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H U Keller - One of the best experts on this subject based on the ideXlab platform.

  • effects of the myosin inhibitor 2 3 butanedione monoxime bdm on cell shape locomotion and fluid Pinocytosis in human polymorphonuclear leucocytes
    Cell Biology International, 2000
    Co-Authors: N Urwyler, P Eggli, H U Keller
    Abstract:

    Abstract We investigated the role of myosin in polymorphonuclear leucocyte (PMN) shape changes, locomotion, and fluid Pinocytosis using the myosin inhibitor 2,3 butanedione monoxime (BDM). Treatment of resting spherical PMNs with BDM produced spheroid cells showing small continuous shape changes (IC50=15.5 m m BDM) and occasionally small blebs. Cell polarity, as induced by the chemotactic peptide fNLPNTL or by colchicine, and locomotion were completely suppressed (IC50=8.4 to 10 m m ). Suppression of fNLPNTL- or colchicine-induced cell polarity produced spheroid cells, suppression of PMA-induced shape changes and fluid Pinocytosis produced non-motile spherical cells (IC50=25 to 30 m m BDM). BDM suppressed formation of lamellipodia but not formation of blebs. Suppression of microvilli by BDM as observed in resting spherical cells was partially antagonized by PMA. The results suggest that myosin is involved in stabilizing the shape of resting spherical cells, including microvilli, and that myosin is required for cell polarity, locomotion, fluid Pinocytosis and for formation of lamellipodia, but not for formation of blebs.

  • selective responses actin polymerization shape changes locomotion Pinocytosis to the pkc inhibitor ro 31 8220 suggest that pkc discriminately regulates functions of human blood lymphocytes
    Journal of Leukocyte Biology, 1995
    Co-Authors: S Trachsel, H U Keller
    Abstract:

    : The results suggest that protein kinase C (PKC) plays a pivotal role in the control of F-actin levels, locomotion, Pinocytosis, and cell shape in lymphocytes. The PKC inhibitor Ro 31-8220 elicits a high proportion of polarized (ED50 = 1.5 x 10(-6) M) and locomoting cells and reduces the relative amount of F-actin (by 29% at 10(-5) M) in initially resting cells. Phorbol myristate acetate (PMA) counterbalances the polarizing effect of Ro 31-8220. This indicates that the spherical shape and the F-actin content of resting cells are maintained by constitutive PKC activity. PMA-induced increases in fluid Pinocytosis, F-actin content, and formation of nonpolar cells with surface protrusion are suppressed by Ro 31-8220 (IC50 = 2-4 x 10(-7) M). Spherical cells and, at higher concentrations (ED50 = 3.3 x 10(-6) M), polarized cells are formed instead. As a result, lymphocyte function switches from fluid Pinocytosis to cell polarity and locomotion. The data indicate that PKC is instrumental in selectively switching lymphocyte function between resting state, locomotor activity, and fluid Pinocytosis. Ro 31-8220 is extremely potent in stimulating lymphocyte polarity and locomotion (B and T cells). It acts faster and/or produces a higher proportion of polarized lymphocytes than other available agonists. It may thus be used as a tool in further experiments requiring locomoting lymphocytes.

  • effects of cytochalasin d on shape and fluid Pinocytosis in human neutrophils as related to cytoskeletal changes actin alpha actinin and microtubules
    European Journal of Cell Biology, 1995
    Co-Authors: H U Keller, V Niggli
    Abstract:

    : We report that cytochalasin D (CD) is not a reliable tool to inhibit all forms of cell motility and actin polymerization in neutrophil granulocytes. In addition to the well-established effects of CD such as altered localization of F-actin, inhibition of surface ruffling, fluid Pinocytosis and actin polymerization in agonist-stimulated cells, we find that in human neutrophils CD can 1) induce another type of continuous shape changes (10(-6) M and 10(-5) M CD), 2) stimulate fluid Pinocytosis (10(-5) M CD), 3) increase actin polymerization (10(-5) to 10(-4) M CD) and alter the localization of F-actin and alpha-actinin (10(-6) to 10(-4) M CD). At 10(-5) M CD F-actin and alpha-actinin are preferentially located in different areas of the cell. At 10(-4) M CD actin and alpha-actinin may colocalize at the membrane but not in cytoplasmic foci. Thus, stimulation of shape changes, Pinocytosis, actin polymerization and differential reorganization of the cytoskeleton occur at CD concentrations which are widely used to inhibit cell motility. The results show that CD is not a reliable tool to inhibit all movements of cells and actin polymerization in general. Shape changes, but not fluid Pinocytosis and the relative redistribution of F-actin and alpha-actinin induced by 10(-5) M cytochalasin D are suppressed by 10(-5) colchicine. This indicates that also microtubules can play a role in determining neutrophil shape and movements.

