scholarly journals Intracellular calcium and calcineurin regulate neutrophil motility on vitronectin through a receptor identified by antibodies to integrins alphav and beta3

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 2038-2048 ◽  
Author(s):  
B Hendey ◽  
M Lawson ◽  
EE Marcantonio ◽  
FR Maxfield
Keyword(s):  
Flow Cytometry ◽  
Intracellular Calcium ◽  
Alpha Subunit ◽  
Intact Cells ◽  
Cell Lysate ◽  
Calcium Calmodulin Dependent ◽  
Quantitative Immunofluorescence ◽  
Peptide Maps ◽  
Neutrophil Cell ◽  
Rule Out

Abstract Buffering of intracellular calcium ([Ca2+]i) or inhibition of the calcium/calmodulin-dependent phosphatase, calcineurin, results in neutrophils being unable to detach from vitronectin with a consequent loss of motility. Treatment of [Ca2+]i-buffered or calcineurin- inhibited neutrophils with monoclonal antibodies (MoAbs) to beta3 or alphav beta3 integrins allowed neutrophils to detach and restored motility. Quantitative immunofluorescence and flow cytometry showed that MoAbs specific for beta3, alphav, or alphav beta3 integrins bind to neutrophils. Immunolocalization studies using antibodies to the highly conserved cytoplasmic domains of alphav and beta3 also identified the receptor on neutrophils. Whereas antibodies to alphav, alphav beta3, and beta3 recognized the receptor in intact cells, only the beta3 MoAb immunoprecipitated the receptor from a neutrophil cell lysate. The alpha subunit co-immunoprecipitated by the beta3 antibody reacted with an antibody to alphav by Western blot. Peptide maps of V8 protease digests showed a strong similarity in alpha and beta chains precipitated by antibodies to beta3 from neutrophils and endothelial cells. These results indicate that [Ca2+]i and calcineurin regulate neutrophil motility on vitronectin through an alphav beta3-like receptor. Although we cannot rule out the possibility that neutrophils have an isoform of alphav, such an isoform would have to be similar enough to react with alphav- and alphav beta3-specific MoAbs in intact cells.

Intracellular calcium and calcineurin regulate neutrophil motility on vitronectin through a receptor identified by antibodies to integrins alphav and beta3

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 2038-2048 ◽  
Author(s):  
B Hendey ◽  
M Lawson ◽  
EE Marcantonio ◽  
FR Maxfield
Keyword(s):  
Flow Cytometry ◽  
Intracellular Calcium ◽  
Alpha Subunit ◽  
Intact Cells ◽  
Cell Lysate ◽  
Calcium Calmodulin Dependent ◽  
Quantitative Immunofluorescence ◽  
Peptide Maps ◽  
Neutrophil Cell ◽  
Rule Out

Buffering of intracellular calcium ([Ca2+]i) or inhibition of the calcium/calmodulin-dependent phosphatase, calcineurin, results in neutrophils being unable to detach from vitronectin with a consequent loss of motility. Treatment of [Ca2+]i-buffered or calcineurin- inhibited neutrophils with monoclonal antibodies (MoAbs) to beta3 or alphav beta3 integrins allowed neutrophils to detach and restored motility. Quantitative immunofluorescence and flow cytometry showed that MoAbs specific for beta3, alphav, or alphav beta3 integrins bind to neutrophils. Immunolocalization studies using antibodies to the highly conserved cytoplasmic domains of alphav and beta3 also identified the receptor on neutrophils. Whereas antibodies to alphav, alphav beta3, and beta3 recognized the receptor in intact cells, only the beta3 MoAb immunoprecipitated the receptor from a neutrophil cell lysate. The alpha subunit co-immunoprecipitated by the beta3 antibody reacted with an antibody to alphav by Western blot. Peptide maps of V8 protease digests showed a strong similarity in alpha and beta chains precipitated by antibodies to beta3 from neutrophils and endothelial cells. These results indicate that [Ca2+]i and calcineurin regulate neutrophil motility on vitronectin through an alphav beta3-like receptor. Although we cannot rule out the possibility that neutrophils have an isoform of alphav, such an isoform would have to be similar enough to react with alphav- and alphav beta3-specific MoAbs in intact cells.

The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface

1997 ◽  
Vol 110 (6) ◽  
pp. 687-694 ◽  
Author(s):  
M. Steegmaier ◽  
E. Borges ◽  
J. Berger ◽  
H. Schwarz ◽  
D. Vestweber
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Surface Proteins ◽  
Intact Cells ◽  
Fgf Receptor ◽  
Cell Surface Biotinylation ◽  
Cell Surface Staining ◽  
Surface Staining ◽  
Selectin Ligand ◽  
Golgi Protein

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.

