l-trans-Pyrrolidine-2,4-Dicarboxylic Acid-Evoked Striatal Glutamate Levels Are Attenuated by Calcium Reduction, Tetrodotoxin, and Glutamate Receptor Blockade

Abstract: l - trans -Pyrrolidine-2,4-dicarboxylic acid ( l - trans -PDC) reverses plasma membrane glutamate transporters and elevates extracellular glutamate levels in vivo. We investigated the possibility that l - trans -PDC-stimulated glutamate levels are mediated partially by increases in transsynaptic activity. Therefore, the degree to which l - trans -PDC-evoked glutamate levels depend on calcium, sodium-channel activation, and glutamate-receptor activation was investigated by infusing via reverse microdialysis (a) 0.1 m M calcium, (b) 1 µ M tetrodotoxin, a selective blocker of voltage-dependent sodium channels, (c) R (−)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a selective NMDA-receptor antagonist, or (d) LY293558, a selective α-amino-3-hydroxy-5-methylisoxazole-4-propionate antagonist. In separate experimental groups, l - trans -PDC-evoked glutamate levels were reduced significantly by 55% in the presence of 0.1 m M calcium and by 46% in the presence of tetrodotoxin. Additionally, CPP and LY293558 significantly attenuated l - trans -PDC-evoked glutamate levels without altering basal glutamate levels. These data suggest that glutamate transporter reversal by l - trans -PDC initially elevates extracellular glutamate levels enough to stimulate postsynaptic glutamate receptors within the striatum. It is proposed that glutamate-receptor stimulation activates a positive feedback loop within the basal ganglia, leading to further glutamate release from corticostriatal and thalamostriatal afferents. Therefore, either extracellular striatal calcium reduction or tetrodotoxin perfusion leads to decreased action potential-dependent glutamate release from these terminals. In addition, blocking glutamate receptors directly reduces medium spiny neuronal firing and indirectly attenuates corticostriatal and thalamostriatal activity, resulting in an overall depression of l - trans -PDC-stimulated glutamate levels.     

Dopamine Autoreceptors Modulate Dopamine Release from the Prefrontal Cortex

Electrical stimulation (at 0.3, 1, or 10 Hz, 120 pulses each) produced a calcium-dependent overflow of radioactivity from slices of the rabbit prefrontal cortex preloaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) in the presence of desipramine. Flat frequency-release curves were observed. Apomorphine and LY-171555 inhibited in a concentration-dependent fashion the evoked overflow of DA, an effect antagonized by haloperidol. Stimulation frequencies comparable to normal firing rates of mesocortical neurons (10 Hz) drastically reduced apomorphine-induced inhibition of DA overflow. Haloperidol produced greater facilitation of DA overflow at 10 than at 1 Hz. Nomifensine, a neuronal uptake inhibitor, enhanced DA overflow. These results indicate that mesocortical DA neurons projecting to the prefrontal cortex have release modulatory autoreceptors of the D2 subtype.     

Entamoeba histolytica: FYVE-finger domains, phosphatidylinositol 3-phosphate biosensors, associate with phagosomes but not fluid filled endosomes

Endocytosis is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Although a number of E. histolytica proteins that regulate this process have been identified, less is known about the role of lipids. In other systems, phosphatidylinositol 3-phosphate (PI3P), a product of phosphatidylinositol 3-kinase (PI 3-kinase), has been shown to be required for endocytosis. FYVE-finger domains are protein motifs that bind specifically to PI3P. Using a PI3P biosensor consisting of glutathione-S-transferase (GST) fused to two tandem FYVE-finger domains, we have localized PI3P to phagosomes but not fluid-phase pinosomes in E. histolytica, suggesting a role for PI3P in phagocytosis. Treatment of cells with PI 3-kinase inhibitors impaired GST-2 x FYVE-phagosome association supporting the authenticity of the biosensor staining. However, treatment with PI 3-kinase inhibitors did not inhibit E. histolytica-particle interaction, indicating that PI3P is not required for the initial step, but is required for subsequent steps of phagocytosis.     

