scholarly journals Quantitative mapping of oxidation-sensitive cysteine residues in SERCA in vivo and in vitro by HPLC–electrospray-tandem MS: selective protein oxidation during biological aging

2006 ◽  
Vol 394 (3) ◽  
pp. 605-615 ◽  
Author(s):  
Victor S. Sharov ◽  
Elena S. Dremina ◽  
Nadezhda A. Galeva ◽  
Todd D. Williams ◽  
Christian Schöneich
Keyword(s):  
High Efficiency ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
Biological Aging ◽  
Cysteine Residues ◽  
Tandem Ms ◽  
Age Dependent ◽  
The Individual

The selective reversible S-glutathiolation of specific SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) cysteine residues represents a novel physiologic pathway of NO (nitric oxide)-dependent arterial smooth muscle relaxation [Adachi, Weisbrod, Pimentel, Ying, Sharov, Schöneich and Cohen (2004) Nat. Med. 10, 1200–1207]. This mechanism may be impaired through the irreversible oxidation of functionally important cysteine residues as a consequence of oxidative stress and aging. To establish whether in vivo aging and in vitro oxidation by peroxynitrite result in the loss of such functionally important cysteine residues of SERCA, we have developed and optimized a quantitative method to monitor the oxidation state of the individual SERCA cysteine residues using a maleimide-based fluorescence dye, TG1 (ThioGlo® 1), as a label for cysteine residues that have not been altered by oxidation and are not involved in disulphide bridges. A high efficiency for TG1 labelling of such residues and the chemical structure of cysteine–TG1 adducts were validated by MS analysis of model peptides, model proteins and rat skeletal muscle SERCA1. Tryptic peptides containing 18 out of a total of 24 cysteine residues were identified by HPLC–ESI (electrospray ionization)–MS/MS (tandem MS). Two cysteine residues, at positions 344 and 349, were detected in the form of an internal disulphide bridge, and another 16 were found to be labelled with TG1. Using HPLC–ESI–MS, we quantitatively mapped peroxynitrite oxidation of eight cysteine residues (positions 364, 417, 420, 498, 525, 674, 675 and 938), some of which are involved in the control of SERCA activity. Biological aging resulted in the partial modification of cysteine residues 377, 498, 525, 561, 614, 636, 674, 675, 774 and 938. Neither peroxynitrite exposure nor biological aging affected the apparent SERCA1 ATP affinity. Our data show an age-dependent loss of cysteine residues (approx. 2.8 mol of cysteine/mol of SERCA1), which may be partially responsible for the age-dependent decrease in the specific Ca2+-ATPase activity (by 40%).

scholarly journals Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle

1999 ◽  
Vol 340 (3) ◽  
pp. 657-669 ◽  
Author(s):  
Rosa I. VINER ◽  
Deborah A. FERRINGTON ◽  
Todd D. WILLIAMS ◽  
Diana J. BIGELOW ◽  
Christian SCHÖNEICH
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Biological Aging ◽  
Tyrosine Nitration ◽  
Age Dependent ◽  
Slow Twitch Muscle ◽  
Slow Twitch ◽  
Fast Twitch

The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (≈ 3.5±0.7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.

scholarly journals Analysis of intrapopulation polymorphism of table carrot varieties for resistance to mycotic rot pathogens of root crops

2020 ◽  
pp. 81-87
Author(s):  
Е. G. Kozar ◽  
I. A. Engalycheva ◽  
S. A. Vetrova ◽  
K. S. Muhina ◽  
T. S. Vjurtts ◽  
...  
Keyword(s):  
High Efficiency ◽  
Plant Protection ◽  
Storage Conditions ◽  
Root Crops ◽  
Different Types ◽  
The Stability ◽  
The Individual ◽  
Federal Center

