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  • The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. 18337823

    Many tumour cells have elevated rates of glucose uptake but reduced rates of oxidative phosphorylation. This persistence of high lactate production by tumours in the presence of oxygen, known as aerobic glycolysis, was first noted by Otto Warburg more than 75 yr ago. How tumour cells establish this altered metabolic phenotype and whether it is essential for tumorigenesis is as yet unknown. Here we show that a single switch in a splice isoform of the glycolytic enzyme pyruvate kinase is necessary for the shift in cellular metabolism to aerobic glycolysis and that this promotes tumorigenesis. Tumour cells have been shown to express exclusively the embryonic M2 isoform of pyruvate kinase. Here we use short hairpin RNA to knockdown pyruvate kinase M2 expression in human cancer cell lines and replace it with pyruvate kinase M1. Switching pyruvate kinase expression to the M1 (adult) isoform leads to reversal of the Warburg effect, as judged by reduced lactate production and increased oxygen consumption, and this correlates with a reduced ability to form tumours in nude mouse xenografts. These results demonstrate that M2 expression is necessary for aerobic glycolysis and that this metabolic phenotype provides a selective growth advantage for tumour cells in vivo.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Localization of M2 muscarinic receptor protein in parvalbumin and calretinin containing cells of the adult rat entorhinal cortex using two complementary methods. 15708496

    We investigated parvalbumin (PV) and calretinin (CR) containing interneurons in the rat entorhinal cortex. RNA amplification following single cell dissection of immunohistochemically labeled cells from layers II to VI revealed that PV cells, in contrast to CR cells, express the m2 muscarinic receptor (M2AchR) protein. Double immunostaining to confirm the results of RNA amplification indicated that the majority of PV cells contain M2AchR protein, whereas only a small proportion of CR cells do. In contrast, a large number of layer I CR cells, which are mostly Cajal-Retzius cells, were positive for M2AchR. RNA amplification following dissection of these cells also revealed that they contain the M2AchR protein. These findings emphasize that there are significant differences in the expression of different proteins, even among similar neuronal types in the same brain region. This highlights the importance of accurately collecting single cells, and knowledge of anatomical details in molecular biological studies.
    Document Type:
    Reference
    Product Catalog Number:
    MAB367
    Product Catalog Name:
    Anti-Muscarinic Acetylcholine Receptor m2 Antibody, clone M2-2-B3

  • Muscarinic M2 and M1 receptors reduce GABA release by Ca2+ channel modulation through activation of PI3K/Ca2+ -independent and PLC/Ca2+ -dependent PKC. 17581851

    We measured pharmacologically isolated GABAergic currents from layer II/III neurons of the rat auditory cortex using patch-clamp recording. Activation of muscarinic receptors by muscarine (1 microM) or oxotremorine (10 microM) decreased the amplitude of electrically evoked inhibitory postsynaptic currents to about one third of their control value. Neither miniature nor exogenously evoked GABAergic currents were altered by the presence of muscarinic agonists, indicating that the effect was spike-dependent and not mediated postsynaptically. The presence of the N- or P/Q-type Ca(2+) channel blockers omega-conotoxin GVIA (1 microM) or omega-AgaTx TK (200 nM) greatly blocked the muscarinic effect, suggesting that Ca(2+)-channels were target of the muscarinic modulation. The presence of the muscarinic M(2) receptor (M(2)R) antagonists methoctramine (5 muM) or AF-DX 116 (1 microM) blocked most of the muscarinic evoked inhibitory postsynaptic current (eIPSC) reduction, indicating that M(2)Rs were responsible for the effect, whereas the remaining component of the depression displayed M(1)R-like sensitivity. Tissue preincubation with the specific blockers of phosphatidyl-inositol-3-kinase (PI(3)K) wortmannin (200 nM), LY294002 (1 microM), or with the Ca(2+)-dependent PKC inhibitor Gö 6976 (200 nM) greatly impaired the muscarinic decrease of the eIPSC amplitude, whereas the remaining component was sensitive to preincubation in the phospholipase C blocker U73122 (10 microM). We conclude that acetylcholine release enhances the excitability of the auditory cortex by decreasing the release of GABA by inhibiting axonal V-dependent Ca(2+) channels, mostly through activation of presynaptic M(2)Rs/PI(3)K/Ca(2+)-independent PKC pathway and-to a smaller extent-by the activation of M(1)/PLC/Ca(2+)-dependent PKC.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Roles of M2 and M4 muscarinic receptors in regulating acetylcholine release from myenteric neurons of mouse ileum. 15574798

