Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M₁ receptors negatively modulates the activity of inhibitory muscarinic M₂ receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involved endogenous adenosine tonically activating A₁ receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [³H]-choline (2.5 μCi/ml). Selective activation of muscarinic M₁ and adenosine A₁ receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 μM) and R-N⁶-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [³H]-ACh release induced by muscarinic M₂ receptor activation with oxotremorine (10 μM). Attenuation of the inhibitory effect of oxotremorine (10 μM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M₁ autoreceptors, suggesting that suppression of M₂-inhibiton by A₁ receptor activation is independent on muscarinic M₁ receptor activity. Conversely, the negative crosstalk between M₁ and M₂ autoreceptors seems to involve endogenous adenosine tonically activating A₁ receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 μM) by McN-A-343 (3 μM) was suppressed by the A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 μM). The results suggest that the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A₁ receptors at the rat motor endplate.     

Involvement of glutamate NMDA and AMPA receptors, glial cells and IL-1β in the spinal hyperalgesia evoked by the chemokine CCL2 in mice

We study here the involvement of excitatory amino acid receptors, glial cell activation and IL-1β release in the spinal hyperalgesia evoked by the chemokine CCL2 (MCP-1). Three hours after the intrathecal administration of CCL2 (1-100 ng), mice exhibit dose-dependent thermal hyperalgesia, that was inhibited by the coadministration of the antagonist of chemokine receptor type 2 (CCR2) RS504393 (0.3-3 μg). To assess the involvement of excitatory amino acid receptor sensitisation, CCL2 was coadministered with CPP (0.3-3 ng) and NBQX (25-250 ng), antagonists of NMDA and AMPA receptors, respectively. Both drugs blocked CCL2-evoked hyperalgesia, strongly suggesting that CCL2 evokes in vivo NMDA and AMPA receptor sensitisation, as previously described in electrophysiological studies. Furthermore, this rapid induction of CCL2-mediated hyperalgesia was blocked by the previous acute administration of the microglial inhibitor minocyclin (4.9 μg) or the astroglial inhibitor l-aminoadipate (1.6 μg). Since IL-1β can be released by activated glial cells we tested whether this cytokine could be underlying the spinal sensitisation induced by CCL2. The administration of the type I IL-1 receptor antagonist, IL-1ra (3-30 μg), partially prevented CCL2-evoked hyperalgesia. Finally, to elucidate if IL-1β could produce NMDA and AMPA receptor sensitisation by itself, we performed experiments in which this cytokine was i.t. administered. Thermal hyperalgesia induced by IL-1β (30 pg) was completely prevented by the coadministration of CPP (3 ng) but unaffected by NBQX (250 ng). Globally, our results suggest that spinal CCL2 induces thermal hyperalgesia by sensitising NMDA and AMPA receptors in a process that involves glial activation and IL-1β release.     

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 μM to 1 mM). ACh increased both intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺ concentrations ([Na⁺]_i) in MPG neurons. The recovery of [Na⁺]_i after the removal of ACh was markedly delayed by ouabain (100 μM), an inhibitor of Na⁺/K⁺ ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2 ± 5.4% (n = 7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca²⁺]_i with BAPTA/AM (20 μM) or the blockade of small-conductance Ca²⁺-activated K⁺ channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na⁺]_i and [Ca²⁺]_i, which activates Na⁺/K⁺ ATPase and Ca²⁺-activated K⁺ channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.     