Relevance. It was to study the variability of the degree of defeat of promising varieties of table carrots by diseases of various etiologies during storage for several years, to determine the level of their polymorphism based on resistance to various types of pathogens and the effectiveness of selection based on an individual immunological assessment of uterine root crops.Materials and methods. The experimental part of the work was performed in 2017-2020 at the FSBSI "Federal Scientific Vegetable Center" with carrot varietal populations of Margosha and Minor. The identification of the species composition of pathogens, the degree of damage, and the level of resistance of variety specimens were carried out in accordance with appropriate methods and determinants. For the immunological assessment of the resistance of carrot cultivars in vitro, the most virulent isolates of different types of fungi were used from the collection of the Laboratory of Immunity and Plant Protection of the Federal Center for Scientific Research.Results. It was established that in the populations of Margosha and Minor under natural storage conditions, the ratio and degree of spread of economically significant diseases differ significantly in different years. Based on an in vitro immunological assessment, a high polymorphism of the individual resistance of genotypes within both populations to the majority of micromycetes was revealed and the most resistant forms to the most dangerous types of pathogens were identified. Immunological analysis of the resulting offspring confirmed the high efficiency of group selection based on a comprehensive assessment of the stability of individual uterine root crops in vivo and in vitro.

Altered endothelial cell-mediated arterial dilation in dogs with D. immitis infection

1987 ◽  
Vol 253 (5) ◽  
pp. H1325-H1329 ◽  
Author(s):  
L. Kaiser ◽  
J. F. Williams ◽  
E. A. Meade ◽  
H. V. Sparks
Keyword(s):  
Arachidonic Acid ◽  
Endothelial Cell ◽  
Vascular Reactivity ◽  
Vascular Endothelial Cells ◽  
Seasonal Pattern ◽  
Muscle Relaxation ◽  
Arachidonic Acid Metabolism ◽  
Smooth Muscle Relaxation

Vascular endothelial cells regulate arterial diameter in vivo and in vitro. Stimulation of endothelial cell muscarinic receptors by acetylcholine results in the production and release of a nonprostaglandin metabolite of arachidonic acid that causes vascular smooth muscle relaxation. We examined femoral artery endothelium-dependent vasodilator responses in normal dogs and dogs with heartworm (Dirofilaria immitis) infection. Endothelium-dependent vascular reactivity was attenuated in dogs with D. immitis infection studied in the spring but not in the fall. The dilator response was inversely related to the number of female worms but not related to the presence of circulating microfilariae. Indomethacin markedly depressed responses to acetylcholine in dogs with D. immitis but did not alter acetylcholine-induced dilation in normal dogs. These data suggest that D. immitis releases substances that alter distal arterial endothelial cell arachidonic acid metabolism. The seasonal pattern may reflect the onset of maximal reproductive activity in the spring and its decline as the vector season ends in the fall.

scholarly journals Acetate intolerance is mediated by enhanced synthesis of nitric oxide by endothelial cells.

1997 ◽  
Vol 8 (9) ◽  
pp. 1431-1436 ◽  
Author(s):  
A Amore ◽  
P Cirina ◽  
S Mitola ◽  
L Peruzzi ◽  
R Bonaudo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Muscle Relaxation ◽  
Smooth Muscle Relaxation ◽  
Powerful Stimulus ◽  
Necrosis Factor

The clinical picture of acetate intolerance strictly mimics the nitric oxide (NO) effect, including smooth muscle relaxation and extreme vasodilation. Because acetate induces production of cAMP, which is a powerful stimulus of NO synthase (NOS), we evaluated the effect of different dialysate solutions with and without acetate on NOS activity in endothelial cells (EC). NOS activity of EC, evaluated as H3-citrulline produced from H3-arginine, was modulated by the dialysate composition (e.g., 38 mmol/L acetate produced an increase of 3.2 +/- 0.39-fold compared with basal values (P < 0.0005), and the small amount of acetate (4 mmol/L) in 35 mmol/L bicarbonate solution increased the NOS activity by 2 +/- 0.49-fold (P < 0.05). Conversely, the acetate-free solution produced no effect on NOS activity. The mRNA encoding for inducible NOS was highly expressed in EC incubated with acetate buffer and also with acetate in bicarbonate dialysis buffer. The EC proliferative index was depressed by acetate (P < 0.0005), and tumor necrosis factor synthesis was increased (P < 0.0005) compared with acetate-free buffer. This study suggests that dialytic "acetate intolerance" can be induced by the activation, through cAMP and tumor necrosis factor release, of NOS. The small amount of acetate in bicarbonate dialysate, although capable of inducing in vitro NOS activation, is likely to be rapidly metabolized, whereas the large amounts of this anion in acetate fluids overwhelm metabolism by the liver. Acetate-free dialysate is the only solution that provides an acceptable level of biocompatibility both in vivo and in vitro.