Stimulation of oxygen uptake by glucagon is oxygen dependent in perfused rat liver

1989 ◽  
Vol 256 (2) ◽  
pp. G369-G376
Author(s):  
Z. Kizaki ◽  
R. G. Thurman
Keyword(s):  
Intracellular Calcium ◽  
High Flow ◽  
Perfused Liver ◽  
Normal Flow ◽  
Sprague Dawley ◽  
O2 Uptake ◽  
Intact Cells ◽  
Flow Rates ◽  
Isolated Mitochondria ◽  
Stimulation Of

Livers from well-fed female Sprague-Dawley rats (100-150 g) were perfused at flow rates of 4 or 8 ml.g liver-1.min-1 to deliver O2 to the organ at various rates. During perfusion at normal flow rates (4 ml.g-1.min-1), glucagon (10 nM) increased O2 uptake in perfused liver by approximately 40 mumol.g-1.h-1. In contrast, glucagon increased O2 uptake by nearly 100 mumol.g-1.h-1 when livers were perfused at high flow rates. Increase in O2 uptake was directly proportional to flow rate and was blocked partially by infusion of phorbol myristate acetate (100 nM) before glucagon. Increase in O2 uptake due to elevated flow was not due to enhanced glucagon delivery, since infusion of 120 nM glucagon at normal flow rates only increased O2 uptake by approximately 40 mumol.g-1.h-1. On the other hand, when O2 tension in the perfusate was manipulated at normal flow rates, the stimulation of O2 uptake by glucagon increased proportional to the average O2 tension in the liver. Infusion of 8-bromo-adenosine 3',5'-cyclic monophosphate (BrcAMP; 25 microM) also increased O2 uptake more than twice as much at high compared with normal flow rates. In the presence of angiotensin II (5 nM), a hormone that increases intracellular calcium, glucagon increased O2 uptake by nearly 100 mumol.g-1.h-1 at normal flow rates. Infusion of glucagon or BrcAMP into livers perfused at normal flow rates increased state 3 rates of O2 uptake of subsequently isolated mitochondria significantly by approximately 25%. In contrast, perfusion with glucagon or BrcAMP at high flow rates increased mitochondrial respiration by 50-60%. Glucagon addition acutely to suspensions of mitochondria, however, had no effect on O2 uptake. These data are consistent with reports that glucagon administration in vivo or treatment of intact cells with glucagon increases O2 uptake of subsequently isolated mitochondria, a phenomenon that can account for the observed increase in O2 uptake in livers perfused at high flow rates with glucagon. Furthermore, these results are consistent with the hypothesis that the effect of glucagon on mitochondria is O2 dependent in the perfused liver. This is most likely due to an effect of intracellular calcium on a mechanism mediated via cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals PB1686 USE OF FLOW CYTOMETRY AND IMMUNOHISTOCHEMISTRY TO RULE OUT CNS RELAPSE OF ACUTE LYMPHOBLASTIC LEUKEMIA

HemaSphere ◽  
2019 ◽  
Vol 3 (S1) ◽  
pp. 777 ◽  
Author(s):  
M. Moschovi ◽  
C. Kelaidi ◽  
A. Zampogiannis ◽  
N. Tourkantoni ◽  
M. Tzanoudaki ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Cns Relapse ◽  
Rule Out

scholarly journals Inhibition of Calcium/Calmodulin-Dependent Protein Kinase Kinase β Is Detrimental in Hypoxia–Ischemia Neonatal Brain Injury

2019 ◽  
Vol 20 (9) ◽  
pp. 2063
Author(s):  
Jia-Wei Min ◽  
Fan Bu ◽  
Li Qi ◽  
Yashasvee Munshi ◽  
Gab Seok Kim ◽  
...  
Keyword(s):  
Brain Injury ◽  
Protein Kinase ◽  
Intracellular Calcium ◽  
Infarct Volume ◽  
Dependent Protein Kinase ◽  
Neonatal Hypoxia ◽  
Calcium Calmodulin Dependent ◽  
Hypoxia Ischemia ◽  
Dependent Protein ◽  
Protein Kinase Kinase

