Lysophosphatidic acid opens a Ca++ channel in human erythrocytes

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2420-2425
Author(s):  
Lu Yang ◽  
Dina A. Andrews ◽  
Philip S. Low
Keyword(s):  
Lysophosphatidic Acid ◽  
Kinase Inhibitors ◽  
Membrane Integrity ◽  
Protein Kinase Inhibitors ◽  
Human Erythrocytes ◽  
Channel Blockers ◽  
Ca Channel ◽  
Active Population ◽  
Fresh Rbcs ◽  
The Impact

Lysophosphatidic acid (LPA) is a lipid-derived second messenger that mobilizes many cells of the circulatory and vascular systems to assist in thrombus development and wound healing. LPA, however, has not been tested on human erythrocytes, largely because erythrocytes are considered to be both biologically inert and inactive in intercellular communication. To test this presumption, we have examined the impact of LPA on signaling reactions within the human red blood cell (RBC). Using both 45Ca++ and a Ca++-sensitive fluorescent probe (Fluo-3), we demonstrated that LPA, but not phosphatidic acid or the closely related sphingosine-1–phosphate, stimulates the influx of micromolar quantities of extracellular Ca++ into fresh RBCs. This Ca++ influx was shown to be channel mediated rather than leak promoted because the influx was observed at LPA concentrations too low to perturb membrane integrity, it was inhibited by P-type but not L-type Ca++ channel blockers, it was inhibited by broad-specificity protein kinase inhibitors, and it was not induced by inactive analogues of LPA. Further characterization reveals that only approximately 25% of the RBCs participate in LPA-induced Ca++ entry and that within this active population, Ca++ gating occurs in an all-or-nothing manner. Because the stimulation of Ca++ uptake occurs at LPA concentrations (1-5 μmol/L) known to occur near a developing thrombus and because the internalized Ca++can potentially promote prothrombic properties in the stimulated RBCs, we conclude that RBCs are not insensitive to signals released from other cells.

Lysophosphatidic acid opens a Ca++ channel in human erythrocytes

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2420-2425 ◽  
Author(s):  
Lu Yang ◽  
Dina A. Andrews ◽  
Philip S. Low
Keyword(s):  
Lysophosphatidic Acid ◽  
Kinase Inhibitors ◽  
Membrane Integrity ◽  
Protein Kinase Inhibitors ◽  
Human Erythrocytes ◽  
Channel Blockers ◽  
Ca Channel ◽  
Active Population ◽  
Fresh Rbcs ◽  
The Impact

Abstract Lysophosphatidic acid (LPA) is a lipid-derived second messenger that mobilizes many cells of the circulatory and vascular systems to assist in thrombus development and wound healing. LPA, however, has not been tested on human erythrocytes, largely because erythrocytes are considered to be both biologically inert and inactive in intercellular communication. To test this presumption, we have examined the impact of LPA on signaling reactions within the human red blood cell (RBC). Using both 45Ca++ and a Ca++-sensitive fluorescent probe (Fluo-3), we demonstrated that LPA, but not phosphatidic acid or the closely related sphingosine-1–phosphate, stimulates the influx of micromolar quantities of extracellular Ca++ into fresh RBCs. This Ca++ influx was shown to be channel mediated rather than leak promoted because the influx was observed at LPA concentrations too low to perturb membrane integrity, it was inhibited by P-type but not L-type Ca++ channel blockers, it was inhibited by broad-specificity protein kinase inhibitors, and it was not induced by inactive analogues of LPA. Further characterization reveals that only approximately 25% of the RBCs participate in LPA-induced Ca++ entry and that within this active population, Ca++ gating occurs in an all-or-nothing manner. Because the stimulation of Ca++ uptake occurs at LPA concentrations (1-5 μmol/L) known to occur near a developing thrombus and because the internalized Ca++can potentially promote prothrombic properties in the stimulated RBCs, we conclude that RBCs are not insensitive to signals released from other cells.

scholarly journals Small-Molecule Protein Kinases Inhibitors and the Risk of Fungal Infections

