils in vitro: role of calcium, G-proteins and tyrosine kinases.

The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to human peripheral blood neutrophils primes phospholipase D (PLD) to subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). The present investigation was directed at the elucidation of the pathway(s) involved in the regulation of the activity of PLD in untreated as well as in GM-CSF-primed neutrophils. Pretreatment with pertussis toxin (PT) totally inhibited fMLP-induced activation of PLD in control or GM-CSF-treated cells. PT did not affect the activation of PLD by PMA but inhibited the priming effect of GM-CSF. Activation of PLD by fMLP was dose-dependently inhibited by erbstatin, an inhibitor of tyrosine kinases. Furthermore, pre-incubation with GM-CSF accelerated the tyrosine phosphorylation response to fMLP (as analysed by protein immunoblot with antiphosphotyrosine antibodies). In PMA-stimulated neutrophils, erbstatin antagonized the priming effect of GM-CSF on PLD without affecting the direct effects of the phorbol ester. Buffering cytoplasmic calcium with the chelator BAPTA inhibited fMLP-induced activation of PLD as monitored by the formation of phosphatidylethanol. The stimulation of PLD by PMA was partially attenuated in BAPTA-loaded cells while the priming effect of GM-CSF was abolished. Thus, priming of human neutrophil PLD by GM-CSF may be mediated by G-proteins, by increases in the levels of cytosolic free calcium, and by stimulation of protein kinase C and/or tyrosine kinase(s).

Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes.

We focus on the mechanisms of regulation of phospholipase D (PLD) activity. Three agonists known to stimulate PLD activity, fMet-Leu-Phe (fMLP), phorbol 12-myristate 13-acetate (PMA) and V4+-OOH, induced a differential translocation of ADP-ribosylation factor (ARF), RhoA, and protein kinase Calpha (PKCalpha), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V4+-OOH only elicited RhoA translocation and PMA induced ARF and PKCalpha translocation. Three tyrosine kinases inhibitors, ST-638, methyl 2,5-dihydroxycinnamate, and genistein reduced fMLP-stimulated PLD activity by up to 80%. Furthermore, tyrosine kinase inhibitors reduced the fMLP-induced increase of GTPgammaS-stimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKCalpha to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKCalpha in the signaling pathway leading to PLD activation by fMLP. RO 31-8220, a specific inhibitor of PKC, reduced PMA-induced PLD activity by 80% in intact HL60 granulocytes but enhanced fMLP-stimulated PLD activity by 60%. Although PMA alone had no effect on RhoA recruitment to the membrane fraction, in the presence of RO 31-8220 the levels of membrane-bound RhoA were increased. The levels of membrane-bound ARF and PKCalpha were unaffected by RO 31-8220 during PMA stimulation. In contrast, fMLP-induced recruitment of ARF and RhoA was insensitive to RO 31-8220 but PKCalpha translocation was increased. We propose that RhoA translocation may be regulated by PKC in an ATP-independent manner. Furthermore, increased fMLP-induced PKCalpha translocation in the presence of RO 31-8220 may partially account for the synergistic activation of PLD observed when both fMLP and RO 31-8220 are used together in intact HL60 cells.

Crystal-induced neutrophil activation. VII. Involvement of Syk in the responses to monosodium urate crystals.

The inflammatory response in acute gouty arthritis is in large part a result of the interaction between neutrophils and monosodium urate (MSU) crystals. The tyrosine kinase Syk, which has been largely associated with the phagocytic response by Fc receptors and with spreading mediated by integrins, has been identified as one of the major proteins tyrosine-phosphorylated in human neutrophils upon stimulation by MSU crystals and is known to be mediated in part by the Fc receptor, CD16. This has led to the present examination of the implication of Syk in the activation pathways used by MSU crystals. The tyrosine-phosphorylation patterns induced by MSU crystals and by the ligation of CD16 were inhibited by piceatannol, which, conversely, only slightly delayed but did not diminish the peak of tyrosine phosphorylation induced by cross-linking CD32 or by the addition of fMet-Leu-Phe. Moreover, piceatannol inhibited the activity of Syk as monitored by in vitro kinase assays, by its in situ tyrosine phosphorylation, and by its activity toward exogenous substrates after stimulation by MSU crystals. We also measured the impact of piceatannol on the mobilization of calcium, the production of superoxide anions, and the activity of PLD stimulated by MSU crystals. We noted a distinct inhibition of all these responses by piceatannol. Finally, the morphological changes observed in neutrophils as characteristic of MSU crystal internalization were diminished significantly by piceatannol. The results obtained show that Syk plays a critical and central role in the signal-transduction pathways called upon by MSU crystals subsequent to their interaction with human neutrophils.