PKB/AKT is involved in resumption of meiosis in mouse oocytes

BACKGROUND INFORMATION: In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. RESULTS: We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. CONCLUSIONS: The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation.     

Pharmacological Characterization of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Inhibitors

  1. The mechanism of anandamide uptake and disposal has been an issue of considerable debate in the cannabinoid field. Several compounds have been reported to inhibit anandamide uptake or fatty acid amide hydrolase (FAAH; the primary catabolic enzyme of anandamide) activity with varying degrees of potency and selectivity. We recently reported the first evidence of a binding site involved in the uptake of endocannabinoids that is independent from FAAH. There are no direct comparisons of purported selective inhibitory compounds in common assay conditions measuring anandamide uptake, FAAH activity and binding activity.2. A subset of compounds reported in the literature were tested in our laboratory under common assay conditions to measure their ability to (a) inhibit [¹⁴C]-anandamide uptake in cells containing (RBL-2H3) or cells lacking (HeLa) FAAH, (b) inhibit purified FAAH hydrolytic activity, and (c) inhibit binding to a putative binding site involved in endocannabinoid transport in both RBL and HeLa cell membranes.3. Under these conditions, nearly all compounds tested inhibited (a) uptake of [¹⁴C]-anandamide, (b) enzyme activity in purified FAAH preparations, and (c) radioligand binding of [³H]-LY2183240 in RBL and HeLa plasma membrane preparations. General rank order potency was preserved within the three assays. However, concentration response curves were right-shifted for functional [¹⁴C]-anandamide uptake in HeLa (FAAH^−/−) cells.4. A more direct comparison of multiple inhibitors could be made in these three assay systems performed in the same laboratory, revealing more information about the selectivity of these compounds and the relationship between the putative endocannabinoid transport protein and FAAH. At least two separate proteins appear to be involved in uptake and degradation of anandamide. The most potent inhibitory compounds were right-shifted when transport was measured in HeLa (FAAH^−/−) cells suggesting a requirement for a direct interaction with the FAAH protein to maintain high affinity binding of anandamide or inhibitors to the putative anandamide transport protein.     

LPS-stimulated MUC5AC production involves Rac1-dependent MMP-9 secretion and activation in NCI-H292 cells

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Previous studies have reported that lipopolysaccharides (LPS) stimulate mucin 5AC (MUC5AC) production via epidermal growth factor receptor (EGFR) in human airway cells. Moreover, this production was shown to depend on the expression and activity of matrix metalloproteinase 9 (MMP-9), which is increased in COPD patients’ serum. In the present study we investigated the signaling pathway mediating LPS-stimulated secretion and activation of MMP-9, and the regulatory effects of this pathway on the production of MUC5AC in the human airway cells NCI-H292. Using specific inhibitors, we found that LPS-stimulated cells secreted and activated MMP-9 via EGFR. Our results also indicate that signaling events downstream of EGFR involved PI3K-dependent activation of Rac1, which mediated the NADPH-generated reactive oxygen species responsible for MMP-9 secretion and activation. Finally, we observed that EGFR/PI3K/Rac1/NADPH/ROS/MMP-9 regulate MUC5AC production in LPS-challenged NCI-H292 cells.     

Niemann–Pick C2 (NPC2) and intracellular cholesterol trafficking

Cholesterol is an important precursor for numerous biologically active molecules, and it plays a major role in membrane structure and function. Cholesterol can be endogenously synthesized or exogenously taken up via the endocytic vesicle system and subsequently delivered to post-endo/lysosomal sites including the plasma membrane and the endoplasmic reticulum. Niemann–Pick C (NPC) disease results in the accumulation of exogenously-derived cholesterol, as well as other lipids, in late endosomes and lysosomes (LE/LY). Identification of the two genes that underlie NPC disease, NPC1 and NPC2, has focused attention on the mechanisms by which lipids, in particular cholesterol, are transported out of the LE/LY compartment. This review discusses the role of the NPC2 protein in cholesterol transport, and the potential for concerted action of NPC1 and NPC2 in regulating normal intracellular cholesterol homeostasis.