2019 ◽  
Vol 13 (4) ◽  
pp. 229-243 ◽  
Author(s):  
Katie Bechman ◽  
James B Galloway ◽  
Kevin L Winthrop
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Fungal Infections ◽  
Break Point ◽  
Pharmacokinetic Profile ◽  
Protein Kinase Inhibitors ◽  
Janus Kinase ◽  
The Impact

Abstract Purpose of Review This review discusses fungal infections associated with licenced small-molecule protein kinase inhibitors. For each major drug class, the mechanism of action and targeted pathways and the impact on host defence against fungi are described. Recent Findings Protein kinase inhibitors are successfully used in the treatment of malignancies and immune-mediated diseases, targeting signalling pathways for a broad spectrum of cytokines and growth-stimuli. These agents predispose to fungal infections by the suppression of integral components of the adaptive and innate immune response. Summary The greatest risk of fungal infections is seen with bruton tyrosine kinase inhibitors, e.g. ibrutinib. Infections are also reported with agents that target mTOR, Janus kinase and break point cluster (Bcr) gene–Abelson (Abl) tyrosine kinase (BCR-ABL). The type of fungal infection fits mechanistically with the specific pathway targeted. Infections are often disseminated and present soon after the initiation of therapy. The pharmacokinetic profile, possibility of off-target kinase inhibition, and underlying disease pathology contribute to infection risk.

Release of dopamine and norepinephrine by hypoxia from PC-12 cells

1998 ◽  
Vol 274 (6) ◽  
pp. C1592-C1600 ◽  
Author(s):  
Ganesh K. Kumar ◽  
Jeffrey L. Overholt ◽  
Gary R. Bright ◽  
Kwong Y. Hui ◽  
Hongwen Lu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Culture Conditions ◽  
Protein Kinase Inhibitors ◽  
Channel Blockers ◽  
Pc 12 Cells ◽  
Stimulus Secretion Coupling ◽  
Carbon Fiber Electrode ◽  
Da Release

We examined the effects of hypoxia on the release of dopamine (DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cells and assessed the involvement of Ca2+ and protein kinases in stimulus-secretion coupling. Catecholamine release was monitored by microvoltammetry using a carbon fiber electrode as well as by HPLC coupled with electrochemical detection (ECD). Microvoltammetric analysis showed that hypoxia-induced catecholamine secretion (Po 2 of medium ∼40 mmHg) occurred within 1 min after the onset of the stimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysis revealed that, at any level of Po 2, the release of NE was greater than the release of DA. In contrast, in response to K+ (80 mM), DA release was ∼11-fold greater than NE release. The magnitude of hypoxia-induced NE and DA releases depended on the passage, source, and culture conditions of the PC-12 cells. Omission of extracellular Ca2+ or addition of voltage-gated Ca2+ channel blockers attenuated hypoxia-induced release of both DA and NE to a similar extent. Protein kinase inhibitors, staurosporine (200 nM) and bisindolylmaleimide I (2 μM), on the other hand, attenuated hypoxia-induced NE release more than DA release. However, protein kinase inhibitors had no significant effect on K+-induced NE and DA releases. These results demonstrate that hypoxia releases catecholamines from PC-12 cells and that, for a given change in Po 2, NE release is greater than DA release. It is suggested that protein kinases are involved in the enhanced release of NE during hypoxia.

Nifedipine inhibits calcium-activated K transport in human erythrocytes

1990 ◽  
Vol 259 (2) ◽  
pp. C332-C339 ◽  
Author(s):  
D. M. Kaji
Keyword(s):  
Human Erythrocytes ◽  
Channel Blockers ◽  
Ca Channel ◽  
Zero Current ◽  
Ca Channel Blockers ◽  
Free Media ◽  
Drug Free ◽  
Dissociation Constant Kd ◽  
K Transport ◽  
External K

