Modulation of human cardiovascular outward rectifying chloride channel by intra- and extracellular ATP

The macroscopic volume-regulated anion current (VRAC) is regulated by both intracellular and extracellular ATP, which has important implications in signaling and regulation of cellular excitability. The outwardly rectifying Cl− channel (ORCC) is a major contributor to the VRAC. This study investigated the effects of intracellular and extracellular ATP on the ORCCs expressed in the human cardiovascular system. With inside-out single-channel patch-clamp techniques, ORCCs were recorded from myocytes isolated from human atrium and septal ventricle and from primary cells originating from human coronary artery endothelium and human coronary artery smooth muscle. ORCCs from all of these tissues had similar biophysical properties, i.e., they were outwardly rectifying in symmetrical Cl− solutions, exhibited a slope conductance of ∼90–100 pS at positive potentials and ∼22 pS at negative potentials, and had a high open probability that was independent of voltage or time. The presence of ATP at the cytosolic face of the membrane increased the number of patches that contained functional ORCC but had no effect on gating. In contrast, “extracellular” ATP (in pipette solution) had no effect on the proportion of patches in which ORCC was detected but strongly reduced the open probability by increasing the closed dwell time. The potency order for nucleotides to affect gating was ATPγS > ATP = UTP > ADP > AMP, which suggests that a negatively charged phosphate group is involved in ORCC block. Our findings are consistent with a role of ORCC in the human cardiovasculature (atrium, ventricle, and coronary arteries). Regulation of ORCC by extracellular ATP suggests that this channel may have an important role in maintaining electrical activity and membrane potential under conditions in which extracellular ATP levels are elevated, such as with ATP release from nerve endings or during pathophysiological conditions.

Download Full-text

Disruption of actin filaments increases the activity of sodium-conducting channels in human myeloid leukemia cells.

Molecular Biology of the Cell ◽

10.1091/mbc.7.12.1857 ◽

1996 ◽

Vol 7 (12) ◽

pp. 1857-1864 ◽

Cited By ~ 32

Author(s):

Y A Negulyaev ◽

E A Vedernikova ◽

A V Maximov

Keyword(s):

Sodium Channels ◽

Actin Filaments ◽

Single Channel ◽

K562 Cells ◽

Channel Measurements ◽

Patch Clamp Technique ◽

Open Probability ◽

Pipette Solution ◽

Whole Cell

With the use of the patch clamp technique, the role of cytoskeleton in the regulation of ion channels in plasma membrane of leukemic K562 cells was examined. Single-channel measurements have indicated that disruption of actin filaments with cytochalasin D (CD) resulted in a considerable increase of the activity of non-voltage-gated sodium-permeable channels of 12 pS unitary conductance. Background activity of these channels was low; open probability (po) did not exceed 0.01-0.02. After CD, po grew at least 10-20 times. Cell-attached and whole-cell recordings showed that activation of sodium channels was elicited within 1-3 min after the addition of 10-20 micrograms/ml CD to the bath extracellular solution or in the presence of 5 micrograms/ml CD in the intracellular pipette solution. Preincubation of K562 cells with CD during 1 h also increased drastically the activity of 12 pS sodium channels. Whole-cell measurements confirmed that CD-activated channels were permeable to monovalent cations (preferentially to Na+ and Li+), but not to bivalent cations (Ca2+, Ba2+). Colchicine (1 microM), which affect microtubules, did not alter background channel activity. Our data indicate that actin filaments organization plays an important role in the regulation of sodium-permeable channels which may participate in providing passive Na+ influx in red blood cells.

