K012 Pharmacological blockade or genetic deletion of AT2R abolishes flow-induced structural remodeling in rat and mice resistance arteries in vivo

Gastric surface pH (pHo) transiently increases in response to focal epithelial damage. The sources of that increase, either from paracellular leakage of interstitial fluid or transcellular acid/base fluxes, have not been determined. Using in vivo microscopy approaches we measured pHowith Cl-NERF, tissue permeability with intravenous fluorescent-dextrans to label interstitial fluid (paracellular leakage), and gastric epithelial intracellular pH (pHi) with SNARF-5F (cellular acid/base fluxes). In response to two-photon photodamage, we found that cell-impermeant dyes entered damaged cells from luminal or tissue compartments, suggesting a possible slow transcellular, but not paracellular, route for increased permeability after damage. Regarding cytosolic acid/base status, we found that damaged cells acidified (6.63 ± 0.03) after photodamage, compared with healthy surface cells both near (7.12 ± 0.06) and far (7.07 ± 0.04) from damage ( P < 0.05). This damaged cell acidification was further attenuated with 20 μM intravenous EIPA (6.34 ± 0.05, P < 0.05) but unchanged by addition of 0.5 mM luminal H2DIDS (6.64 ± 0.08, P > 0.05). Raising luminal pH did not realkalinize damaged cells, suggesting that the mechanism of acidification is not attributable to leakiness to luminal protons. Inhibition of apical HCO3−secretion with 0.5 mM luminal H2DIDS or genetic deletion of the solute-like carrier 26A9 (SLC26A9) Cl−/HCO3−exchanger blocked the pHoincrease normally observed in control animals but did not compromise repair of damaged tissue. Addition of exogenous PGE2significantly increased pHoin wild-type, but not SLC26A9 knockout, animals, suggesting that prostaglandin-stimulated HCO3−secretion is fully mediated by SLC26A9. We conclude that cellular HCO3−secretion, likely through SLC26A9, is the dominant mechanism whereby surface pH transiently increases in response to photodamage.

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Neuronal pannexin-1 channels are not molecular routes of water influx during spreading depolarization-induced dendritic beading

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x16639328 ◽

2016 ◽

Vol 37 (5) ◽

pp. 1626-1633 ◽

Cited By ~ 7

Author(s):

Jeremy Sword ◽

Deborah Croom ◽

Phil L Wang ◽

Roger J Thompson ◽

Sergei A Kirov

Keyword(s):

Pyramidal Neurons ◽

Transport Mechanisms ◽

Coupled Transport ◽

Pannexin 1 ◽

Two Photon ◽

Spreading Depolarization ◽

Water Influx ◽

Membrane Bound ◽

Genetic Deletion

Spreading depolarization-induced focal dendritic swelling (beading) is an early hallmark of neuronal cytotoxic edema. Pyramidal neurons lack membrane-bound aquaporins posing a question of how water enters neurons during spreading depolarization. Recently, we have identified chloride-coupled transport mechanisms that can, at least in part, participate in dendritic beading. Yet transporter-mediated ion and water fluxes could be paralleled by water entry through additional pathways such as large-pore pannexin-1 channels opened by spreading depolarization. Using real-time in vivo two-photon imaging in mice with pharmacological inhibition or conditional genetic deletion of pannexin-1, we showed that pannexin-1 channels are not required for spreading depolarization-induced focal dendritic swelling.

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Enhanced resistance artery sensitivity to agonists under isobaric compared with isometric conditions

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1994.266.1.h147 ◽

1994 ◽

Vol 266 (1) ◽

pp. H147-H155 ◽

Cited By ~ 13

Author(s):

W. R. Dunn ◽

G. C. Wellman ◽

J. A. Bevan

Keyword(s):

Membrane Potential ◽

Angiotensin Ii ◽

Myogenic Tone ◽

Isometric Contractions ◽

Fundamental Interaction ◽

Resistance Artery ◽

Resistance Arteries ◽

Enhanced Resistance ◽

Increased Sensitivity

We have compared the responsiveness of rabbit mesenteric resistance arteries with agonists under isometric and isobaric conditions. When pressurized (60 mmHg), arteries spontaneously reduced their diameter by 18.1%. An equivalent isometric stress did not generate force in a “wire” myograph. Subsequently, much higher concentrations of norepinephrine (NE) and histamine were required to cause isometric contractions than were needed to reduce vascular diameter of pressurized vessels, whereas angiotensin II produced a maintained response only in pressurized arteries. Reducing transmural pressure to 20 mmHg abolished pressure-induced myogenic tone and decreased arterial sensitivity to NE. Under isometric conditions, partial depolarization with KCl increased sensitivity to NE and histamine to within the concentration range effective in pressurized vessels and also "revealed" responses to angiotensin II. The membrane potential of the vascular smooth muscle cells under partially depolarized conditions was similar to that found in vivo and in vessels studied isobarically. These observations demonstrate a fundamental interaction between pressure-induced myogenic tone and the sensitivity of resistance arteries to vasoactive stimuli. This influence was mimicked in isometrically mounted vessels by partial depolarization, indicating a possible pivotal role for membrane potential in determining the reactivity of the resistance vasculature.