The effect of nifedipine on K transport across human erythrocytes was investigated. Nifedipine had no effect on K influx mediated by the Na-K pump, Na-K-2Cl cotransport, or the passive residual K flux. However, it inhibited the K and water loss from ATP-depleted cells in the presence of external Ca (Cao). Similar inhibition of Ca-activated K [K(Ca)] efflux was observed in fresh cells exposed to Cao and A23187 or ionomycin. The inhibition was observed even when nifedipine was added after initiation of the K(Ca) efflux and was not readily reversed by washing cells with drug-free media. When K(Ca) efflux was plotted as a function of external free Ca, nifedipine reduced the maximum K(Ca) efflux but had no effect on the Ca concentration required for half-maximum K(Ca) efflux. The inhibition of K(Ca) efflux by nifedipine was not consequent to its effect on conductive Cl permeability, because valinomycin-induced K efflux in Cl media was enhanced rather than reduced by nifedipine and because the inhibition was also seen with SCN, a nonlimiting anion. Nifedipine inhibited the K(Ca) efflux with a dissociation constant (Kd) of 4 microM. The inhibitory capacity of nifedipine was reduced by increasing external K. Nifedipine reduced not only the basic conductance but also the zero-current K conductance with a Kd of 23 microM. Other Ca-channel blockers, such as verapamil and diltiazem, did not inhibit K(Ca) efflux, but other dihydropyridines, including BAY K 8644, a Ca-channel agonist, were effective in inhibiting K(Ca) efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Synthesis of 5-(ethylsulfonyl)-2-methoxyaniline: An important pharmacological fragment of VEGFR2 and other inhibitors

2013 ◽  
Vol 9 ◽  
pp. 173-179 ◽  
Author(s):  
Miroslav Murár ◽  
Gabriela Addová ◽  
Andrej Boháč
Keyword(s):  
Kinase Inhibitors ◽  
Biological Activities ◽  
Sulfonyl Chloride ◽  
Protein Kinase Inhibitors ◽  
Channel Blockers ◽  
Cardiovascular Agents ◽  
Ion Channel Blockers ◽  
Convenient Synthesis ◽  
Antiangiogenic Drugs ◽  
Synthetic Intermediates

Background: 5-(Ethylsulfonyl)-2-methoxyaniline (5) is part of the structure in 131 compounds possessing different biological activities. In most cases, they have antitumor properties (112 compounds). Other compounds are described as cardiovascular agents, ion-channel blockers, nervous-system blockers, anti-inflammatory agents, or antidiabetic, antiosteoporotic and hypolipemic species. Compound 5 is a precursor of different protein-kinase inhibitors or enzyme modulators (EGFR, PDGFR, ckit, CDK 2 and 4, MMPs 2, 3, 9 and 13, etc.). The structure of 5 represents a fragment for several powerful inhibitors of VEGFR2, a key angiogenic receptor. Antiangiogenic inhibitors slow down or stop new blood-vessel formation from pre-existing vasculature. Some antiangiogenic drugs inhibiting the VEGFR2 receptor are successfully used in clinics for the treatment of several types of tumours in synergy with chemotherapy (e.g., Nexavar® from Bayer, Sutent® from Pfizer and Votrient® from GlaxoSmithKline, approved by the FDA in 2005, 2006 and 2009, respectively). The structure of 5 is an important pharmacophoric fragment of potent VEGFR2 inhibitors (e.g., AAZ from PDB complex 1Y6A, enzymatic IC50 = 22 nM). Up to now, 25 VEGFR2 inhibitors possessing a fragment of 5 can be found in the literature. Despite the high significance of 5-(ethylsulfonyl)-2-methoxyaniline (5) its preparation has not yet been described. Results: Here we have developed a convenient synthesis of important polyheterosubstituted aniline 5 starting from commercially available 4-methoxybenzene-1-sulfonyl chloride (1) in four steps and 59% overall yield. The target 5-(ethylsulfonyl)-2-methoxyaniline (5) and its synthetic intermediates 2–4 together with a new compound 5-(ethylsulfonyl)-2-methoxy-1,3-dinitrobenzene (4a) have been precisely physicochemically characterised.