Diperoxovanadate can substitute for H(2)O(2) at much lower concentration in inducing features of premature cellular senescence in mouse fibroblasts (NIH3T3)

Stress induced premature senescence (SIPS) in mammalian cells is an accelerated ageing response and experimentally obtained on treatment of cells with high concentrations of H₂O₂, albeit at sub-lethal doses, because H₂O₂ gets depleted by abundant cellular catalase. In the present study diperoxovanadate (DPV) was used as it is known to be stable at physiological pH, to be catalase-resistant and to substitute for H₂O₂ in its activities at concentrations order of magnitudes lower. On treating NIH3T3 cells with DPV, SIPS-like morphology was observed along with an immediate response of rounding of the cells by disruption of actin cytoskeleton and transient G2/M arrest. DPV could bring about growth arrest and senescence associated features at 25 μM dose, which were not seen with similar doses of either H₂O₂ or vanadate. A minimal dose of 150 μM of H₂O₂ was required to induce similar affects as 25 μM DPV. Increase in senescent associated markers such as p21, HMGA2 and PAI-1 was more prominent in DPV treated cells compared to similar dose of H₂O₂. DPV-treated cells showed marked relocalization of Cyclin D1 from nucleus to cytoplasm. These results indicate that DPV, stable inorganic peroxide, is more efficient in inducing SIPS at lower concentrations compared to H₂O₂.     

Inflammatory mediators potentiate high affinity GABAA currents in rat dorsal root ganglion neurons

Following acute tissue injury action potentials may be initiated in afferent processes terminating in the dorsal horn of the spinal cord that are propagated back out to the periphery, a process referred to as a dorsal root reflex (DRR). The DRR is dependent on the activation of GABA_A receptors. The prevailing hypothesis is that DRR is due to a depolarizing shift in the chloride equilibrium potential (E_Cl) following an injury-induced activation of the Na⁺–K⁺–Cl⁻-cotransporter. Because inflammatory mediators (IM), such as prostaglandin E₂ are also released in the spinal cord following tissue injury, as well as evidence that E_Cl is already depolarized in primary afferents, an alternative hypothesis is that an IM-induced increase in GABA_A receptor mediated current (I_GABA) could underlie the injury-induced increase in DRR. To test this hypothesis, we explored the impact of IM (prostaglandin E₂ (1 μM), bradykinin (10 μM), and histamine (1 μM)) on I_GABA in dissociated rat dorsal root ganglion (DRG) neurons with standard whole cell patch clamp techniques. IM potentiated I_GABA in a subpopulation of medium to large diameter capsaicin insensitive DRG neurons. This effect was dependent on the concentration of GABA, manifest only at low concentrations (<10 μM). THIP evoked current were also potentiated by IM and GABA (1 μM) induced tonic currents enhanced by IM were resistant to gabazine (20 μM). The present data are consistent with the hypothesis that an acute increase in I_GABA contributes to the emergence of injury-induced DRR.     

Methylmercury-induced IL-6 release requires phospholipase C activities

Methylmercury (MeHg) is a neurotoxin capable of causing severe damage to the CNS, especially in the developing fetus. Glia in the CNS release a number of cytokines that are important for proper CNS development and function. We reported earlier that MeHg could induce interleukin-6 (IL-6) release in primary mouse glia. This finding is significant considering previous reports indicating that sustained IL-6 exposure could be detrimental to cerebellar granule neurons, one of the major cellular targets of MeHg cytotoxicity. By using pharmacological antagonists against phophatidycholine- and phosphoinositol-specific phospholipase C, the current study indicated that phospholipase C activity was necessary for MeHg-induced IL-6 release. Results from pharmacological antagonists further suggested that the calcium signaling initiated by phospholipase C appeared essential for this event. In contrast, protein kinase C activity did not appear to be important. Even though mitogen-activated protein kinases were important for IL-6 release in some experimental systems, these enzymes did not appear to be required for MeHg-induced IL-6 release in glia. Based on these data and those reported by us and others, there is a possibility that MeHg-induced phospholipase C activation initiates a calcium signaling that causes phospholipase A₂ activation. This, in turn, leads to arachidonic acid and lysophosphatidyl choline generation, both of which are potent inducers for IL-6 release.     