  • selective effects of the pkc inhibitors ro 31 8220 and cgp 41 251 on pmn locomotion cell polarity and Pinocytosis
    Journal of Cellular Physiology, 1994
    Co-Authors: H U Keller, V Niggli
    Abstract:

    Using two newly synthesized inhibitors, Ro 31-8220 and CGP 41 251, of protein kinase C (PKC), we analyzed: (1) how distinct PMN functions (shape changes, locomotion, Pinocytosis) are regulated, and (2) the role of protein phosphorylation and PKC in this process. We were able to transform: (1) resting PMNs into locomoting cells using fNLPNTL, (2) locomoting cells into non-locomoting highly pinocytic cells using PMA, and (3) PMA-stimulated cells showing marked Pinocytosis into locomoting or into resting cells using Ro 31-8220. It is thus possible to selectively manipulate PMN function (resting state, locomotion, marked Pinocytosis), indicating that there are different regulatory pathways. It was not possible to induce locomotion and marked Pinocytosis simultaneously, indicating crosstalk between pathways. Ro 31-8220 inhibited PMA-induced shape changes (nonpolar cells) and Pinocytosis, but not fNLPNTL-induced shape changes (polarity) and Pinocytosis. At higher concentrations, Ro 31-8220 alone elicited cell polarity and chemokinesis, indicating that a constitutively active protein kinase is involved in maintaining the spherical shape of resting PMNs. Functional effects of another PKC inhibitor, CGP 41 251, on neutrophil function were strikingly different. CGP 41 251 selectively inhibited fNLPNTL-induced polarity and locomotion (but not colchicine or Ro 31-8220-induced polarity), and it failed to inhibit PMA-induced, stimulated Pinocytosis and shape changes. Although the effects of Ro 31-8220 vs. CGP 41 251 on PMN function were strikingly different, the inhibition of profiles for constitutive and for fNLPNTL- or PMA-induced protein phosphorylation in intact PMNs showed only small differences, which could not yet be conclusively related to cell function. © 1994 Wiley-Liss, Inc.

  • the pkc inhibitor ro 31 8220 selectively suppresses pma and diacylglycerol induced fluid Pinocytosis and actin polymerization in pmns
    Biochemical and Biophysical Research Communications, 1993
    Co-Authors: H U Keller, Verena Niggli
    Abstract:

    Abstract The PKC-inhibitor Ro 31-8220 inhibits stimulated fluid Pinocytosis of human PMNs induced by the PKC-activators phorbol myristate acetate (PMA, IC 50 = 1.35×10 −6 M) or diacylglycerols (OAG, diC8) by 95%, whereas Ro 31-8220 has no effect on D 2 O- or fNLPNTL-induced Pinocytosis and enhances cytochalasin D-induced Pinocytosis. Also formation of F-actin induced by PMA or diacylglycerols is selectively inhibited. The results indicate that a Ro 31-8220-sensitive PKC is involved in signal transduction for enhanced Pinocytosis and F-actin formation in response to one class of stimuli (classical activators of PKC) but not to others.

Howard S Kruth - One of the best experts on this subject based on the ideXlab platform.

  • measurement of aortic cell fluid phase Pinocytosis in vivo by flow cytometry
    Journal of Vascular Research, 2017
    Co-Authors: Joshua J Anzinger, Clovis S Palmer, Pradeep K Dagur, Manoj Kumar Barthwal, Howard S Kruth
    Abstract:

    Objective: Fluid-phase Pinocytosis is a receptor-independent mechanism of endocytosis that occurs in all mammalian cells and may be a mechanism for the uptake of LDL by macrophages. As there are currently no methods for the measurement of fluid-phase Pinocytosis by individual aortic cells in vivo, we sought to identify a suitable method. Methods: ApoE-/- mice were retro-orbitally injected with AngioSPARK fluorescent nanoparticles specifically designed to not interact with cells. After 24 h, mice were sacrificed, and the aortas were isolated and then digested to analyze aortic cell uptake of AngioSPARK by flow cytometry. Results: CD11b-expressing aortic macrophages from mice injected with AngioSPARK showed high levels of fluid-phase Pinocytosis compared to aortic cells not expressing CD11b (4,393.7 vs. 408.3 mean fluorescence intensity [MFI], respectively). Conclusion: This new technique allows for the measurement of fluid-phase Pinocytosis by aortic cells in vivo, making it possible to examine the cell-signaling molecules and drugs that affect this process. Published by S. Karger AG, Basel