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Aβ42 oligomers trigger synaptic loss through CAMKK2-AMPK-dependent effectors coordinating mitochondrial fission and mitophagy

10.1101/637199 ◽

2019 ◽

Cited By ~ 1

Author(s):

Annie Lee ◽

Chandana Kondapalli ◽

Daniel M. Virga ◽

Tommy L. Lewis ◽

So Yeon Koo ◽

...

Keyword(s):

Genome Engineering ◽

Pyramidal Neurons ◽

Es Cells ◽

Mitochondrial Fission ◽

Significant Loss ◽

Synaptic Loss ◽

Structural Remodeling ◽

Swedish Mutation ◽

Human Neurons

AbstractDuring the early stages of Alzheimer’s disease (AD) in both mouse models and human patients, soluble forms of Amyloid-β1-42 oligomers (Aβ42o) trigger loss of excitatory synapses (synaptotoxicity) in cortical and hippocampal pyramidal neurons (PNs) prior to the formation of insoluble Aβ plaques. We observed a spatially restricted structural remodeling of mitochondria in the apical tufts of CA1 PNs dendrites in the hAPPSWE,IND transgenic AD mouse model (J20), corresponding to the dendritic domain receiving presynaptic inputs from the entorhinal cortex and where the earliest synaptic loss is detected in vivo. We also observed significant loss of mitochondrial biomass in human neurons derived from a new model of human ES cells where CRISPR-Cas9-mediated genome engineering was used to introduce the ‘Swedish’ mutation bi-allelically (APPSWE/SWE). Recent work uncovered that Aβ42o mediates synaptic loss by over-activating the CAMKK2-AMPK kinase dyad, and that AMPK is a central regulator of mitochondria homeostasis in non-neuronal cells. Here, we demonstrate that Aβ42o-dependent over-activation of CAMKK2-AMPK mediates synaptic loss through coordinated MFF-dependent mitochondrial fission and ULK2-dependent mitophagy in dendrites of PNs. We also found that the ability of Aβ42o-dependent mitochondrial remodeling to trigger synaptic loss requires the ability of AMPK to phosphorylate Tau on Serine 262. Our results uncover a unifying stress-response pathway triggered by Aβo and causally linking structural remodeling of dendritic mitochondria to synaptic loss.

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Genetic Deletion or Pharmacological Blockade of Nociceptin/Orphanin FQ Receptors in the Ventral Tegmental Area Attenuates Nicotine‐Motivated Behaviour

British Journal of Pharmacology ◽

10.1111/bph.15762 ◽

2021 ◽

Author(s):

Ana Domi ◽

Veronica Lunerti ◽

Michele Petrella ◽

Esi Domi ◽

Anna Maria Borruto ◽

...

Keyword(s):

Ventral Tegmental Area ◽

Orphanin Fq ◽

Pharmacological Blockade ◽

Genetic Deletion ◽

Ventral Tegmental

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Genetic deletion of TRPA1 receptor attenuates amyloid beta- 1-42 (Aβ1-42)-induced neurotoxicity in the mouse basal forebrain in vivo

Mechanisms of Ageing and Development ◽

10.1016/j.mad.2020.111268 ◽

2020 ◽

Vol 189 ◽

pp. 111268

Author(s):

M. Payrits ◽

E. Borbely ◽

S. Godo ◽

D. Ernszt ◽

A. Kemeny ◽

...

Keyword(s):

Amyloid Beta ◽

Basal Forebrain ◽

Genetic Deletion ◽

Mouse Basal Forebrain

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Two Phosphoglucomutase Paralogs Facilitate Ionophore-Triggered Secretion of the Toxoplasma Micronemes

mSphere ◽

10.1128/msphere.00521-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 7

Author(s):

Sudeshna Saha ◽

Bradley I. Coleman ◽

Rashmi Dubey ◽

Ira J. Blader ◽

Marc-Jan Gubbels

Keyword(s):