Activation of α2-adrenoceptors in the lateral hypothalamus reduces pilocarpine-induced salivation in rats

Anti-hypertensive drugs that act on central α₂-adrenoceptors and imidazoline receptors usually cause dry mouth in patients. A central area important for the control of salivary secretion and also for the effects of α₂-adrenoceptor activation is the lateral hypothalamus (LH). Therefore, in the present study we investigated the effects of the injections of moxonidine (an α₂-adrenoceptor and imidazoline agonist) alone or combined with RX 821002 (α₂-adrenoceptor antagonist) into the LH on the salivation induced by intraperitoneal (i.p.) pilocarpine (cholinergic muscarinic agonist). Male Holtzman rats with stainless steel cannula implanted into the LH were used. Saliva was collected using pre-weighted small cotton balls inserted into the animal’s mouth under ketamine anesthesia. Salivation induced by i.p. pilorcarpine (4 μmol/kg of body weight) was reduced by the injection of moxonidine (10 and 20 nmol/0.5 μl) into the LH (222 ± 46 and 183 ± 19 mg/7 min, vs. vehicle: 480 ± 30 mg/7 min). The inhibitory effect of moxonidine on pilocarpine-induced salivation was abolished by prior injections of RX 821002 (160 and 320 nmol/0.5 μl) into the LH (357 ± 25 and 446 ± 38 mg/7 min). Injections of the α₁-adrenoceptor antagonist prazosin (320 nmol/0.5 μl) into the LH did not change the effects of moxonidine. The results show that activation of α₂-adrenoceptors in the LH inhibits pilocarpine-induced salivation, suggesting that LH is one of the possible central sites involved in the anti-salivatory effects produced by the treatment with α₂-adrenoceptor agonists.     

Role of Phospholipase A2 Activation in Histamine Release from Human Basophils

The present experiments were undertaken to investigate the role of phospholipase A₂ (PLA₂) activation in histamine release from human basophils. A PLA₂ inhibitor, P-bromophenacyl bromide (BPB), inhibited IgE-mediated anti-IgE-induced histamine release from human basophils with a concentration of drug required to produce 50% inhibition (IC₅₀) of 1.5 × 10^?6 m when leukocytes were preincubated with this agent for 15 min. Histamine release induced by calcium ionophore A23187 and formyl-l-methionyl-l-leucyl-l-phenylalanine was also blocked by BPB with IC₅₀ of 4.1 × 10^?6 m, and 3.5 × 10^?6 m, respectively. A PLA₂ activator, 12–0-tetradecanoylphorbol-13-acetate (TPA) caused basophil histamine release with a dose-dependent fashion. BPB inhibited TPA-induced histamine release (IC₅₀: 2.5 × 10^?6 m). However, another PLA₂ activator, melittin, and PLA₂ did not release histamine through non-cytotoxic mechanisms. Collectively, these results suggest that PLA₂ activation plays a central role in histamine release from human basophils via generation of lysophosphatidylcholine or products of the lipoxygenase pathway of arachidonic acid metabolism.     

Novel 8-(furan-2-yl)-3-benzyl thiazolo [5,4-e][1,2,4] triazolo [1,5-c] pyrimidine-2(3H)-thione as selective adenosine A2A receptor antagonist

Adenosine A_2A receptor (A_2AR) antagonists have emerged as potential drug candidates to alleviate progression and symptoms of Parkinson's disease (PD), and reduce the dopaminergic side effects. The synthesis of novel compound 8-(furan-2-yl)-3-benzyl thiazolo [5,4-e][1,2,4] triazolo [1,5-c] pyrimidine-2-(3H)-thione (BTTP) was carried out to evaluate the potential of BTTP as A_2AR antagonist using SCH58261, a standard A_2AR antagonist. The strong interaction of BTTP with A_2AR (ΔG = −12.46 kcal/mol and K_i = 0.6 nM) in silico analysis was confirmed by radioligand receptor binding studies showing high affinity (K_i = 0.004 nM) and selectivity with A_2AR (A_2A/A₁ = 1155-fold). The effect of CGS21680 (selective A_2AR agonist) induced cAMP concentration (0.1 pmol/ml) in HEK293 cells was antagonized with BTTP (0.065 pmol/ml) and SCH58261 (0.075 pmol/ml). Furthermore, BTTP pre-treated (5, 10 and 20 mg/kg) haloperidol-induced mice demonstrated significant attenuation in catalepsy and akinesia. BTTP induced elevation in the striatal dopamine concentration (2.90 μM/mg of tissue) was comparable to SCH58261 (2.92 μM/mg of tissue) at the dose of 10 mg/kg. The results firmly articulate that BTTP possesses potential A_2AR antagonist activity and can be further explored for the treatment of PD.     

