scholarly journals The Chloride Conductance Inhibitor NS3623 Enhances the Activity of a Non-selective Cation Channel in Hyperpolarizing Conditions

2021 ◽  
Vol 12 ◽  
Author(s):  
David Monedero Alonso ◽  
Laurent Pérès ◽  
Aline Hatem ◽  
Guillaume Bouyer ◽  
Stéphane Egée
Keyword(s):  
Membrane Potential ◽  
Functional Characterization ◽  
Anion Channel ◽  
Cytoskeletal Proteins ◽  
Cation Channel ◽  
Chloride Conductance ◽  
Dependent Manner ◽  
Cation Conductance ◽  
Gardos Channel

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed Pcat. Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se.

scholarly journals Role of Calcium in Phosphatidylserine Externalisation in Red Blood Cells from Sickle Cell Patients

Anemia ◽  
2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Erwin Weiss ◽  
David Charles Rees ◽  
John Stanley Gibson
Keyword(s):  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Phosphatidylserine Exposure ◽  
Channel Inhibition ◽  
Cation Conductance ◽  
Gardos Channel ◽  
Cation Permeability

Phosphatidylserine exposure occurs in red blood cells (RBCs) from sickle cell disease (SCD) patients and is increased by deoxygenation. The mechanisms responsible remain unclear. RBCs from SCD patients also have elevated cation permeability, and, in particular, a deoxygenation-induced cation conductance which mediates entry, providing an obvious link with phosphatidylserine exposure. The role of was investigated using FITC-labelled annexin. Results confirmed high phosphatidylserine exposure in RBCs from SCD patients increasing upon deoxygenation. When deoxygenated, phosphatidylserine exposure was further elevated as extracellular [] was increased. This effect was inhibited by dipyridamole, intracellular chelation, and Gardos channel inhibition. Phosphatidylserine exposure was reduced in high saline. levels required to elicit phosphatidylserine exposure were in the low micromolar range. Findings are consistent with entry through the deoxygenation-induced pathway (), activating the Gardos channel. [] required for phosphatidylserine scrambling are in the range achievablein vivo.

scholarly journals Evolutionary selection of a 19-stranded mitochondrial β-barrel scaffold bears structural and functional significance

2020 ◽  
Vol 295 (43) ◽  
pp. 14653-14665
Author(s):  
Shashank Ranjan Srivastava ◽  
Radhakrishnan Mahalakshmi
Keyword(s):  
Functional Significance ◽  
Anion Channel ◽  
Dependent Manner ◽  
Structural Adaptation ◽  
Voltage Dependent Anion Channel ◽  
Voltage Dependent ◽  
Gated Channel ◽  
Engineered Structures ◽  
Transmembrane Transporters

Transmembrane β-barrels of eukaryotic outer mitochondrial membranes (OMMs) are major channels of communication between the cytosol and mitochondria and are indispensable for cellular homeostasis. A structurally intriguing exception to all known transmembrane β-barrels is the unique odd-stranded, i.e. 19-stranded, structures found solely in the OMM. The molecular origins of this 19-stranded structure and its associated functional significance are unclear. In humans, the most abundant OMM transporter is the voltage-dependent anion channel. Here, using the human voltage-dependent anion channel as our template scaffold, we designed and engineered odd- and even-stranded structures of smaller (V216, V217, V218) and larger (V220, V221) barrel diameters. Determination of the structure, dynamics, and energetics of these engineered structures in bilayer membranes reveals that the 19-stranded barrel surprisingly holds modest to low stability in a lipid-dependent manner. However, we demonstrate that this structurally metastable protein possesses superior voltage-gated channel regulation, efficient mitochondrial targeting, and in vivo cell survival, with lipid-modulated stability, all of which supersede the occurrence of a metastable 19-stranded scaffold. We propose that the unique structural adaptation of these transmembrane transporters exclusively in mitochondria bears strong evolutionary basis and is functionally significant for homeostasis.

scholarly journals Functional characterization of a voltage-gated anion channel from teleost fish intestinal epithelium

1988 ◽  
Vol 136 (1) ◽  
pp. 383-403 ◽  
Author(s):  
C. A. Loretz ◽  
C. R. Fourtner
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Anion Channel ◽  
Intestinal Epithelial ◽  
Channel Conductance ◽  
Patch Clamp Technique ◽  
Voltage Gated