Download Full-text

The signaling role of extracellular ATP in co-culture of Shiraia sp. S9 and Pseudomonas fulva SB1 for enhancing hypocrellin A production

Microbial Cell Factories ◽

10.1186/s12934-021-01637-9 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Xin Ping Li ◽

Lu Lu Zhou ◽

Yan Hua Guo ◽

Jian Wen Wang

Keyword(s):

Atp Release ◽

Specific Inhibitor ◽

Extracellular Atp ◽

Oxidase Inhibitor ◽

Disulfonic Acid ◽

Early Event ◽

Fungal Metabolites ◽

Extracellular Signaling ◽

Hypocrellin A

Abstract Background Adenosine 5′-triphosphate (ATP) plays both a central role as an intracellular energy source, and a crucial extracellular signaling role in diverse physiological processes of animals and plants. However, there are less reports concerning the signaling role of microbial extracellular ATP (eATP). Hypocrellins are effective anticancer photodynamic therapy (PDT) agents from bambusicolous Shiraia fungi. The co-culture of Shiraia sp. S9 and a bacterium Pseudomonas fulva SB1 isolated from Shiraia fruiting bodies was established for enhanced hypocrellin A (HA) production. The signaling roles of eATP to mediate hypocrellin biosynthesis were investigated in the co-culture. Results The co-culture induced release of eATP at 378 nM to the medium around 4 h. The eATP release was interdependent on cytosolic Ca2+ concentration and reactive oxygen species (ROS) production, respectively. The eATP production could be suppressed by the Ca2+ chelator EGTA or abolished by the channel blocker La3+, ROS scavenger vitamin C and NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). The bacterium-induced H2O2 production was strongly inhibited by reactive blue (RB), a specific inhibitor of membrane purinoceptors, but dependent on the induced Ca2+ influx in the co-culture. On the other hand, the application of exogenous ATP (exATP) at 10–300 µM to Shiraia cultures also promoted fungal conidiation and HA production, both of which were blocked effectively by the purinoceptor inhibitors pyridoxalphosphate-6-azophenyl-2′, 4′-disulfonic acid (PPADS) and RB, and ATP hydrolase apyrase. Both the induced expression of HA biosynthetic genes and HA accumulation were inhibited significantly under the blocking of the eATP or Ca2+ signaling, and the scavenge of ROS in the co-culture. Conclusions Our results indicate that eATP release is an early event during the intimate bacterial–fungal interaction and eATP plays a signaling role in the bacterial elicitation on fungal metabolites. Ca2+ and ROS are closely linked for activation of the induced ATP release and its signal transduction. This is the first report on eATP production in the fungal–bacterial co-culture and its involvement in the induced biosynthesis of fungal metabolites. Graphic abstract

Download Full-text

Ryanodine receptor dysfunction in porcine stunned myocardium

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.273.2.h796 ◽

1997 ◽

Vol 273 (2) ◽

pp. H796-H804 ◽

Cited By ~ 3

Author(s):

C. Valdivia ◽

J. O. Hegge ◽

R. D. Lasley ◽

H. H. Valdivia ◽

R. Mentzer

Keyword(s):

Coronary Artery ◽

Lipid Bilayers ◽

Ryanodine Receptor ◽

Myocardial Stunning ◽

Single Channel ◽

Stunned Myocardium ◽

Wall Thickening ◽

Planar Lipid Bilayers ◽

Open Probability ◽

Single Channel Recordings

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

Download Full-text

Abstract 950: Direct Evidence for an Important Role of Agonist-independent Constitutive I K,ACh Channel Activity in Atrial Tachycardia Remodeling

Circulation ◽

10.1161/circ.116.suppl_16.ii_187-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Niels Voigt ◽

Ange Maguy ◽

Yung-Hsin Yeh ◽

Xiao-Yan Qi ◽

Ursula Ravens ◽

...