Methylmercury causes glial IL-6 release

Methylmercury (MeHg) is an environmental toxin that causes severe neurological complications in humans and experimental animals. MeHg caused IL-6 release from the rat C6 glioma cells, the human U251HF glioma cells and the human retina pigment epithelial (ARPE-19) cells. These results plus those we reported earlier using mouse N9 microglia cells indicate that IL-6 induction may be a general property of MeHg among various glial cell types across species. MeHg caused a concentration-dependent increase of cellular oxidation with a maximal level reached by approximately 10 microM MeHg, which was similar to that caused by 30 microM H2O2 or t-butyl hydroperoxide (tBH). The ability of MeHg to induce IL-6 release was not affected by exogenously added H2O2 or t-butyl hydroperoxide. Furthermore, IL-6 release was not accompanied by other cytokine release. Given the reports by others that IL-6 could modulate neuronal survival, glia may affect MeHg neurotoxicity by their IL-6 release when exposed to this neurotoxin.     

JAK-STAT pathway modulates the roles of iNOS and COX-2 in the cytoprotection of early phase of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress

Our previous studies have demonstrated that preconditioning with hydrogen peroxide (H₂O₂) activated the JAK-STAT pathway that played an important role in the cytoprotection, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mediated the late phase of cytoprotection induced by high concentration of H₂O₂ after preconditioning. Here we sought to identify the downstream targets of the JAK-STAT axis that mediated H₂O₂ preconditioning and the expression of iNOS and COX-2 in the early phase of H₂O₂ preconditioning. It was shown that (1) Preconditioning with H₂O₂ at 100 μmol/L for 90 min in PC12 cells induced significant expression of iNOS and COX-2. (2) Pretreatment with the iNOS inhibitor AG (10 μmol/L) or the COX-2 inhibitor NS-398 (10 μmol/L) respectively 20 min before H₂O₂ preconditioning not only inhibits the increased expression of iNOS or COX-2 but also abrogates the protective effects of H₂O₂ preconditioning against apoptosis induced by oxidative stress. (3) Pretreatment with the JAK inhibitor AG-490 (10 μmol/L) 20 min before H₂O₂ preconditioning obviously inhibits the up-regulation of iNOS or COX-2 induced by H₂O₂ preconditioning. These results suggested that JAK-STAT pathway modulates the roles of iNOS and COX-2 in the cytoprotection of early phase of H₂O₂ preconditioning.     

Quercetin regulates oxidized LDL induced inflammatory changes in human PBMCs by modulating the TLR-NF-κB signaling pathway

