Do Cysteine Residues Regulate Transient Receptor Potential Canonical Type 6 Channel Protein Expression? (original) (raw)

Gene and Protein Responses of Human Monocytes to Extracellular Cysteine Redox Potential

Toxicological Sciences, 2009

The redox potential of the major thiol/disulfide couple, cysteine (Cys) and its disulfide cystine (CySS), in plasma (E h Cys) is oxidized in association with oxidative stress, and oxidized E h Cys is associated with cardiovascular disease risk. In vitro exposure of monocytes to oxidized E h Cys increases expression of the proinflammatory cytokine, interleukin-1b (IL-1b), suggesting that E h Cys could be a mechanistic link between oxidative stress and chronic inflammation. Because cell membranes contain multiple Cys-rich proteins, which could be sensitive to E h Cys, we sought to determine whether E h Cys specifically affects proinflammatory signaling or has other effects on monocytes. We used microarray analysis and mass spectrometry-based proteomics to evaluate global changes in protein redox state, gene expression, and protein abundance in monocytes in response to E h Cys. Pathway analysis results revealed that in addition to IL-1b-related pathways, components of stress/detoxification and cell death pathways were increased by oxidized E h Cys, while components of cell growth and proliferation pathways were increased by a reduced potential. Phenotypic studies confirmed that a cell stress response occurred with oxidized E h and that cell proliferation was stimulated with reduced E h . Therefore, plasma E h Cys provides a control over monocyte phenotype, which could contribute to cardiovascular disease risk and provide a novel therapeutic target for disease prevention.

Canonical Transient Receptor Potential 6 (TRPC6), a Redox-regulated Cation Channel

Journal of Biological Chemistry, 2010

This study examined the effect of H 2 O 2 on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H 2 O 2 significantly stimulated Ca 2؉ entry in a dose-dependent manner. Electrophysiological experiments showed that H 2 O 2 significantly increased TRPC6 channel open probability and whole-cell currents. H 2 O 2 also evoked a robust inward current in A7r5 vascular smooth muscle cells, which was nearly abolished by knockdown of TRPC6 using a small interfering RNA. Catalase substantially attenuated arginine vasopressin (AVP)-induced Ca 2؉ entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H 2 O 2 response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide-and H 2 O 2 -induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-snglycerol effect on TRPC6 was significantly greater in the presence of H 2 O 2 . Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H 2 O 2 . Similarly, in cells transfected with TRPC6-EGFP, confocal microscopy showed a significant increase in fluorescence intensity in the region of the cell membrane and adjacent to the membrane. AVP also increased the fluorescence intensity on the surface of the cells co-transfected with TRPC6-EGFP and V1 receptor, and this response was inhibited by catalase. These data indicate that H 2 O 2 activates TRPC6 channels via modification of thiol groups of intracellular proteins. This cysteine oxidation-dependent pathway not only stimulates the TRPC6 channel by itself but also sensitizes the channels to diacylglycerol and promotes TRPC6 trafficking to the cell surface.

Inhibition of ATP-induced calcium influx by homocysteine in human umbilical vein endothelial cells

Cell Biology International

Mechanisms involved in the association between hyperhomocysteinemia and vascular occlusive diseases remain unclear. Homocysteine (Hcy) may disturb calcium (Ca(2+) ) cytosolic regulation in endothelial cells, a process that can directly affect the synthesis of vasoactive substances, such as nitric oxide (NO). We have investigated the effect of acute and chronic incubation with high concentrations of Hcy (100 and 500 μmoles/L) on the changes in the intracellular Ca(2+) concentration ([Ca(2+) ](i) ) induced by ATP, using primary cultures of human umbilical vein endothelial cells (HUVEC). The changes in [Ca(2+) ](i,) expressed as ▵F(t) /F(b) , were measured using the microspectrofluorimetric technique with Fluo-3 as Ca(2+) indicator. HUVEC acutely exposed to Hcy did not produce significant effects on any of the parameters studied. However, chronic exposition (24 h) caused a significant decrease in the speed of store-mediated Ca(2+) entry, expressed as (▵F(t) /F(b) )/t (s(-1) ). Exposure...

N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

The Korean Journal of Physiology & Pharmacology, 2016

N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca 2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca 2+ ] і) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca 2+ ] і in human neutrophils. We observed that NAC (1 μM ~ 1 mM) and cysteine (10 μM ~ 1 mM) increased [Ca 2+ ] і in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucinephenylalanine (fMLP)-induced increase in [Ca 2+ ] i in human neutrophils was observed. In Ca 2+-free buffer, NAC-and cysteine-induced [Ca 2+ ] i increase in human neutrophils completely disappeared, suggesting that NAC-and cysteine-mediated increase in [Ca 2+ ] i in human neutrophils occur through Ca 2+ influx. NAC-and cysteine-induced [Ca 2+ ] і increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 μM) and ruthenium red (20 μM). In Na +-free HEPES, both NAC and cysteine induced a marked increase in [Ca 2+ ] i in human neutrophils, arguing against the possibility that Na +-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca 2+ ] і increasing activity. Our results show that NAC and cysteine induce [Ca 2+ ] і increase through Ca 2+ influx in human neutrophils via SKF96365-and ruthenium reddependent way.