Keyword(s):

Atrial Tachycardia ◽

Single Channel ◽

Channel Activity ◽

Open Probability ◽

Direct Demonstration ◽

Open Time ◽

Therapy Target ◽

Channel Properties ◽

Constitutively Active

Background: Although atrial tachycardia (AT) appears to promote agonist-independent constitutively active I K,ACh that increases susceptibility to AF, direct demonstration of dysregulated I K,ACh channel function is lacking. We studied AT effects on single I K,ACh channel activity in dog atria. Methods: I K,ACh channel activity was recorded with cell-attached patch clamp in isolated atrial myocytes of control (CTL) and AT (7 days, 400 min −1 ) dogs. Results : AT prolonged inducible AF duration from 44±22 to 413±167 s; N=9 dogs/gp, P<0.001. In the absence of cholinergic stimulation, single-channel openings with typical I K,ACh conductance and rectification were observed in CTL and AT (Figure ). AT produced prominent agonist-independent I K,ACh activity due to 7-fold increased opening frequency (f o ) and 10-fold increased open probability (P o ) vs CTL (P<0.01 for each), but unaltered open time and single channel conductance. With maximum I K,ACh activation (10 μm carbachol, CCh), f o was 38% lower, open time constant 25% higher, and P o and unitary conductance unchanged for AT vs CTL. The selective Kir3 blocker tertiapin (100 nM) reduced f o and P o by 48% and 51% (P<0.05 each) without altering other channel properties, confirming the identity of I K,ACh. Conclusions : AT produces prominent agonist-independent constitutive single-channel I K,ACh activity, providing a molecular basis for previously-observed AT-enhanced macroscopic I K,ACh , as well as associated AP-shortening and tertiapin-suppressible AF promotion. These results suggest an important role for constitutively active I K,ACh channels in AT-remodeling and support their interest as a potential novel AF-therapy target.

Download Full-text

Role of AIF in human coronary artery endothelial cell apoptosis

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00579.2003 ◽

2004 ◽

Vol 286 (1) ◽

pp. H354-H358 ◽

Cited By ~ 18

Author(s):

Wenguang Zhang ◽

Dayuan Li ◽

Jawahar L. Mehta

Keyword(s):

Coronary Artery ◽

Large Scale ◽

Cell Injury ◽

Density Lipoprotein ◽

Immunoblot Analysis ◽

Terminal Deoxynucleotidyl Transferase ◽

Human Coronary Artery ◽

Complementary Sequence ◽

Fourfold Increase

Apoptosis-inducing factor (AIF), which exerts its effect via a caspase-independent pathway, has been suggested to be a mediator of cell injury. We have recently identified the expression of AIF in human coronary artery endothelial cells (HCAECs). The present study was designed to determine the pathophysiological role of AIF in oxidized low-density lipoprotein (ox-LDL)-induced apoptosis of HCAECs. The cells were cultured and treated with ox-LDL (40 μg/ml) for 24 h. Ox-LDL increased AIF expression, caused apoptosis of HCAECs (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and large-scale DNA fragmentation), and induced translocation of AIF from the cytoplasm to the nucleus (fluorescence immunocytochemistry). Pretreatment of HCAECs with a caspase inhibitor (ZVAD-fmk) did not influence AIF-mediated apoptosis in response to ox-LDL. We developed a specific antisense oligonucleotide targeted to the 5′-TCG CCG AAA TGT TCC GGT GTG GA-3′ portion of the human AIF mRNA sequence (AIF-AS) to bind a complementary sequence overlapping the translational start site. Pretreatment of cells with the AIF-AS for 24 h resulted in suppression of ox-LDL-upregulated AIF protein, as measured by immunoblot analysis. AIF-AS also reduced apoptosis and AIF translocation ( P < 0.01 vs. ox-LDL alone). Next, we constructed a recombinant AIF plasmid by inserting whole-length AIF cDNA into the expression vector pcDNA3.1 with a cytomegalovirus promoter. HCAECs transfected with plasmid showed a two- to fourfold increase in AIF expression, extensive apoptosis, and translocation of AIF from the cytoplasm to the nucleus. These results from two approaches indicate that AIF plays an important role in ox-LDL-induced endothelial injury.