Toll-like receptors (TLRs) have been shown to play a pivotal role in both innate and adaptive immune responses. TLR family is the essential recognition and signaling component of mammalian host defense. Both genetic and biochemical data support a common signaling pathway that finally leads to the activation of NF-κB and induction of the cytokines and co-stimulatory molecules required for the activation of the adaptive immune response. The present study was designed to examine the involvement of TLR2 and TLR4 in the oxidized LDL induced inflammation in human PBMCs and the effect of flavonoid quercetin on TLR-NF-κB signaling mechanism. LDL was isolated from human plasma and oxidation of LDL was done by incubating with 10 μM CuSO₄ overnight at 37 °C. The isolated human PBMCs in culture were used as the model system. 50 μg/ml ox-LDL treatment significantly up regulated TLR2 and TLR4 expression in isol human PBMCs after 24 h of culture and this was down regulated by quercetin at 25 μM concentration. ox-LDL caused a significant activation of NF-κB as evidenced by the detection of enhanced p65 subunit in nuclear extracts. Supplementation of quercetin significantly modulates the NF-κB p65 nuclear translocation. The cytokine IL-6 production was significantly increased in ox-LDL treated group and was decreased by quercetin treatment. Quercetin mediated reduction of TLR2 and TLR4 expression and the inhibition of nuclear translocation of NF-κB p65 in turn decreased the inflammatory enzymes like 5-LOX and COX and also decreased the mRNA expression of inducible enzymes like COX-2 and iNOS. Quercetin inhibited the ox-LDL induced TLR2 and TLR4 expression at mRNA level and modulated the TLR-NF-κB signaling pathway thereby inhibited the cytokine production and down regulated the activity of inflammatory enzymes thus have protective effect against the ox-LDL induced inflammation in PBMCs.     

5-HT1A-like receptor activation inhibits abstinence-induced methamphetamine withdrawal in planarians

No pharmacological therapy is approved to treat methamphetamine physical dependence, but it has been hypothesized that serotonin (5-HT)-enhancing drugs might limit the severity of withdrawal symptoms. To test this hypothesis, we used a planarian model of physical dependence that quantifies withdrawal as a reduction in planarian movement. Planarians exposed to methamphetamine (10 μM) for 60 min, and then placed (tested) into drug-free water for 5 min, displayed less movement (i.e., withdrawal) than either methamphetamine-naïve planarians tested in water or methamphetamine-exposed planarians tested in methamphetamine. A concentration-related inhibition of withdrawal was observed when methamphetamine-exposed planarians were placed into a solution containing either methamphetamine and 5-HT (0.1–100 μM) or methamphetamine and the 5-HT_1A receptor agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) (10, 20 μM). Planarians with prior methamphetamine exposure displayed enhanced withdrawal when tested in a solution of the 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (WAY 100635) (1 μM). Methamphetamine-induced withdrawal was not affected by the 5-HT_2B/2C receptor agonist meta-chlorophenylpiperazine (m-CPZ) (0.1–20 μM). These results provide pharmacological evidence that serotonin-enhancing drugs inhibit expression of methamphetamine physical dependence in an invertebrate model of withdrawal, possibly through a 5-HT_1A-like receptor-dependent mechanism.     

IL-1β acutely increases pulmonary SP and permeability without associated changes in airway resistance and ventilation in anesthetized rats

Bronchopulmonary C-fibers (PCFs), when activated, promote substance P (SP) release, increase microvascular leak, and produce bronchoconstriction and apnea. IL-1β administered systemically or locally into the pulmonary parenchyma stimulates PCFs. Thus, we tested whether right atrial bolus injection or aerosol inhalation of IL-1β, to mainly stimulate PCFs, would acutely affect pulmonary SP level and vascular permeability, airway resistance (R_L), and ventilation in anesthetized rats. Our results showed that 30 min after IL-1β injection (2–6 μg kg⁻¹), SP levels and Evans blue extravasation in bronchoalveolar lavage fluid were markedly increased and these responses were eliminated or largely reduced in neonatal capsaicin-treated rats. In contrast, neither injection nor inhalation of IL-1β (5–15 μg ml⁻¹) significantly altered R_L and ventilation. Additionally, the capsaicin-evoked (4 μg kg⁻¹, i.v.) apneic response was unaffected by IL-1β treatment. Our data suggest that IL-1β, as administered in this study, can acutely increase pulmonary SP and vascular permeability, likely via stimulating PCFs, with little impact on R_L and ventilation.     