Agonist-specific tyrosine phosphorylation of Cbl in human neutrophils.

The effects of soluble and particulate agonists on the tyrosine phosphorylation levels of the proto-oncogene Cbl in human neutrophils were examined. Experimental conditions allowing the maintenance of Cbl as well as of its tyrosine phosphorylation status were first established. Their use allowed us to observe that Cbl was tyrosine phosphorylated in response to some (FcgammaRII ligation, opsonized bacteria and zymosan, granulocyte-macrophage colony-stimulating factor, monosodium urate, and calcium pyrophosphate microcrystals), but not all (fMet-Leu-Phe, interleukin-8) neutrophil agonists. Cbl was also shown to account for a varying proportion of the 120-kDa phosphoprotein(s) observed in response to the above stimuli. These data establish that Cbl is present in human neutrophils and that its level of tyrosine phosphorylation is modulated by some of these cells' agonists, and in particular by phagocytic particles. Furthermore, the signaling pathways activated by chemotactic factors and the other neutrophil stimuli tested in this investigation diverge at or downstream from the tyrosine phosphorylation of Cbl.

CGRP receptor blockade by MK-8825 alleviates allodynia in infraorbital nerve-ligated rats.

BACKGROUND: Previous data showed that, in rats, anti-migraine drugs (triptans, olcegepant) significantly reduced mechanical allodynia induced by infraorbital nerve (ION) ligation but not that evoked by sciatic nerve (SN) ligation. Whether this also occurs with MK-8825, a novel anti-migraine drug also acting through CGRP receptor blockade (but chemically unrelated to olcegepant) was tested in the present study, which also investigated possible anti-neuroinflammatory effects of this drug. METHODS: Adult male Sprague-Dawley rats underwent unilateral chronic constriction injury (CCI) to either the ION or the SN, and mechanical allodynia was assessed 2 weeks later within the ipsilateral vibrissae territory or hindpaw, respectively. Transcripts of neuroinflammatory markers were quantified by real-time quantitative RT-PCR in ipsilateral trigeminal ganglion and spinal trigeminal nucleus in CCI-ION rats. RESULTS: Acute as well as repeated (for 4 days) administration of MK-8825 (30-100 mg/kg, i.p.) significantly reduced CCI-ION-induced mechanical allodynia but was ineffective in CCI-SN rats. CCI-ION was associated with marked up-regulation of neuronal and glial inflammatory markers (ATF3, IL6, iNOS, COX2) in ipsilateral trigeminal ganglion but not spinal trigeminal nucleus. MK-8825-induced inhibition of iNOS mRNA up-regulation probably underlay its anti-allodynic effect because pharmacological blockade of iNOS by AMT (6 mg/kg, s.c.) mimicked this effect. CONCLUSIONS: These data further support the idea that CGRP receptor blockade might be a valuable approach to alleviate trigeminal, but not spinal, neuropathic pain through, at least partly, an inhibitory effect on neuropathic pain-associated increase in NO production in trigeminal ganglion.

Altered nociception in mice with genetically induced hypoglutamatergic tone.