  • fluid phase Pinocytosis of ldl by macrophages a novel target to reduce macrophage cholesterol accumulation in atherosclerotic lesions
    Current Pharmaceutical Design, 2013
    Co-Authors: Howard S Kruth
    Abstract:

    Circulating low-density lipoprotein (LDL) that enters the blood vessel wall is the main source of cholesterol that accumulates within atherosclerotic plaques. Much of the deposited cholesterol accumulates within plaque macrophages converting these macrophages into cholesterol-rich foamy looking cells. Cholesterol accumulation in macrophages contributes to cholesterol retention within the vessel wall, and promotes vessel wall inflammation and thrombogenicity. Thus, how macrophages accumulate cholesterol and become foam cells has been the subject of intense investigation. It is generally believed that macrophages accumulate cholesterol only through scavenger receptor-mediated uptake of modified LDL. However, an alternative mechanism for macrophage foam cell formation that does not depend on LDL modification or macrophage receptors has been elucidated. By this alternative mechanism, macrophages show receptor-independent uptake of unmodified native LDL that is mediated by fluid-phase Pinocytosis. In receptor-independent, fluid-phase Pinocytosis, macrophages take up LDL as part of the fluid that they ingest during microPinocytosis within small vesicles called micropinosomes, and by macroPinocytosis within larger vacuoles called macropinosomes. This produces cholesterol accumulation in macrophages to levels characteristic of macrophage foam cells in atherosclerotic plaques. Fluid-phase Pinocytosis of LDL is a plausible mechanism that can explain how macrophages accumulate cholesterol and become disease-causing foam cells. Fluid-phase Pinocytosis of LDL is a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis. Recent studies show that phosphoinositide 3-kinase (PI3K), liver X receptors (LXRs), the macrophage colony-stimulating factor (M-CSF) receptor, and protein kinase C (PKC) mediate macrophage macroPinocytosis of LDL, and thus, these may be relevant targets to inhibit macrophage cholesterol accumulation in atherosclerosis.

  • murine bone marrow derived macrophages differentiated with gm csf become foam cells by pi3kγ dependent fluid phase Pinocytosis of native ldl
    Journal of Lipid Research, 2012
    Co-Authors: Joshua J Anzinger, Manoj Kumar Barthwal, Janet Chang, Qing Xu, Thomas Bohnacker, Matthias P Wymann, Howard S Kruth
    Abstract:

    Accumulation of cholesterol by macrophage uptake of LDL is a key event in the formation of atherosclerotic plaques. Previous research has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) is present in atherosclerotic plaques and promotes aortic lipid accumulation. However, it has not been determined whether murine GM-CSF-differentiated macrophages take up LDL to become foam cells. GM-CSF-differentiated macrophages from LDL receptor-null mice were incubated with LDL, resulting in massive macrophage cholesterol accumulation. Incubation of LDL receptor-null or wild-type macrophages with increasing concentrations of ¹²?I-LDL showed nonsaturable macrophage LDL uptake that was linearly related to the amount of LDL added, indicating that LDL uptake was mediated by fluid-phase Pinocytosis. Previous studies suggest that phosphoinositide 3-kinases (PI3K) mediate macrophage fluid-phase Pinocytosis, although the isoform mediating this process has not been determined. Because PI3K? is known to promote aortic lipid accumulation, we investigated its role in mediating macrophage fluid-phase Pinocytosis of LDL. Wild-type macrophages incubated with LDL and the PI3K? inhibitor AS605240 or PI3K?-null macrophages incubated with LDL showed an ?50% reduction in LDL uptake and cholesterol accumulation compared with wild-type macrophages incubated with LDL only. These results show that GM-CSF-differentiated murine macrophages become foam cells by fluid-phase Pinocytosis of LDL and identify PI3K? as contributing to this process.