Mineral intake independent from gastric irritation or pica by cell-dehydrated rats

Gavage of 2 M NaCl (IG 2 M NaCl), a procedure to induce cell-dehydration—and water and 0.15 M NaCl intake in a two-bottle choice test—is also a potential gastric irritant. In this study, we assessed whether mineral intake induced by IG 2 M NaCl is associated with gastric irritation or production of pica in the rat. We first determined the amount of mineral solution (0.15 M NaCl, 0.15 M NaHCO₃, 0.01 M KCl and 0.05 mM CaCl₂) and water ingested in response to IG 2 M NaCl in a five-bottle test. Then, we used mineral solutions (0.01 M KCl and 0.15 M NaHCO₃), whose intakes were significantly increased compared to controls, and water in three-bottle tests to test the gastric irritation hypothesis. The IG 2 M NaCl induced KCl and NaHCO₃ intake that was not inhibited by gavage with gastric protectors Al(OH)₃ or NaHCO₃. IG 2 M NaCl or gavage of 0.6 N acetic acid induced mild irritation, hyperemia, of the glandular part of the stomach. A gavage of 50% ethanol induced strong irritation seen as pinpoint ulcerations. Neither ethanol nor acetic acid induced any fluid intake. Neither IG 2 M NaCl nor acetic acid induced kaolin intake, a marker of pica in laboratory rats. Ethanol did induce kaolin intake. These results suggest that IG 2 M NaCl induced a mineral fluid intake not selective for sodium and independent from gastric irritation or pica.     

Role of Arachidonic Acid and Its Metabolites in the Priming of NADPH Oxidase in Human Polymorphonuclear Leukocytes by Peritoneal Dialysis Effluent

Peritoneal dialysis effluent (PDE) contains a low-molecular-weight solute that will activate and prime the NADPH oxidase of human neutrophils via a phospholipase A₂ (PLA₂)-dependent mechanism. Since the products of PLA₂ are known to activate and prime the oxidase we have investigated their role in the dialysis effluent-mediated activation and priming of human neutrophils. NADPH oxidase activity of PDE-primed and -unprimed neutrophils was measured by lucigenin-enhanced chemiluminescence in the presence of known inhibitors of the arachidonic acid cascade. Incubation of neutrophils with the nonselective PLA₂ inhibitor quinacrine (0 to 100 μM) reduced oxidase activity in both primed and unprimed cells. Furthermore, primed cells were more sensitive to the action of quinacrine than were unprimed cells. We were unable to determine the relative roles of secretory PLA₂ (sPLA₂) and cytosolic PLA₂ (cPLA₂) since the selective sPLA₂ inhibitor scalaradial (0 to 100 μM) inhibited oxidase activity in both groups of cells by similar degrees, while the specific cPLA₂ inhibitor AACO-CF₃ (0 to 50 μM) failed to affect activity in either group. Inhibition of platelet-activating factor (PAF), cycloxygenase, and 5-lipoxygenase-activating protein by hexanolamino-PAF (0 to 25 μM), flurbiprofen (0 to 25 μM), and MK886 (0 to 5 μM), respectively, had no effect upon oxidase activity. However, the direct inhibition of 5-lipoxygenase by caffeic acid or lipoxin A₄ resulted in a similar concentration-dependent attenuation of oxidase activity in both primed and unprimed cells. Leukotriene B₄ (LTB₄) release from primed neutrophils was comparable to that from unprimed cells with the exception of phorbol myristate acetate-stimulated cells, which released fivefold more LTB₄ than control. Taken together, these results suggest that it is arachidonic acid per se, and not its metabolites, that is important in priming of the neutrophil NADPH oxidase by dialysis effluent.     