Extensive pharmacological evidence supports the idea that glutamate plays a key role in both acute and chronic pain. In the present study, we investigated the implication of the excitatory amino acid in physiological nociception by using mutant mice deficient in phosphate-activated glutaminase type 1 (GLS1), the enzyme that synthesizes glutamate in central glutamatergic neurons. Because homozygous GLS1-/- mutants die shortly after birth, assays for assessing mechanical, thermal and chemical (formalin) nociception were performed on heterozygous GLS1+/- mutants, which present a clear-cut decrease in glutamate synthesis in central neurons. As compared to paired wild-type mice, adult male GLS1+/- mutants showed decreased responsiveness to mechanical (von Frey filament and tail-pressure, but not tail-clip, tests) and thermal (Hargreaves' plantar, tail-immersion and hot-plate tests) nociceptive stimuli. Genotype-related differences were also found in the formalin test for which GLS1+/- mice exhibited marked decreases in the nociceptive responses (hindlimb lift, lick and flinch) during both phase 1 (0-5 min) and phase 2 (16-45 min) after formalin injection. On the other hand, acute treatment with memantine (1mg/kg i.p.), an uncompetitive antagonist at NMDA glutamate receptors, reduced nociception responses in wild-type but not GLS1+/- mice. Conversely, antinociceptive response to acute administration of a low dose (1mg/kg s.c.) of morphine was significantly larger in GLS1+/- mutants versus wild-type mice. Our findings indicate that genetically driven hypoactivity of central glutamatergic neurotransmission renders mice hyposensitive to nociceptive stimulations, and promotes morphine antinociception, further emphasizing the critical role of glutamate in physiological nociception and its opioid-mediated control.

Interference of HIV-1 Nef in the sphingomyelin transduction pathway activated by tumour necrosis factor-alpha in human glial cells.

OBJECTIVE: The HIV-1 nef gene product, thought to interact with mediators of cell signalling, is overexpressed during the restricted HIV-1 infection of human astrocytes. This infection can be reactivated following exposure to tumour necrosis factor (TNF)-alpha. We examined the possibility that Nef alters the TNF-alpha-induced cell signalling in astroglioma cells through the sphingomyelin pathway. METHODS: Sphingomyelinase activation by TNF-alpha was analysed in U251MG glial cells constitutively expressing Nef and compared with U251MG cells stably transfected with the expression vector alone. The consequent effect on the cellular proliferative response and induction of nuclear factor NF-kappa B and AP-1 binding activities were examined. RESULTS: A marked enhancement in the levels of ceramide, a product of the sphingomyelin hydrolysis, was observed in U251MG-Nef upon stimulation with TNF-alpha. In contrast, ceramide levels in control cells were barely increased under similar conditions. A concomitant reduction of sphingomyelin level occurred in U251MG-Nef cells. In addition, the reduced survival rate of U251MG cells resulting from TNF-alpha activation was prevented in the presence of Nef. Furthermore, electrophoretic mobility shift assays indicated that nef expression inhibits AP-1 activation without altering the induction of NF-kappa B. CONCLUSION: These results strongly suggest that nef expression in U251MG cells modulates the sphingomyelinase signalling pathway triggered by TNF-alpha, thus leading to important modifications in the activation and proliferation of glial cells. They also provide new insights to explain the widespread reactive astrogliosis observed in AIDS-associated neuropathological disorders.

Role of protein kinase C alpha, Arf, and cytoplasmic calcium transients in phospholipase D activation by sodium fluoride in osteoblast-like cells.

The effect of fluoride on phospholipase D (PLD) activation was studied using in vitro culture of Saos-2, MG-63 osteosarcoma cells, and normal osteoblast-like cells derived from human bone explants. Millimolar concentrations of NaF induced a significant accumulation of phosphatidylethanol (PEt) in Saos-2 cells but not in MG-63 and normal osteoblast-like cells. PLD activation was evident at 15 mM and concentration-dependent up to 50 mM. This stimulation was inhibited by deferoxamine, a chelator of Al3+, suggesting that PLD activation involves fluoride-sensitive G proteins. A good correlation was found between the levels of intracellular free Ca2+ and the activation of PLD. The time courses of the two responses were nearly identical. The ability of NaF to induce both responses was largely dependent on the presence of extracellular calcium. The calcium ionophore A23187 reproduced the effect of NaF, and this effect was antagonized by EGTA, suggesting that PLD activation was, at least in part, a calcium-regulated event. Phorbol 12-myristate 13-acetate (PMA) also stimulated PLD activity in human bone cells. Protein kinase C alpha (PKC alpha) and epsilon were expressed in Saos-2 cells. Acute pretreatment of cells with PMA reduced concomitantly the amounts of PKC alpha, but not of PKC epsilon, and the subsequent activation of PLD elicited by PKC activators. The PLD response to NaF was not attenuated but rather enhanced by down-regulation of PKC alpha. Therefore, PKC-alpha-induced PLD activation is unlikely to mediate the effect of NaF. Moreover, PMA and NaF showed a supraadditive effect on PLD activation in Saos-2 cells. This stimulation, in contrast to NaF alone, was not reduced by EGTA. Hence, mobilization of calcium by NaF cannot account for the enhanced PLD activation in response to PMA stimulation. Membrane Arf and RhoA contents were assessed by Western immunoblot analyses. Membranes derived from NaF-stimulated Saos-2 cells contained more Arf and RhoA when compared with membranes derived from control or PMA-stimulated cells. Translocation of the small GTPases was calcium-independent. We conclude that PLD activation by NaF in Saos-2 cells includes a fluoride-sensitive G protein, increases in the levels of intracellular calcium, and Arf/RhoA redistribution to membranes. The results also indicate that the NaF-induced Arf/RhoA translocation exerts in concert with PMA-activated PKC alpha a synergistic effect on the activation of PLD in Saos-2 cells.