scholarly journals Cytokine-induced iNOS and ERK1/2 inhibit adenylyl cyclase type 5/6 activity and stimulate phosphodiesterase 4D5 activity in intestinal longitudinal smooth muscle

2014 ◽  
Vol 307 (4) ◽  
pp. C402-C411 ◽  
Author(s):  
Sunila Mahavadi ◽  
Ancy D. Nalli ◽  
Divya P. Kumar ◽  
Wenhui Hu ◽  
John F. Kuemmerle ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
Muscle Relaxation ◽  
Muscle Cells ◽  
Smooth Muscle Relaxation ◽  
Longitudinal Smooth Muscle ◽  
Tnf Α ◽  
Camp Formation

This study identified a distinctive pattern of expression and activity of adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms in mouse colonic longitudinal smooth muscle cells and determined the changes in their expression and/or activity in response to proinflammatory cytokines (IL-1β and TNF-α) in vitro and 2,4,6 trinitrobenzene sulphonic acid (TNBS)-induced colonic inflammation in vivo. AC5/6 and PDE4D5, expressed in circular muscle cells, were also expressed in longitudinal smooth muscle. cAMP formation was tightly regulated via feedback phosphorylation of AC5/6 and PDE4D5 by PKA. Inhibition of PKA activity by myristoylated PKI blocked phosphorylation of AC5/6 and PDE4D5 and enhanced cAMP formation. TNBS treatment in vivo and IL-1β and TNF-α in vitro induced inducible nitric oxide synthase (iNOS) expression, stimulated ERK1/2 activity, caused iNOS-mediated S-nitrosylation and inhibition of AC5/6, and induced phosphorylation of PDE4D5 and stimulated its activity. The resultant decrease in AC5/6 activity and increase in PDE4D5 activity decreased cAMP formation and smooth muscle relaxation. S-nitrosylation and inhibition of AC5/6 activity were reversed by the iNOS inhibitor 1400W, whereas phosphorylation and activation of PDE4D5 were reversed by the phosphatidylinositol 3-kinase inhibitor LY294002 and the ERK1/2 inhibitor PD98059. The effects of IL-1β or TNF-α on forskolin-stimulated cAMP formation and smooth muscle relaxation reflected inhibition of AC5/6 activity and activation of PDE4D5 and were partly reversed by 1400W or PD98059 and completely reversed by a combination of the two inhibitors. The changes in the cAMP/PKA signaling and smooth muscle relaxation contribute to colonic dysmotility during inflammation.

scholarly journals Beetroot, A Remarkable Vegetable: Its Nitrate and Phytochemical Contents Can be Adjusted in Novel Formulations to Benefit Health and Support Cardiovascular Disease Therapies

Antioxidants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 960
Author(s):  
Diego dos S. Baião ◽  
Davi V. T. da Silva ◽  
Vania M. F. Paschoalin
Keyword(s):  
Patient Adherence ◽  
Ischemia Reperfusion ◽  
Muscle Relaxation ◽  
Supportive Therapy ◽  
Smooth Muscle Relaxation ◽  
Blood Pressures ◽  
Cardioprotective Effects ◽  
Myocardial Ischemia Reperfusion

The cardioprotective effects of dietary nitrate from beetroot in healthy and hypertensive individuals are undeniable and irrefutable. Nitrate and nitrate-derived nitrite are precursors for nitric oxide synthesis exhibiting an effect on cardiomyocytes and myocardial ischemia/reperfusion, improving endothelial function, reducing arterial stiffness and stimulating smooth muscle relaxation, decreasing systolic and diastolic blood pressures. Beetroot phytochemicals like betanin, saponins, polyphenols, and organic acids can resist simulated gastrointestinal digestion, raising the hypothesis that the cardioprotective effects of beetroots result from the combination of nitrate/nitrite and bioactive compounds that limit the generation of reactive oxygen species and modulate gene expression. Nitrate and phytochemical concentrations can be adjusted in beet formulations to fulfill requirements for acute or long-term supplementations, enhancing patient adherence to beet intervention. Based on in vitro, in vivo, and clinical trials, beet nitrate and its bioactive phytochemicals are promising as a novel supportive therapy to ameliorate cardiovascular diseases.

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