L-type calcium channel α-subunit and protein kinase inhibitors modulate Rem-mediated regulation of current

2006 ◽  
Vol 291 (4) ◽  
pp. H1959-H1971 ◽  
Author(s):  
Shawn M. Crump ◽  
Robert N. Correll ◽  
Elizabeth A. Schroder ◽  
William C. Lester ◽  
Brian S. Finlin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Small Gtpases ◽  
Protein Kinase Inhibitors ◽  
Ca Channel ◽  
Α Subunit ◽  
Hek 293 ◽  
Channel Current ◽  
Channel Inhibition ◽  
Ca Channel Current

Cardiac voltage-gated L-type Ca channels (CaV) are multiprotein complexes, including accessory subunits such as CaVβ2 that increase current expression. Recently, members of the Rad and Gem/Kir-related family of small GTPases have been shown to decrease current, although the mechanism remains poorly defined. In this study, we evaluated the contribution of the L-type Ca channel α-subunit (CaV1.2) to CaVβ2-Rem inhibition of Ca channel current. Specifically, we addressed whether protein kinase A (PKA) modulation of the Ca channel modifies CaVβ2-Rem inhibition of Ca channel current. We first tested the effect of Rem on CaV1.2 in human embryonic kidney 293 (HEK-293) cells using the whole cell patch-clamp configuration. Rem coexpression with CaV1.2 reduces Ba current expression under basal conditions, and CaVβ2a coexpression enhances Rem block of CaV1.2 current. Surprisingly, PKA inhibition by 133 nM H-89 or 50 μM Rp-cAMP-S partially relieved the Rem-mediated inhibition of current activity both with and without CaVβ2a. To test whether the H-89 action was a consequence of the phosphorylation status of CaV1.2, we examined Rem regulation of the PKA-insensitive CaV1.2 serine 1928 (S1928) to alanine mutation (CaV1.2-S1928A). CaV1.2-S1928A current was not inhibited by Rem and when coexpression with CaVβ2a was not completely blocked by Rem coexpression, suggesting that the phosphorylation of S1928 contributes to Rem-mediated Ca channel modulation. As a model for native Ca channel complexes, we tested the ability of Rem overexpression in HIT-T15 cells and embryonic ventricular myocytes to interfere with native current. We find that native current is also sensitive to Rem block and that H-89 pretreatment relieves the ability of Rem to regulate Ca current. We conclude that Rem is capable of regulating L-type current, that release of Rem block is modulated by cellular kinase pathways, and that the CaV1.2 COOH terminus contributes to Rem-dependent channel inhibition.

STAT5 and NF-κB Induced Oncogenic Mir-155 Directly Targets PU.1 in FLT3-ITD Associated AML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3515-3515
Author(s):  
Dennis Gerloff ◽  
Rebekka Grundler ◽  
Daniela Braeuer-Hartmann ◽  
Christiane Katzerke ◽  
Jens-Uwe Hartmann ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
In Silico Analysis ◽  
Protein Kinase Inhibitors ◽  
Luciferase Assay ◽  
U937 Cells ◽  
Differentiation Block ◽  
The Impact ◽  
Acute Myeloid