Acupuncture-like stimulation induces a heterosegmental release of Met-enkephalin-like material in the rat spinal cord.

In order to investigate the effects induced by acupuncture on the activity of enkephalinergic neurons in the spinal cord, either the lumbar or the cervico-trigeminal area was perfused with artificial cerebrospinal fluid (CSF) (0.1 ml/min) in halothane-anaesthetized rats, and Met-enkephalin-like material (MELM) was measured in 0.5 ml fractions of the perfusates. The effects of manual acupuncture performed by a traditional Chinese acupuncturist at the 'Zusanli' point on the right hind limb were compared to the effects induced by acupuncture applied at a non-acupoint next to 'Zusanli.' The manipulation of needles either at the 'Zusanli' point or at the non-acupoint had no effect on the release of MELM from the lumbar area but significantly increased the release from the cervico-trigeminal zone. It is concluded that manual acupuncture triggers a heterosegmental activation of enkephalinergic neurones within the spinal cord and that this effect is non-specific in terms of the location of the stimulated point.

Subcutaneous formalin induces a segmental release of Met-enkephalin-like material from the rat spinal cord.

The possible influence of a noxious chemical stimulus on the activity of spinal enkephalinergic neurones was examined by measuring the outflow of Met-enkephalin-like material (MELM) in CSF perifusates from the lumbar and cervico-trigeminal zones in halothane-anaesthetized rats. Following subcutaneous injection of 50 microliters of a 10% formalin solution in saline into the muzzle, MELM outflow increased at the cervico-trigeminal level but not at the lumbar level. Conversely, a significant enhancement in MELM outflow occurred at the lumbar but not at the cervico-trigeminal level when formalin was injected into a hind paw. In both cases, the increase in MELM release took place 5-10 min after the treatment and was of short duration (5-10 min). It is concluded that noxious chemical stimuli can induce a transient activation of enkephalinergic neurones within spinal zones receiving the nociceptive inputs. The marked differences in the characteristics of spinal MELM overflow due to noxious chemical, thermal and mechanical stimuli further support the notion that each type of nociceptive stimulus probably activates spinal enkephalinergic systems by triggering distinct neuronal mechanisms.

Spontaneous and evoked release of met-enkephalin-like material from the spinal cord of arthritic rats in vivo.

Perfusion of the intrathecal space with artificial CSF was achieved in control and arthritic rats under halothane anaesthesia in order to collect the met-enkephalin-like material (MELM) released from the whole spinal cord. On the fourth week following the intradermal injection of Freund's adjuvant to induce arthritis, a marked reduction (-56%) in the spontaneous outflow of MELM was noted in arthritic rats. This effect did not involve changes in the degradation process of MELM, since it persisted when kelatorphan was added to the perfusing fluid in order to inhibit completely the peptidases acting on met-enkephalin. Raising the K+ concentration in the perfusing fluid from 2.4 to 40 mM, as well as moving the hind paws, produced a significant enhancement of MELM release which was (at least) as pronounced in arthritic as in control rats. These results suggest that the basal activity of spinal enkephalinergic neurones, but not that triggered by various stimuli, is reduced in arthritic rats.