An anion channel was isolated, using patch-clamp technique, from the basolateral membrane of goby intestinal epithelial cells. Single-channel conductance varied over a range from 20 to 90 pS. The channel was voltage-gated over the physiological range of cell membrane potential with depolarization increasing the proportion of time in the open state. There was no Ca2+ sensitivity. The selectivity sequence was SO4(2-) greater than Cl- greater than Mes-. The channel may function in vivo as one of several avenues of basolateral membrane Cl- exit with the voltage-gating property serving to match basolateral Cl- exit to apical entry.

scholarly journals Paraquat Exposure Induces Pulmonary Cell Mitophagy by Enhancing the PINK1/Parkin Signaling

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Kaixiang Liu ◽  
Zhipeng Zhan ◽  
Wei Gao ◽  
Jie Feng ◽  
Xisheng Xie
Keyword(s):  
Oxidative Stress ◽  
Acute Lung Injury ◽  
Membrane Potential ◽  
Pulmonary Fibrosis ◽  
Mitochondrial Membrane ◽  
A549 Cells ◽  
Time Dependent ◽  
Dependent Manner ◽  
Factor Expression

Paraquat (PQ) poisoning can cause oxidative stress, acute lung injury (ALI), and fibrosis. Excess oxidative stress can induce mitophagy. However, whether PQ exposure can induce mitophagy, contributing to the development of ALI and pulmonary fibrosis in vivo has not been clarified. Here, we show that PQ exposure induces ALI and fiber accumulation in the lung of rats in a time-dependent manner, accompanied by upregulating fibronectin (FN) and Collagen I (COL-I) expression. PQ exposure increased mitophagosome formation and PINK1 and Parkin expression in the lungs of rats. Similarly, PQ exposure reduced the viability and mitochondrial membrane potential, but enhanced FN, COL-I, PINK1, and Parkin expression in A549 cells. In contrast, PINK1 silencing significantly mitigated the PQ-upregulated Parkin, FN, and COL-I expression in A549 cells. Hence, PQ exposure induced ALI and fibrosis in rats by enhancing the PINK1/Parkin signaling and profibrotic factor expression in the lungs. Therefore, our findings suggest that the PINK1/Parkin signaling may be new therapeutic targets and may provide new insights in the pathogenesis of PQ-related ALI in rats.

Probenecid Inhibits Platelet Responses to Aggregating Agents in Vitro and Has a Synergistic Inhibitory Effect with Penicillin G

1996 ◽  
Vol 76 (02) ◽  
pp. 239-244 ◽  
Author(s):  
M A Packham ◽  
M L Rand ◽  
D W Perry ◽  
D H Ruben ◽  
R L Kinlough-Rathbone
Keyword(s):  
Platelet Activating Factor ◽  
Anion Channel ◽  
Inhibitory Effect ◽  
Penicillin G ◽  
Dependent Manner ◽  
Fura 2 ◽  
Synergistic Inhibitory Effect ◽  
Dose Dependent

SummaryProbenecid is an anion channel blocker and uricosuric agent, originally developed to slow the rate of excretion of penicillin. It is now also administered with many other drugs to reduce their required dosages. Recently, probenecid (2.5 mM) has been used to prevent leakage of fura-2 or fluo-3 when these indicators of cytosolic Ca2+ levels have been introduced into cells. However, we found that probenecid markedly inhibited the increases in cytosolic Ca2+ caused by ADP, thrombin, the thrombin receptor-activating peptide (SFLLRN, TRAP), ADP, sodium arachidonate, the thromboxane A2 (TXA2) mimetic U46619, and platelet-activating factor (PAF). This finding precluded the use of probenecid with platelets in measurements of cytosolic Ca2+ with indicators such as fura-2. We then investigated the effects of probenecid on aggregation and release of 14C-serotonin from prelabeled platelets. Responses to all the agonists were inhibited by 2.5 mM probenecid, but concentrations as low as 0.25-0.5 mM inhibited responses to agonists that act largely via TXA2 (collagen, sodium arachidonate and U46619). Collagen-induced TXA2 formation was inhibited in a dose-dependent manner. Responses of aspirin-pretreated platelets to thrombin, SFLLRN, U46619 and PAF were also inhibited by probenecid, indicating that prevention of TXA2 formation does not account for all the inhibitory effects. The combination of probenecid with penicillin G produced additive or synergistic inhibition of platelet responses; responses dependent on TXA2 were synergistically inhibited by concentrations of the drugs that are reached in vivo. The synergistic inhibitory effect of probenecid on platelet functions could further impair hemostasis if it has already been partially compromised by the administration of other drugs.

Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells

2002 ◽  
Vol 283 (6) ◽  
pp. G1249-G1256 ◽  
Author(s):  
Wei Qu ◽  
Kenichi Ikejima ◽  
Zhi Zhong ◽  
Michael P. Waalkes ◽  
Ronald G. Thurman
Keyword(s):  
Chloride Channels ◽  
Low Dose ◽  
Anion Channel ◽  
Isolated Hepatocytes ◽  
Dependent Manner ◽  
Sprague Dawley ◽  
Renal Tubular Cells ◽  
Parenchymal Cells ◽  
Dose Dependent

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE2, which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca2+ concentration ([Ca2+]i) in hepatic parenchymal cells by agonists released during stress, such as with PGE2 and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca2+]i in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE2 caused a dose-dependent increase in [Ca2+]i from basal values of 130 ± 10 to maximal levels of 434 ± 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca2+ dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca2+ from intracellular stores, also partially blocked the increase in [Ca2+]i caused by PGE2, suggesting that intracellular Ca2+ pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca2+ pools are involved in the increase in [Ca2+]i caused by PGE2. Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca2+]i due to PGE2 in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl− was omitted, glycine was much less effective in preventing the increase in [Ca2+]i due to PGE2. Phenylephrine, an α1-type adrenergic receptor agonist, also increased [Ca2+]i, as expected, from 159 ± 20 to 432 ± 43 nM. Glycine also blocked the increase in [Ca2+]i due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca2+]i in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.

scholarly journals Structural and functional characterization of the USP11 deubiquitinating enzyme, which interacts with the RanGTP-associated protein RanBPM

2002 ◽  
Vol 367 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Haruko IDEGUCHI ◽  
Atsuhisa UEDA ◽  
Masatsugu TANAKA ◽  
Jun YANG ◽  
Takashi TSUJI ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Proteasome Inhibitors ◽  
Immunofluorescence Assay ◽  
Dependent Manner ◽  
Yeast Two Hybrid ◽  
Cell Library ◽  
Specific Protease ◽  
Two Hybrid

RanBPM is a RanGTP-binding protein required for correct nucleation of microtubules. To characterize the mechanism, we searched for RanBPM-binding proteins by using a yeast two-hybrid method and isolated a cDNA encoding the ubiquitin-specific protease USP11. The full-length cDNA of USP11 was cloned from a Jurkat cell library. Sequencing revealed that USP11 possesses Cys box, His box, Asp and KRF domains, which are highly conserved in many ubiquitin-specific proteases. By immunoblotting using HeLa cells, we concluded that 921-residue version of USP11 was the predominant form, and USP11 may be a ubiquitous protein in various human tissues. By immunofluorescence assay, USP11 primarily was localized in the nucleus of non-dividing cells, suggesting an association between USP11 and RanBPM in the nucleus. Furthermore, the association between USP11 and RanBPM in vivo was confirmed not only by yeast two-hybrid assay but also by co-immunoprecipitation assays using exogenously expressed USP11 and RanBPM. We next revealed proteasome-dependent degradation of RanBPM by pulse—chase analysis using proteasome inhibitors. In fact, ubiquitinated RanBPM was detected by both in vivo and in vitro ubiquitination assays. Finally, ubiquitin conjugation to RanBPM was inhibited in a dose-dependent manner by the addition of recombinant USP11. We conclude that RanBPM was the enzymic substrate for USP11 and was deubiquitinated specifically.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat

1996 ◽  
Vol 76 (01) ◽  
pp. 111-117 ◽  
Author(s):  
Yasuto Sasaki ◽  
Junji Seki ◽  
John C Giddings ◽  
Junichiro Yamamoto
Keyword(s):  
Blood Flow ◽  
Thrombus Formation ◽  
Dependent Manner ◽  
Red Cell ◽  
Cell Velocity ◽  
Red Cell Velocity ◽  
The Mean ◽  
Wall Shear ◽  
Dose Dependent

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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