Neonatal hypoxia–ischemia (HI) is a major cause of death and disability in neonates. HI leads to a dramatic rise in intracellular calcium levels, which was originally thought to be detrimental to the brain. However, it has been increasingly recognized that this calcium signaling may also play an important protective role after injury by triggering endogenous neuroprotective pathways. Calcium/calmodulin-dependent protein kinase kinase β (CaMKK β) is a major kinase activated by elevated levels of intracellular calcium. Here we evaluated the functional role of CaMKK β in neonatal mice after HI in both acute and chronic survival experiments. Postnatal day ten wild-type (WT) and CaMKK β knockout (KO) mouse male pups were subjected to unilateral carotid artery ligation, followed by 40 min of hypoxia (10% O2 in N2). STO-609, a CaMKK inhibitor, was administered intraperitoneally to WT mice at 5 minutes after HI. TTC (2,3,5-triphenyltetrazolium chloride monohydrate) staining was used to assess infarct volume 24 h after HI. CaMKK β KO mice had larger infarct volume than WT mice and STO-609 increased the infarct volume in WT mice after HI. In chronic survival experiments, WT mice treated with STO-609 showed increased tissue loss in the ipsilateral hemisphere three weeks after HI. Furthermore, when compared with vehicle-treated mice, they showed poorer functional recovery during the three week survival period, as measured by the wire hang test and corner test. Loss of blood–brain barrier proteins, a reduction in survival protein (Bcl-2), and an increase in pro-apoptotic protein Bax were also seen after HI with CaMKK β inhibition. In conclusion, inhibition of CaMKK β exacerbated neonatal hypoxia–ischemia injury in mice. Our data suggests that enhancing CaMKK signaling could be a potential target for the treatment of hypoxic–ischemic brain injury.

Alpha-subunit of calcium/calmodulin-dependent protein kinase II enhances gamma-aminobutyric acid and inhibitory synaptic responses of rat neurons in vitro

1995 ◽  
Vol 73 (5) ◽  
pp. 2099-2106 ◽  
Author(s):  
R. A. Wang ◽  
G. Cheng ◽  
M. Kolaj ◽  
M. Randic
Keyword(s):  
Protein Kinase ◽  
Gabaa Receptor ◽  
Aminobutyric Acid ◽  
Alpha Subunit ◽  
Gamma Aminobutyric Acid ◽  
Patch Clamp Technique ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein

1. Here we report that in acutely isolated rat spinal dorsal horn neurons, the gamma-aminobutyric acid-A (GABAA) receptor can be regulated by calcium/calmodulin-dependent protein kinase II (CaM-KII). Intracellularly applied, the alpha-subunit of CaM-KII enhanced GABAA-receptor-activated current recorded with the use of the whole cell patch-clamp technique. This effect was associated with reduced desensitization of GABA responses. 2. GABA-induced currents are also potentiated by calyculin A, an inhibitor of protein phosphatases 1 and 2A. 3. Conventional intracellular recordings were made from hippocampal CA1 neurons in slices to determine the effect of intracellular application of CaM-KII on inhibitory synaptic potentials evoked by electrical stimulation of the stratum oriens/alveus. The inhibitory synaptic potential was enhanced by CaM-KII; this mechanism may contribute to long-term enhancement of inhibitory synaptic transmission and may also play a role in other forms of plasticity in the mammalian brain.

Dual regulation of PTH-stimulated adenylate cyclase activity by GTP

1986 ◽  
Vol 251 (5) ◽  
pp. F858-F864 ◽  
Author(s):  
A. P. Teitelbaum ◽  
R. A. Nissenson ◽  
L. A. Zitzner ◽  
K. Simon
Keyword(s):  
Adenylate Cyclase ◽  
Pertussis Toxin ◽  
Regulatory Protein ◽  
Inhibitory Effect ◽  
Alpha Subunit ◽  
Kidney Cells ◽  
Intact Cells ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Nucleotide Regulation

Guanyl nucleotide regulation of parathyroid hormone (PTH)-activated adenylate cyclase was studied in membrane preparations of cultured opossum kidney cells. Guanosine triphosphate (GTP) (100 microM) decreased PTH-stimulated activity by 70%. Pertussis toxin enhanced PTH stimulation in intact cells and membranes, completely blocked the inhibitory effect of GTP, and catalyzed the [32P]ADP-ribosylation of a 38,000-dalton protein migrating in the position of the alpha-subunit of the inhibitory GTP-regulatory protein Ni. Cholera toxin was used to identify the alpha-subunit of the stimulatory GTP-binding protein Ns, a 42,000-dalton protein. We tested the idea that Ni may be involved in mediating the reduced response of opossum kidney cells to PTH after pretreatment with the hormone (desensitization). GTP inhibited PTH-stimulated activity to approximately the same degree in membranes from PTH-pretreated cells and control cells whether or not the cells had also received pertussis toxin. We conclude that GTP inhibits PTH action in opossum kidney cells through Ni but that PTH-induced desensitization is not mediated by Ni.