Toxoplasma Gondii ◽

Phosphatidic Acid ◽

Host Cell ◽

Lytic Cycle ◽

Ionophore A23187 ◽

Host Cell Invasion ◽

Content Type ◽

Apicomplexan Parasites ◽

Genetic Deletion

ABSTRACT Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice. Paralogs of the widely prevalent phosphoglucomutase (PGM) protein called parafusin function in calcium (Ca2+)-mediated exocytosis across eukaryotes. In Toxoplasma gondii, the parafusin-related protein 1 (PRP1) has been associated with Ca2+-dependent microneme organelle secretion required for essential processes like host cell invasion and egress. Using reverse genetics, we observed PRP1 to be dispensable for completion of the lytic cycle, including host cell invasion and egress by the parasite. However, the absence of the gene affected increased microneme release triggered by A23187, a Ca2+ ionophore used to raise the cytoplasmic Ca2+ concentration mimicking the physiological role of Ca2+ during invasion and egress. The basal levels of constitutive microneme release in extracellular parasites and phosphatidic acid-triggered microneme secretion were unaffected in the mutant. The phenotype of the deletion mutant of the second PGM-encoding gene in Toxoplasma, PGM2, was similar to the phenotype of the PRP1 deletion mutant. Furthermore, the ability of the tachyzoites to induce acute infection in the mice remained normal in the absence of both PGM paralogs. Our data thus reveal that the microneme secretion upon high Ca2+ flux is facilitated by the Toxoplasma PGM paralogs, PRP1 and PGM2. However, this protein-mediated release is neither essential for lytic cycle completion nor for acute virulence of the parasite. IMPORTANCE Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice.

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IL1RAP potentiates multiple oncogenic signaling pathways in AML

Journal of Experimental Medicine ◽

10.1084/jem.20180147 ◽

2018 ◽

Vol 215 (6) ◽

pp. 1709-1727 ◽

Cited By ~ 18

Author(s):

Kelly Mitchell ◽

Laura Barreyro ◽

Tihomira I. Todorova ◽

Samuel J. Taylor ◽

Iléana Antony-Debré ◽

...

Keyword(s):

Tyrosine Kinases ◽

Interleukin 1 ◽

Oncogenic Signaling ◽

Genetic Deletion ◽

Mechanistic Basis ◽

Oncogenic Signaling Pathways ◽

Receptor Accessory Protein

The surface molecule interleukin-1 receptor accessory protein (IL1RAP) is consistently overexpressed across multiple genetic subtypes of acute myeloid leukemia (AML) and other myeloid malignancies, including at the stem cell level, and is emerging as a novel therapeutic target. However, the cell-intrinsic functions of IL1RAP in AML cells are largely unknown. Here, we show that targeting of IL1RAP via RNA interference, genetic deletion, or antibodies inhibits AML pathogenesis in vitro and in vivo, without perturbing healthy hematopoietic function or viability. Furthermore, we found that the role of IL1RAP is not restricted to the IL-1 receptor pathway, but that IL1RAP physically interacts with and mediates signaling and pro-proliferative effects through FLT3 and c-KIT, two receptor tyrosine kinases with known key roles in AML pathogenesis. Our study provides a new mechanistic basis for the efficacy of IL1RAP targeting in AML and reveals a novel role for this protein in the pathogenesis of the disease.

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Downregulation of miR-503 in Activated Kidney Fibroblasts Disinhibits KCNN4 in an in Vitro Model of Kidney Fibrosis

Kidney & Blood Pressure Research ◽

10.1159/000498875 ◽

2019 ◽

Vol 44 (1) ◽

pp. 113-122 ◽

Cited By ~ 2

Author(s):

Christoph Mann ◽

Brajesh Pratap Kaistha ◽

Michael Kacik ◽

Thorsten Stiewe ◽

Joachim Hoyer

Keyword(s):

Kidney Fibrosis ◽

Calcium Dependent ◽

Channel Current ◽

Channel Expression ◽

Pharmacological Blockade ◽

Vitro Model ◽

End Stage ◽

Activated Fibroblasts

Background/Aims: Activated fibroblasts are key controllers of extracellular matrix turnover in kidney fibrosis, the pathophysiological end stage of chronic kidney disease. The proliferation of activated fibroblasts depends on the expression of the calcium-dependent potassium channel KCNN4. Expression of this ion channel is upregulated in fibrotic kidneys. Genetic and pharmacological blockade of KCNN4 inhibits fibrosis in vitro and in vivo. Methods: We studied the regulation of KCNN4 and possible involvement of miRNAs in an in-vitro fibrosis model using murine kidney fibroblasts. We tested fibroblast proliferation, channel function, channel expression and expression regulation after FGF-2 stimulation. Results: Proliferation was significantly increased by FGF-2, channel current and expression were almost doubled (+ 91% and +125%, respectively). MiRNA microarray identified upregulation of miRNA-503, which targets RAF1 and thereby controls KCNN4-expression via disinhibition of the Ras/Raf/MEK/ ERK-cascade. Conclusion: This data show a) a profound upregulation of KCNN4 in stimulated fibroblast and b) identifies miR-503 as a regulator of KCNN4 expression.

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