Aspirin-triggered lipoxin induces CB1-dependent catalepsy in mice

Evidence are that inhibition of cyclooxygenase 2 (COX-2) enhances endocannabinoid signaling, indicating a crosstalk between these two eicosanoid pathways. Aspirin, a non-selective COX inhibitor, acetylates COX-2 with generation of a lipoxygenase (LOX) substrate, whose end product is the 15-epi-lipoxin A₄ (15-epi-LXA₄), an aspirin-triggered lipoxin. Our objective was to investigate whether 15-epi-LXA₄ would potentiate in vivo effects of the endocannabinoid anandamide (AEA). Catalepsy was selected as a behavioral parameter and tested 5 min after AEA injection in all experiments. AEA induced dose-dependent (200 pmol/2 μl, i.c.v.) catalepsy. A sub-dose of AEA (10 pmol/2 μl, i.c.v.) was potentiated by aspirin (300 mg/kg, p.o.) via a 5-LOX-dependent step. The cataleptic effect induced by the interaction between sub-doses of 15-epi-LXA₄ (0.01 pmol/2 μl, i.c.v.) and AEA (10 pmol/2 μl, i.c.v.) was prevented by the cannabinoid CB₁ receptors antagonist SR141716A (1 mg/kg, i.p.), but not by the antagonist of lipoxin ALX receptors Boc-2 (10 μg/kg, i.p.). While previous studies have shown that COX inhibition itself may enhance endocannabinoid effects, here we add another piece of evidence revealing that a LOX-derivative produced in consequence of COX-2 acetylation participates in this process.     

In vitro activation of murine peritoneal macrophages by recombinant YopJ: production of nitric oxide, proinflammatory cytokines and chemokines

Recently it was reported that 3 μg/ml of recombinant YopJ induced apoptosis in murine peritoneal macrophages in vitro. However, in this study, we report the activation of murine peritoneal macrophages in vitro on treatment with sub-apoptotic dose of recombinant YopJ protein (1 μg/ml). The activation involves enhanced production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), IL-12, and IL-6. Production of NO and IL-6 was found to peak at 24 h of rYopJ treatment, whereas IL-12 and IFN-γ production peaked at 18 h of rYopJ treatment. Increased mRNAs expression of nitric oxide, IL-12, IL-6 and IFN-γ molecules, was also observed in rYopJ-treated macrophages by RT-PCR. rYopJ induced the enhanced activity of protein tyrosine kinases which was inhibited by pharmacological inhibitor genestein, wortmanin and H-7 suggesting the role of tyrosine kinases, PI3K and PKC in the above process. rYopJ also induced increased enhanced production chemokines MIP-1α, MCP-1, and RANTES in macrophages. Significantly, increased expression of TLR-2, TLR-6, MyD 88 and IRAK-1 was also observed by immunoblotting in rYopJ-treated macrophages. rYopJ induced production of NO, TNF-α and IL-6 was significantly inhibited in macrophages pretreated with pharmacological inhibitor wortmanin, genestein and H-7 demonstrating the probable involvement of protein tyrosine kinases in the above process.     

Interleukin-1β Inhibits Glutamate Release in Hippocampus of Young,But Not Aged,Rats

The proinflammatory cytokine, interleukin-1, is synthesized in neuronal and glial cells and is released in response to stress/injury. IL-1 exerts profound effects on the central nervous system, which include an inhibitory effect on synaptic activity in hippocampus, a brain area expressing a high density of IL-1 receptors. We report that IL-1β has an inhibitory effect on KCl-stimulated release of glutamate and KCl-stimulated [⁴⁵Ca] influx in synaptosomes prepared from hippocampus of 4-month-old rats. These effects were inhibited by the endogenous receptor antagonist, IL-1ra, and by the phospholipase A₂ (PLA₂) inhibitor, quinacrine, suggesting that IL-1 receptor activation is coupled to PLA₂. An inhibitory effect of IL-1β on protein kinase C activity was also observed. KCl-induced calcium-dependent release and calcium influx, and protein kinase C activity were significantly decreased in hippocampal synaptosomes prepared from 22-month-old compared to 4-month-old animals. In contrast to the inhibitory effect of IL-1β in synaptosomes prepared from young adult animals, no effect was observed on release, calcium influx, or protein kinase C activity in synaptosomes prepared from aged animals. We report that there is an age-related increase in expression of IL-1β in hippocampus and propose that this change may underlie the attenuated responses to IL-1β in hippocampus of aged animals.