    We investigated the subtype of presynaptic muscarinic receptors associated with inhibition of acetylcholine (ACh) release in the mouse small intestine. We measured endogenous ACh released from longitudinal muscle with myenteric plexus (LMMP) preparations obtained from M1-M5 receptor knockout (KO) mice. Electrical field stimulation (EFS) increased ACh release in all LMMP preparations obtained from M1-M5 receptor single KO mice. The amounts of ACh released in all preparations were equal to that in the wild-type mice. Atropine further increased EFS-induced ACh release in the wild-type mice. Unexpectedly, atropine also increased, to a similar extent, EFS-induced ACh release to the wild-type mice in all M1-M5 receptor single KO mice. In M2 and M4 receptor double KO mice, the amount of EFS-induced ACh release was equivalent to an atropine-evoked level in the wild-type mouse, and further addition of atropine had no effect. M2 receptor immunoreactivity was located in both smooth muscle cells and enteric neurons. M4 receptor immunoreactivity was located in the enteric neurons, being in co-localization with M2 receptor immunoreactivity. These results indicate that both M2 and M4 receptors mediate the muscarinic autoinhibition in ACh release in the LMMP preparation of the mouse ileum, and loss of one of these subtypes can be compensated functionally by a receptor that remained. M1, M3, and M5 receptors do not seem to be involved in this mechanism.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. 23872599

    The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia and macrophages, which can be polarized to distinct functional phenotypes: pro-inflammatory (M1) and anti-inflammatory or immunoregulatory (M2). We found that a switch from an M1- to an M2-dominant response occurred in microglia and peripherally derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 cell conditioned media and impaired in vivo following intra-lesional M2 cell depletion. M2 cell densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger mouse and in multiple sclerosis lesions that normally show remyelination. Blocking M2 cell-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Thus, our results indicate that M2 cell polarization is essential for efficient remyelination and identify activin-A as a therapeutic target for CNS regeneration.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • M2 and M3 muscarinic receptors are involved in enteric nerve-mediated contraction of the mouse ileum: Findings obtained with muscarinic-receptor knockout mouse. 17008557

    The involvement of muscarinic receptors in neurogenic responses of the ileum was studied in wild-type and muscarinic-receptor (M-receptor) knockout (KO) mice. Electrical field stimulation to the wild-type mouse ileum induced a biphasic response, a phasic and sustained contraction that was abolished by tetrodotoxin. The sustained contraction was prolonged for an extended period after the termination of electrical field stimulation. The phasic contraction was completely inhibited by atropine. In contrast, the sustained contraction was enhanced by atropine. Ileal strips prepared from M2-receptor KO mice exhibited a phasic contraction similar to that seen in wild-type mice and a sustained contraction that was larger than that in wild-type mice. In M3-receptor KO mice, the phasic contraction was smaller than that observed in wild-type mice. Acetylcholine exogenously administrated induced concentration-dependent contractions in strips isolated from wild-type, M2- and M3-receptor KO mice. However, contractions in M3-receptor KO mice shifted to the right. The sustained contraction was inhibited by capsaicin and neurokinin NK2 receptor antagonist, suggesting that it is mediated by substance P (SP). SP-induced contraction of M2-receptor KO mice did not differ from that of wild-type mice. SP immunoreactivity was located in enteric neurons, colocalized with M2 receptor immunoreactivity. These results suggest that atropine-sensitive phasic contraction is mainly mediated via the M3 receptor, and SP-mediated sustained contraction is negatively regulated by the M2 receptor at a presynaptic level.
    Document Type:
    Reference
    Product Catalog Number:
    MAB367
    Product Catalog Name:
    Anti-Muscarinic Acetylcholine Receptor m2 Antibody, clone M2-2-B3

  • Coupling of the muscarinic m2 receptor to G protein-activated K(+) channels via Galpha(z) and a receptor-Galpha(z) fusion protein. Fusion between the receptor and Galpha( ... 10660578