Download Full-text

Interleukin-37: The Effect of Anti-Inflammatory Response in Human Coronary Artery Endothelial Cells

Mediators of Inflammation ◽

10.1155/2019/2650590 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

Xianfeng Yan ◽

Bin Xie ◽

Guihai Wu ◽

Jing Hu ◽

Di Wang ◽

...

Keyword(s):

Innate Immunity ◽

Endothelial Cells ◽

Coronary Artery ◽

Inflammatory Response ◽

Atherosclerotic Plaque ◽

Inflammatory Responses ◽

Plaque Formation ◽

Human Coronary Artery ◽

Atherosclerotic Plaque Formation

Interleukin-37 (IL-37) is unique in the IL-1 family since it broadly suppresses innate immunity and elevates in humans with inflammatory and autoimmune diseases. IL-37 shows definite groups and transcripts for human IL37 gene, but it is still not completely understood the effect and mechanisms of inflammatory response in endothelial cells. It is well accepted that endothelial dysfunction caused by inflammation is a key initiating event in atherosclerotic plaque formation, which leads to the occurrence and development of the cardiovascular adverse events in clinical since the inflammatory responses of endothelial cells could induce and enhance the deposition of extensive lipid and the formation of atherosclerotic plaque in the intima. Thus, it is essential to investigate the role and potential mechanisms in endothelial inflammatory response to prevent the formation and development of many cardiovascular diseases including atherosclerosis. So far, the recent studies have revealed that IL-37 is able to inhibit inflammatory response by suppressing the TLR2-NF-κB-ICAM-1 pathway intracellularly in human coronary artery endothelial cells (HCAECs). Further, the role of IL-37 may be related to the IL-18 pathway extracellularly and involved in the adhesion and transmigration of neutrophils in HCAECs.

Download Full-text

Eicosanoids modulate apical Ca(2+)-dependent K+ channels in cultured rabbit principal cells

AJP Renal Physiology ◽

10.1152/ajprenal.1992.263.1.f116 ◽

1992 ◽

Vol 263 (1) ◽

pp. F116-F126 ◽

Cited By ~ 6

Author(s):

B. N. Ling ◽

C. L. Webster ◽

D. C. Eaton

Keyword(s):

Single Channel ◽

Primary Cultures ◽

Cell Volume Regulation ◽

Principal Cell ◽

Resting Membrane Potential ◽

Open Probability ◽

Channel Activation ◽

K Channels ◽

Pg E2

Patch clamp technology was utilized to study the effects of apical phospholipase A2 (PLA2) metabolites on “maxi K” channels in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures (B. N. Ling, C. F. Hinton, and D. C. Eaton. Kidney Int. 40: 441–452, 1991). At resting membrane potential, this channel is quiescent in the cell-attached configuration. Apical application of the PLA2 agonist melittin (1 microgram/ml) for 10 min increased single-channel open probability (Po) from 0.0004 +/- 0.0010 to 0.11 +/- 0.05. Similarly, apical exposure to 50 microM arachidonic acid (AA) or 0.5 microM prostaglandin (PG) E2, but not 0.5 microM PGF2 alpha, also increased channel activity. Conversely, 10 microM of the PLA2 antagonist quinacrine applied apically decreased Po. Removal of apical bath Ca2+ did not prevent melittin-, AA-, or PGE2-induced channel activation. We then examined the role of maxi K channels and eicosanoids in principal cell volume regulation. Within seconds of reducing basolateral bath tonicity (285 to 214 mosmol/kgH2O), NPo (i.e., no. of channels x Po) initially increased approximately 200%, followed by a delayed but prolonged activation phase that was attenuated by removal of apical bath Ca2+. Pretreatment with 10 microM quinacrine, 100 microM indomethacin (cyclooxygenase inhibitor), or 0.25 microM thapsigargin (to deplete intracellular Ca2+ stores) abolished the initial phase of swelling-induced channel activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text