Cytosolic free Calcium and Intracellular Calcium Stores in Neutrophils from Hypertensive Subjects

1985 ◽  
Vol 69 (2) ◽  
pp. 227-230 ◽  
Author(s):  
P. Daniel Lew ◽  
Laurent Favre ◽  
Francis A. Waldvogel ◽  
Michel B. Vallotton
Keyword(s):  
Essential Hypertension ◽  
Intracellular Calcium ◽  
Calcium Ionophore ◽  
Calcium Handling ◽  
Free Calcium ◽  
Calcium Stores ◽  
Cytosolic Free Calcium ◽  
Intact Cells ◽  
Intracellular Calcium Stores ◽  
Calcium Indicator

1. Alterations in intracellular calcium have been implicated in the pathogenesis of essential hypertension. To see whether this is a generalized phenomenon we assessed cytosolic free calcium and intracellular calcium stores in neutrophils from normo- and hyper-tensive subjects, by trapping the fluorescent calcium indicator quin2 in intact cells. 2. Ten patients with untreated essential hypertension were compared with 10 age- and sex-matched normotensive subjects. The levels of cytosolic free calcium and intracellular calcium stores releasable by the calcium ionophore ionomycin did not differ. No significant relationship was found between blood pressure and the calcium parameters in all 20 subjects studied. 3. The results indicate that essential hypertension is not associated with a membrane defect in calcium handling of all human cell systems, leading to generalized increases in resting values of cytosolic free calcium. 4. Neutrophils do not appear to be a good model for intracellular calcium handling in vascular smooth muscle.

“Tasting the Microenvironment”: Human Bone Marrow Stromal Cells Have Microenvironment Chemosensory Capacity Via Taste Receptor Expression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 938-938
Author(s):  
Troy Lund ◽  
Diane Lidke ◽  
Amanda Kobs ◽  
Bruce R. Blazar ◽  
Jakub Tolar
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Intracellular Calcium ◽  
Environmental Changes ◽  
Bitter Taste ◽  
Taste Receptor ◽  
Receptor Expression ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Bitter Taste Receptor

Abstract Abstract 938 The ability of cells to detect molecules in the microenvironment is important in cell signaling and cell responsiveness to environmental changes. Through an isobaric tag for relative and absolute quantitation (iTRAQ) based proteomic screen of human bone marrow stroma-derived cells (MSC) at the third passage we identified the novel expression of a bitter taste receptor, TAS2R46 in undifferentiated MSC and MSC differentiated into osteocytes, adipocytes, and cartilage. The relative amount of the receptor by iTRAQ and qRT-PCR was equivalent in all cell types. TAS2R46 expression was verified by flow cytometry, immunohistochemistry, and RT-PCR. Expression of TAS2R46 was also found in freshly obtained bone marrow mononuclear cells, and sorting by flow cytometry showed that the taste receptor positive cells had the ability to become MSC in vitro, while significantly fewer cells became MSC in the taste receptor negative/low fraction. Other members of the bitter taste receptor family including TAS2R4, TAS2R5, TAS2R1, TAS2R38 were negative by flow cytometry and iTRAQ. Bitter compounds, of which the prototypical molecule is denatonium, were found to increase intracellular human MSC calcium levels in a dose-responsive manner up to a level of 200% above baseline in fluorescent intracellular calcium detection assays. Other bitter compounds tested were caffeine, thujone, and salicin, and quinine gave rise to increased intracellular calcium levels. The specificity of the calcium response was verified through transgenic overexpression experiments, antibody inhibition, and a novel newly developed direct labeling method in which we were to directly label denatonium and show binding to the cell surface. Evaluation of downstream signaling events showed that interaction with this receptor caused a decrease in cAMP levels of 40% with exposure to 3 mM denatonium. Finally, to reveal the potential that this receptor may have, a native physiologically relevant function related to small peptide binding, a casein hydrosylate was used as a substrate. Remarkably, casein hydrosylate caused a similar increase in intracellular calcium as denatonium. Furthermore, this effect was augmented when TAS2R46 was overexpressed as a transgene. Current studies are working towards revealing the specific peptide that binds to this receptor. This is the first description of chemosensory detection by MSC. Our data show that this environmental detection occurs through a novel expression of a bitter taste receptor, TAS2R46. This ability may allow MSC to sample changes in their microenvironment triggered by small molecules, which could include either toxins or hormones as true natural ligands of this receptor class. The expression of bitter taste receptors may influence the response of MSCs to noxious materials or pathological insults to the organism that are detected as bitter substrates. Future studies will examine the directed mobility of MSCs to such stimuli which may offer new insights as to how MSCs sense organism injury. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document