Interaction of n-acetylcysteine and cysteine in human plasma

Journal of Pharmaceutical Sciences, 2012

N-acetyl-L-cysteine (NAC), a well-known antioxidant, has been successfully used as adjuvant therapy for late-stage childhood cerebral adrenoleukodystrophy (c-ALD); however, the mechanisms of NAC action are poorly understood. Previous research indicates that NAC serves as a precursor to L-cysteine (Cys), the rate-limiting substrate in the biosynthesis of glutathione (GSH), a potent, endogenous antioxidant. We hypothesized that NAC acts by liberating protein-bound Cys in plasma in an NAC concentration-dependent manner, which increases unbound Cys available for GSH biosynthesis. Human plasma was incubated for 1 h with varying, clinically relevant concentrations of NAC (0-1000 :g/mL). The effect of this interaction over time was evaluated by incubating plasma for 5-90 min with 100 :g/mL NAC. Unbound and bound Cys and NAC were separated by ultrafiltration, and concentrations were measured using high-performance liquid chromatography-mass spectrometry. Significant increases in unbound Cys were observed with increasing NAC concentrations. Also, Cys plasma protein binding decreased from 85% (10 :g/mL NAC) to approximately 0% (1000 :g/mL). Total endogenous Cys was 66% unbound at 5 min after incubation. These results demonstrate that NAC liberates endogenous, protein-bound Cys in human plasma at clinically relevant NAC concentrations. A greater understanding of NAC actions will aid in the optimization of NAC therapy including its use in c-ALD.

Plasma reduced homocysteine concentrations are increased in end-stage renal disease

Kidney International, 2001

Plasma reduced homocysteine concentrations are increased in mol/L [1, 2]. Mild hyperhomocysteinemia (15 to 30 end-stage renal disease. mol/L) occurs in subclinical folic acid or vitamin B 12 Background. Plasma total homocysteine (tHcy) concentradeficiency and in most people with chronic renal failure tions Ͼ15 mol/L are associated with an increased risk of [3-5]. Hyperhomocysteinemia is a strong independent cardiovascular disease. This is especially the case in end-stage predictor of atherosclerotic vascular events in end-stage renal disease (ESRD), in which tHcy concentrations commonly range between 20 and 30 mol/L. Adverse vascular or prorenal disease (ESRD) [6-8]. However, the epidemiologic thrombotic effects associated with hyperhomocysteinemia are evidence relating tHcy and vascular disease does not assumed to be mediated by the free sulfhydryl (reduced) form establish causality. This requires a plausible pathologic of the molecule (rHcy), but data based on fluorescence highmechanism or mechanisms by which homocysteine medipressure liquid chromatography (HPLC) indicate that rHcy ates vascular damage or thrombosis and, ultimately, concentrations are not increased in ESRD despite two-to threefold elevations in tHcy. demonstration in prospective clinical trials that effective Methods. We developed a sensitive method for measuring homocysteine-lowering treatments can reduce vascular plasma rHcy concentrations in which freshly drawn blood is risk [3, 9]. Such proof is currently lacking. incubated with sodium iodoacetate, and the resulting S-carboxy-Current hypotheses accounting for homocysteinemethylhomocysteine is analyzed by gas chromatography mass spectrometry. mediated vascular or thrombotic effects depend on the Results. Unlike with the earlier methodology, we found reactivity of its free (reduced) sulfhydryl group [1, 3, 9]. plasma rHcy concentrations two to four times higher than Approximately 2% of circulating tHcy is in the reduced normal in ESRD. These concentrations were lowered by hemoform (rHcy), approximately 70% being bound via disuldialysis and were proportional to plasma tHcy over the range fide bonds to plasma albumin [10] and another 25 to of tHcy concentrations that has been associated with increased cardiovascular risk (r 2 ϭ 0.39, P Ͻ 0.0001).

Role of TRPC6 in kidney damage after acute ischemic kidney injury

Scientific Reports, 2022

Transient receptor potential channel subfamily C, member 6 (TRPC6), a non-selective cation channel that controls influx of Ca2+ and other monovalent cations into cells, is widely expressed in the kidney. TRPC6 gene variations have been linked to chronic kidney disease but its role in acute kidney injury (AKI) is unknown. Here we aimed to investigate the putative role of TRPC6 channels in AKI. We used Trpc6−/− mice and pharmacological blockade (SH045 and BI-749327), to evaluate short-term AKI outcomes. Here, we demonstrate that neither Trpc6 deficiency nor pharmacological inhibition of TRPC6 influences the short-term outcomes of AKI. Serum markers, renal expression of epithelial damage markers, tubular injury, and renal inflammatory response assessed by the histological analysis were similar in wild-type mice compared to Trpc6−/− mice as well as in vehicle-treated versus SH045- or BI-749327-treated mice. In addition, we also found no effect of TRPC6 modulation on renal arterial myoge...