    G protein-activated K(+) channel (GIRK), which is activated by the G(betagamma) subunit of heterotrimeric G proteins, and muscarinic m2 receptor (m2R) were coexpressed in Xenopus oocytes. Acetylcholine evoked a K(+) current, I(ACh), via the endogenous pertussis toxin (PTX)-sensitive G(i/o) proteins. Activation of I(ACh) was accelerated by increasing the expression of m2R, suggesting a collision coupling mechanism in which one receptor catalytically activates several G proteins. Coexpression of the alpha subunit of the PTX-insensitive G protein G(z), Galpha(z), induced a slowly activating PTX-insensitive I(ACh), whose activation kinetics were also compatible with the collision coupling mechanism. When GIRK was coexpressed with an m2R x Galpha(z) fusion protein (tandem), in which the C terminus of m2R was tethered to the N terminus of Galpha(z), part of I(ACh) was still eliminated by PTX. Thus, the m2R of the tandem activates the tethered Galpha(z) but also the nontethered G(i/o) proteins. After PTX treatment, the speed of activation of the m2R x Galpha(z)-mediated response did not depend on the expression level of m2R x Galpha(z) and was faster than when m2R and Galpha(z) were coexpressed as separate proteins. These results demonstrate that fusing the receptor and the Galpha strengthens their coupling, support the collision-coupling mechanism between m2R and the G proteins, and suggest a noncatalytic (stoichiometric) coupling between the G protein and GIRK in this model system.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Subcellular distribution of M2 muscarinic receptors in relation to dopaminergic neurons of the rat ventral tegmental area. 16927256

    Acetylcholine can affect cognitive functions and reward, in part, through activation of muscarinic receptors in the ventral tegmental area (VTA) to evoke changes in mesocorticolimbic dopaminergic transmission. Among the known muscarinic receptor subtypes present in the VTA, the M2 receptor (M2R) is most implicated in autoregulation and also may play a heteroreceptor role in regulation of the output of the dopaminergic neurons. We sought to determine the functionally relevant sites for M2R activation in relation to VTA dopaminergic neurons by examining the electron microscopic immunolabeling of M2R and the dopamine transporter (DAT) in the VTA of rat brain. The M2R was localized to endomembranes in DAT-containing somatodendritic profiles but showed a more prominent, size-dependent plasmalemmal location in nondopaminergic dendrites. M2R also was located on the plasma membrane of morphologically heterogenous axon terminals contacting unlabeled as well as M2R- or DAT-labeled dendrites. Some of these terminals formed asymmetric synapses resembling those of cholinergic terminals in the VTA. The majority, however, formed symmetric, inhibitory-type synapses or were apposed without recognized junctions. Our results provide the first ultrastructural evidence that the M2R is expressed, but largely not available for local activation, on the plasma membrane of VTA dopaminergic neurons. Instead, the M2R in this region has a distribution suggesting more indirect regulation of mesocorticolimbic transmission through autoregulation of acetylcholine release and changes in the physiological activity or release of other, largely inhibitory transmitters. These findings could have implications for understanding the muscarinic control of cognitive and goal-directed behaviors within the VTA.
    Document Type:
    Reference
    Product Catalog Number:
    MAB369
    Product Catalog Name:
    Anti-Dopamine Transporter Antibody, NT, clone DAT-Nt

  • Interleukin-17 Indirectly Promotes M2 Macrophage Differentiation through Stimulation of COX-2/PGE2 Pathway in the Cancer Cells. 25038765

    Interleukin-17 (IL-17) is a proinflammatory cytokine that plays important roles in inflammation, autoimmunity, and cancer. The purpose of this study was to determine if IL-17 indirectly regulates macrophage differentiation through up-regulation of cyclooxygenase-2 (COX-2) expression in the cancer cell lines.Human cervical cancer HeLa, human lung cancer A549, and mouse prostate cancer Myc-CaP/CR cell lines were treated with recombinant IL-17; Western blot analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction analysis were utilized to examine the cellular responses.IL-17 up-regulated expression of COX-2 mRNA and protein in HeLa, A549, and Myc- CaP/CR cell lines. IL-17's effects were mediated through nuclear factor-κB and ERK1/2 signaling pathways as the inhibitors of these pathways could inhibit IL-17- induced COX-2 expression. The conditional medium obtained from the cancer cells contained prostaglandin E2, the levels of which were increased by IL-17 treatment. When treated with the conditional medium, particularly with the IL-17-induced conditional medium, mouse RAW264.7 macrophages and human THP-1 monocytes expressed higher levels of IL-10 (a marker of M2 macrophages) than inducible nitric oxide synthase or tumor necrosis factor α (markers of M1 macrophages). In contrast, when RAW264.7 and THP-1 cells were treated directly with IL-17, expression of these marker genes was not markedly changed.The results of this study suggest that IL-17 indirectly promotes M2 macrophage differentiation through stimulation of the COX-2/PGE2 pathway in the cancer cells, thus IL-17 plays an indirect role in regulating the tumor immune microenvironment.
    Document Type:
    Reference
    Product Catalog Number:
    MAB374
    Product Catalog Name:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5