  • fluorescent pegylated nanoparticles demonstrate fluid phase Pinocytosis by macrophages in mouse atherosclerotic lesions
    Journal of Clinical Investigation, 2009
    Co-Authors: Chiara Buono, Joshua J Anzinger, Marcelo Amar, Howard S Kruth
    Abstract:

    The uptake of lipoproteins by macrophages is a critical step in the development of atherosclerotic lesions. Cultured monocyte-derived macrophages take up large amounts of native LDL by receptor-independent fluid-phase Pinocytosis, either constitutively or in response to specific activating stimuli, depending on the macrophage phenotype. We therefore sought to determine whether fluid-phase Pinocytosis occurs in vivo in macrophages in atherosclerotic lesions. We demonstrated that fluorescent pegylated nanoparticles similar in size to LDL (specifically nontargeted Qtracker quantum dot and AngioSPARK nanoparticles) can serve as models of LDL uptake by fluid-phase Pinocytosis in cultured human monocyte–derived macrophages and mouse bone marrow–derived macrophages. Using fluorescence microscopy, we showed that atherosclerosis-prone Apoe-knockout mice injected with these nanoparticles displayed massive accumulation of the nanoparticles within CD68+ macrophages, including lipid-containing foam cells, in atherosclerotic lesions in the aortic arch. Similar results were obtained when atherosclerotic mouse aortas were cultured with nanoparticles in vitro. These results show that macrophages within atherosclerotic lesions can take up LDL-sized nanoparticles by fluid-phase Pinocytosis and indicate that fluid-phase Pinocytosis of LDL is a mechanism for macrophage foam cell formation in vivo.

  • liver x receptors inhibit human monocyte derived macrophage foam cell formation by inhibiting fluid phase Pinocytosis of ldl
    Journal of Lipid Research, 2007
    Co-Authors: Chiara Buono, Yifu Li, Stephen W Waldo, Howard S Kruth
    Abstract:

    Liver X receptors (LXRs) are ligand-activated transcription factors involved in the control of lipid metab- olism and inflammation. Several studies have recently shown that LXRs promote reverse cholesterol transport and inhibit atherosclerosis. Our study investigated whether LXRs affect macrophage uptake of LDL by human monocyte-derived macrophages. We have previously shown that human mono- cytes differentiated into macrophages with macrophage- colony-stimulating factor (M-CSF) constitutively take up large amounts of native LDL by receptor-independent, fluid- phase Pinocytosis. In the research reported here, human monocytes were differentiated to macrophages in the pres- ence of M-CSF with or without the LXR agonists T0901317 or 22(R)-hydroxycholesterol. Then, macrophages were incu- bated with native 125 I-LDL to determine LDL uptake. T0901317 and 22(R)-hydroxycholesterol inhibited 125 I-LDL uptake by 68 6 1% and 69 6 2%, respectively, and decreased pinocytotic vacuoles in the macrophages. 125 I-BSA uptake, a measure of fluid-phase Pinocytosis, and 125 I-LDL uptake were the same, and T0901317 treatment inhibited uptake of both to the same degree. T0901317 did not affect receptor- mediated uptake of acetylated LDL, showing that the LXR effect is specific for fluid-phase Pinocytosis of lipoproteins. Our results show that LXRs downregulate macrophage pino- cytosis of LDL. The findings reveal an additional new mechanism by which LXR agonists may inhibit macrophage cholesterol accumulation and atherosclerosis, namely, by inhibiting macrophage uptake of LDL.—Buono, C., Y. Li, S. W. Waldo, and H. S. Kruth. Liver Xr eceptors inhibit hu- man monocyte-derived macrophage foam cell formation by inhibiting fluid-phase Pinocytosis of LDL. J. Lipid Res. 2007. 48: 2411-2418.

V Niggli - One of the best experts on this subject based on the ideXlab platform.

  • effects of cytochalasin d on shape and fluid Pinocytosis in human neutrophils as related to cytoskeletal changes actin alpha actinin and microtubules
    European Journal of Cell Biology, 1995
    Co-Authors: H U Keller, V Niggli
    Abstract:

    : We report that cytochalasin D (CD) is not a reliable tool to inhibit all forms of cell motility and actin polymerization in neutrophil granulocytes. In addition to the well-established effects of CD such as altered localization of F-actin, inhibition of surface ruffling, fluid Pinocytosis and actin polymerization in agonist-stimulated cells, we find that in human neutrophils CD can 1) induce another type of continuous shape changes (10(-6) M and 10(-5) M CD), 2) stimulate fluid Pinocytosis (10(-5) M CD), 3) increase actin polymerization (10(-5) to 10(-4) M CD) and alter the localization of F-actin and alpha-actinin (10(-6) to 10(-4) M CD). At 10(-5) M CD F-actin and alpha-actinin are preferentially located in different areas of the cell. At 10(-4) M CD actin and alpha-actinin may colocalize at the membrane but not in cytoplasmic foci. Thus, stimulation of shape changes, Pinocytosis, actin polymerization and differential reorganization of the cytoskeleton occur at CD concentrations which are widely used to inhibit cell motility. The results show that CD is not a reliable tool to inhibit all movements of cells and actin polymerization in general. Shape changes, but not fluid Pinocytosis and the relative redistribution of F-actin and alpha-actinin induced by 10(-5) M cytochalasin D are suppressed by 10(-5) colchicine. This indicates that also microtubules can play a role in determining neutrophil shape and movements.

  • selective effects of the pkc inhibitors ro 31 8220 and cgp 41 251 on pmn locomotion cell polarity and Pinocytosis
    Journal of Cellular Physiology, 1994
    Co-Authors: H U Keller, V Niggli
    Abstract:

    Using two newly synthesized inhibitors, Ro 31-8220 and CGP 41 251, of protein kinase C (PKC), we analyzed: (1) how distinct PMN functions (shape changes, locomotion, Pinocytosis) are regulated, and (2) the role of protein phosphorylation and PKC in this process. We were able to transform: (1) resting PMNs into locomoting cells using fNLPNTL, (2) locomoting cells into non-locomoting highly pinocytic cells using PMA, and (3) PMA-stimulated cells showing marked Pinocytosis into locomoting or into resting cells using Ro 31-8220. It is thus possible to selectively manipulate PMN function (resting state, locomotion, marked Pinocytosis), indicating that there are different regulatory pathways. It was not possible to induce locomotion and marked Pinocytosis simultaneously, indicating crosstalk between pathways. Ro 31-8220 inhibited PMA-induced shape changes (nonpolar cells) and Pinocytosis, but not fNLPNTL-induced shape changes (polarity) and Pinocytosis. At higher concentrations, Ro 31-8220 alone elicited cell polarity and chemokinesis, indicating that a constitutively active protein kinase is involved in maintaining the spherical shape of resting PMNs. Functional effects of another PKC inhibitor, CGP 41 251, on neutrophil function were strikingly different. CGP 41 251 selectively inhibited fNLPNTL-induced polarity and locomotion (but not colchicine or Ro 31-8220-induced polarity), and it failed to inhibit PMA-induced, stimulated Pinocytosis and shape changes. Although the effects of Ro 31-8220 vs. CGP 41 251 on PMN function were strikingly different, the inhibition of profiles for constitutive and for fNLPNTL- or PMA-induced protein phosphorylation in intact PMNs showed only small differences, which could not yet be conclusively related to cell function. © 1994 Wiley-Liss, Inc.

Shumin Duan - One of the best experts on this subject based on the ideXlab platform.

  • p2y4 receptor mediated Pinocytosis contributes to amyloid beta induced self uptake by microglia
    Molecular and Cellular Biology, 2013
    Co-Authors: Huiquan Li, Cong Chen, Hangjun Wu, Jianmin Zhang, Xiaoming Li, Hao Wang, Shumin Duan
    Abstract:

    Brain disturbances, like injuries or aberrant protein deposits, evoke nucleotide release or leakage from cells, leading to microglial chemotaxis and ingestion. Recent studies have identified P2Y12 purinergic receptors as triggers for microglial chemotaxis and P2Y6 receptors as mediators for phagocytosis. However, Pinocytosis, known as the internalization of fluid-phase materials, has received much less attention. We found that ATP efficiently triggered Pinocytosis in microglia. Pharmacological analysis and knockdown experiments demonstrated the involvement of P2Y4 receptors and the phosphatidylinositol 3-kinase/Akt cascade in the nucleotide-induced Pinocytosis. Further evidence indicated that soluble amyloid beta peptide 1-42 induced self-uptake in microglia through Pinocytosis, a process involving activation of P2Y4 receptors by autocrine ATP signaling. Our results demonstrate a previously unknown function of ATP as a “drink me” signal for microglia and P2Y4 receptors as a potential therapeutic target for the treatment of Alzheimer's disease.

Verena Niggli - One of the best experts on this subject based on the ideXlab platform.