Abstract Abstract 3515 Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane of FLT3. This mutation leads to constitutive activated FLT3 signalling, which also includes altered FLT3 targets like STAT5. The dysregulation of pathways causes a differentiation block and plays a role in inhibition of hematopoietic transcription factors like PU.1 and C/EBPalpha. MicroRNA (miRNA) expression is also targeted by FLT3-ITD signaling. MiRNAs are small (19 – 22 nt) non coding RNAs, which regulate protein expression by binding to the 3'UTR of their target mRNAs. MicroRNA-155 (miR-155) was found to be highly expressed in FLT3-ITD associated AML patient samples. Thus, we hypothesized that miR-155 is an oncomiR which is regulated by FLT3-ITD signalling. Here we report that FLT3-ITD signaling induces the oncogenic miR-155 by NF-κB and STAT5 pathways. Furthermore we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Analysis of FLT3-ITD positive patient samples show an approximately 2 fold higher miR-155 expression compared to FLT3-WT or FLT3-TKD associated AMLs. In addition we compared the miR-155 expression between 32D cells stably expressing either FLT3-WT or FLT3-ITD. The data show, that miR-155 expression is approximately 10 fold higher in the FLT3-ITD expressing 32D cells. Besides, the overexpression of FLT3-ITD in myeloid U937 cells increases miR-155 expression 2,2 fold. In contrast, a block of FLT3-ITD signalling in FLT3-ITD associated cell line MV4;11 by protein kinase inhibitors (PKIs), PKC412, SU5614 and CEP701, results in an 80% decreased miR-155 expression. In further experiments, we analyzed the role of FLT3-ITD downstream targets STAT5 and NF-κB (p65) in miR-155 regulation. We show that siRNA mediated knockdown of STAT5 or NF-κB (p65) in MV4;11 cells correlates with reduced miR-155 expression. A knockdown of NF-κB (p65) and STAT5 is able to overcome the FLT3-ITD overexpression induced miR-155 expression in U937 cells. To investigate the STAT5 impact on miR-155 expression in FLT3-ITD associated AML in vivo, we analyzed the miR-155 expression of irradiated C57BL/6 mice, transplanted with bone marrow from MX-Cre STAT5flox/flox mice stably expressing FLT3-ITD, after induced STAT5 knockout. The expression of miR-155 was significantly decreased to 0,4 fold in the STAT5−/− mice compared to STAT5flox/flox. These data reveal that STAT5 plays a pivotal role in FLT3-ITD induced miR-155 expression. In addition we demonstrate that p65 binds to the miR-155 promoter in MV4;11 cells while the treatment with the PKI CEP701 results in a p65 dissociation from the miR-155 promoter. Furthermore, we prove that miR-155 overexpression in PMA (phorbol 12-myristate 13-acetate) treated U937 cells reduces macrophage differentiation about 50%. In in silico analysis we found PU.1 as a putative miR-155 target. Interestingly, we could show that overexpression of FLT3-ITD as well as overexpression of miR-155 reduces the PU.1 protein level in U937 cells to 0.5 fold. However, a block of miR-155 by locked nucleic acids (LNAs) in MV4;11 cells, leads to PU.1 protein increase. Further, by luciferase assay analyses we prove that miR-155 directly targets PU.1 3'UTR. Altogether we conclude that FLT3-ITD induces the oncogenic miR-155 via STAT5 and NF-κB (p65). The increased miR-155 expression targets PU.1 and enhances the myeloproliferative disorder in acute myeloid leukemia blasts. These data reveal the impact of deregulated miRNAs in cancer development and may provide novel biomarkers and therapeutic targets in leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Impact of Selected Small-Molecule Kinase Inhibitors on Lipid Membranes

2021 ◽  
Vol 14 (8) ◽  
pp. 746
Author(s):  
Meike Luck ◽  
Markus Fischer ◽  
Maximilian Werle ◽  
Holger A. Scheidt ◽  
Peter Müller
Keyword(s):  
Small Molecule ◽  
Lipid Membranes ◽  
Kinase Inhibitors ◽  
Lipid Membrane ◽  
Membrane Integrity ◽  
Experimental Studies ◽  
Lipid Vesicles ◽  
Protein Kinase Inhibitors ◽  
Log P ◽  
P Value

Small-molecule protein kinase inhibitors are used for the treatment of various diseases. Although their effect(s) on the respective kinase are generally quite well understood, surprisingly, their interaction with membranes is only barely investigated; even though these drugs necessarily come into contact with the plasma and intracellular membranes. Using biophysical methods such as NMR, ESR, and fluorescence spectroscopy in combination with lipid vesicles, we studied the membrane interaction of the kinase inhibitors sunitinib, erlotinib, idelalisib, and lenvatinib; these drugs are characterized by medium log p values, a parameter reflecting the overall hydrophobicity of the molecules, which is one important parameter to predict the interaction with lipid membranes. While all four molecules tend to embed in a similar region of the lipid membrane, their presence has different impacts on membrane structure and dynamics. Most notably, sunitinib, exhibiting the lowest log p value of the four inhibitors, effectively influences membrane integrity, while the others do not. This shows that the estimation of the effect of drug molecules on lipid membranes can be rather complex. In this context, experimental studies on lipid membranes are necessary to (i) identify drugs that may disturb membranes and (ii) characterize drug–membrane interactions on a molecular level. Such knowledge is important for understanding the efficacy and potential side effects of respective drugs.

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